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0th Course of the European School of Neuroimmunology 623 Regeneration and the immune system

Abstract
Scar-free regeneration of tissues and organs occurs in many fish and amphibians, but in mammals is restricted to certain organs during embryonic and early fetal stages. Regeneration of amputated appendages in zebrafish and urodele amphibians (salamanders and newts) involves cellular reprogramming and proliferation at the injury site, with patterning, growth of a regeneration blastema and redifferentiation of the missing tissues. Unlike organ development in the embryo, regeneration resembles wound repair in requiring transient inflammation produced by injury. Limb blastema growth also requires factors derived from re-growing nerve axons. Why the repair process in amputated mammalian limbs does not transition to the developmental program for a new limb remains a key question in regenerative biology.
Anuran amphibians (frogs and toads) can regenerate developing hindlimbs during premetamorphosis, but this ability is lost as the limbs complete development and the larvae approach metamorphosis. In the frog Xenopus laevis the gradual loss of regenerative capacity begins as cells of the adaptive immune system become functional. Analyses of gene expression and proteomic changes in amputated hindlimbs of Xenopus larvae indicated that inflammation and its resolution are up-regulated more strongly and persistently at regeneration-incompetent stages than in younger, regenerating limbs and indicated that changes in the inflammatory response may interfere with important aspects of limb regeneration.
Immunosuppressive agents or knockdown of immune cell differentiation have been shown to restore the transient loss of regenerative ability in Xenopus tails. Similar agents improve regenerative ability of larval hindlimbs. Brief exposure of amputated, regenerationcompetent limbs to solutions of beryllium, a persistent immune adjuvant, inhibits regeneration. Beryllium causes a stronger and more persistent up-regulation of genes related to both inflammation and resolution compared to control, regenerating limbs. Limb stumps treated with this adjuvant also have normal expression levels of several markers for cellular dedifferentiation and reprogramming (such as Sall4), but fail to express key genes required for normal patterning of the regeneration blastema. This work will be reviewed, along with other important studies in the growing body of evidence that in vertebrates cells of the developing immune system determine the capacity for organ and appendage regeneration.
Genetic approaches for neurological disorders Jagodic Maja ⁎

Scar-free regeneration of tissues and organs occurs in many fish and amphibians, but in mammals is restricted to certain organs during embryonic and early fetal stages. Regeneration of amputated appendages in zebrafish and urodele amphibians (salamanders and newts) involves cellular reprogramming and proliferation at the injury site, with patterning, growth of a regeneration blastema and redifferentiation of the missing tissues. Unlike organ development in the embryo, regeneration resembles wound repair in requiring transient inflammation produced by injury. Limb blastema growth also requires factors derived from re-growing nerve axons. Why the repair process in amputated mammalian limbs does not transition to the developmental program for a new limb remains a key question in regenerative biology.
Anuran amphibians (frogs and toads) can regenerate developing hindlimbs during premetamorphosis, but this ability is lost as the limbs complete development and the larvae approach metamorphosis. In the frog Xenopus laevis the gradual loss of regenerative capacity begins as cells of the adaptive immune system become functional. Analyses of gene expression and proteomic changes in amputated hindlimbs of Xenopus larvae indicated that inflammation and its resolution are up-regulated more strongly and persistently at regeneration-incompetent stages than in younger, regenerating limbs and indicated that changes in the inflammatory response may interfere with important aspects of limb regeneration.
Immunosuppressive agents or knockdown of immune cell differentiation have been shown to restore the transient loss of regenerative ability in Xenopus tails. Similar agents improve regenerative ability of larval hindlimbs. Brief exposure of amputated, regenerationcompetent limbs to solutions of beryllium, a persistent immune adjuvant, inhibits regeneration. Beryllium causes a stronger and more persistent up-regulation of genes related to both inflammation and resolution compared to control, regenerating limbs. Limb stumps treated with this adjuvant also have normal expression levels of several markers for cellular dedifferentiation and reprogramming (such as Sall4), but fail to express key genes required for normal patterning of the regeneration blastema. This work will be reviewed, along with other important studies in the growing body of evidence that in vertebrates cells of the developing immune system determine the capacity for organ and appendage regeneration.
Genetic approaches for neurological disorders Jagodic Maja ⁎ Karolinska Institutet, Dept. Clinical Neuroscience, Center for Molecular Medicine, Stockholm, Sweden A number of cellular and molecular processes lead to pathogenic neuroinflammation in multiple sclerosis (MS). However, pinpointing the interactions that are causing disease is challenging. Unbiased genetic approaches can define evolutionary conserved gene variants and pathways, which are crucial for disease pathogenesis. Nevertheless, during three decades of genetic research only a few risk genes have been identified. In recent years, marked progress has been made in human genetics of complex diseases. This included sequencing of the human genome, identification and cataloging of variation, development of high-throughput affordable typing methods and collection of large and well annotated case-control materials. Similarly to other complex diseases, this led to a robust identification of several dozen MS risk genes. Thus, a number of genetically regulated pathways that are emerging in MS, mainly involving immune response reactions, offer better understanding of disease pathogenesis, therapeutic targets and biomarkers.
Due to the complexity of human disease, experimental models such as experimental autoimmune encephalomyelitis have been valuable for understanding disease mechanisms and development of therapies. They have a special value in dissecting the genetic control of neuroinflammation owing to a possibility to employ controlled and designed breeding strategies, unlimited samples sizes and to limit genetic heterogeneity. A number of genome-wide linkage scans identified over 50 genomic loci that regulate experimental neuroinflammation. Specially conducted crosses revealed further complexity involving gene-gene and gene-environment interactions as well as parent-of-origin effects. Recently, more advanced approaches have been that in addition to T cells, B-cell function and microglia activation as well as glutamate pathway are major players in the pathogenesis of MS. Moreover, the identification of antibodies against Aquaporin 4 in Neuromyelitis Optica is a good example how the identification of an antigen and the immune mediator can provide new understanding about the pathogenesis of CNS white matter autoimmunity.
Autoimmunity in grey matter diseases Graus Francesc ⁎
Neuroimmunological research has provided robust evidence that neurons may be the target of an autoimmune attack and that in some instances antibodies against synaptic proteins are responsible for neurological syndromes that may or may not be associated with cancer (paraneoplastic). Limbic encephalitis (LE) is presently considered the model of autoimmune grey matter disorder. LE was initially described as a clinico-pathological entity characterized by an inflamma-tory process mainly involving the hippocampi and amygdala. At present, LE are divided in two broad categories depending on the target antigens. 1. Onconeural, including Hu, Ma2 , and other intracellular antigens not related with cancer including GAD or adenylate kinase 5. Pathological studies of these LE types usually show prominent brain infiltrates of T-cells and patients have a poor prognosis with bad response to immunotherapy. 2. Cell surface antigens of the synapse that include subunits of the glutamate receptors (NMDA and AMPA), GABA receptors, and a secreted synaptic protein LGI1 (previously coprecipitated with voltage-gated potassium channels). Unlike the LE of the first group, the antibodies against these antigens are patrhogenic and patients respond better to immunotherapy. Patients with anti-LGI1 are predominantly men that develop a non paraneoplastic LE. In contrast, patients with AMPA receptor antibodies are female and many develop lung or breast cancer. Patients with LE and GABA receptor antibodies also present in many cases an associated lung cancer. Of particular interest is the encephalitis associated with antibodies to extracellular epitopes of the NR1 subunit of the NMDA receptor. Patients with this syndrome present with severe psychiatric symptoms, seizures, dyskinesias, autonomic instability and hypoventilation. This encephalitis with a profile different from LE suggests that probably there are other grey matter syndromes awaiting its characterization as autoimmune. Under physiological conditions astrocytes, the most abundant cell in the central nervous system (CNS), play critical roles in regulating CNS homeostasis, synapse formation and development and synaptic plasticity. However, following injury or disease astrocytes become reactive, an event often characterized by over expression of glial fibrillary acidic protein (GFAP) and activation of transcription factors such as nuclear factor-kappa B (NF-kB) and the production of immunoregulatory molecules, such as cytokines, chemokines and reactive oxygen species. It is in this capacity that astrocytes form an important component of CNS innate immunity and play an important role in both neuro-recovery and neurodestruction. Using mice in which NF-kB has been selectively inactivated in GFAP expressing cells we investigated the contribution of astroglial-NF-B to CNS pathophysiology and functional recovery in models or spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) . In this lecture, I will present evidence obtained from our group showing that inhibition of astroglial-NF-kB attenuates neuroinflammation, reduces neuropathology and improves functional recovery in models of SCI and EAE. We will discuss potential mechanisms of action and possible therapeutic implications of these studies.
Mechanisms of gp130 signal transduction and the regulation of astroglial and microglial function Campbell Iain ⁎ The University of Sydney, Sydney, Australia
The gp130 signal transduction protein is utilized by a family of cytokines including IL-6, LIF, CNTF and OSM. IL-6 binds membrane bound IL-6Ra or soluble (s)IL-6Ra and forms oligomers with gp130 activating the JAK/STAT1/STAT3 and SHP2/MAPK pathways. IL-6 is implicated in the pathogenesis of various neurological disorders and is a potent activator of astroglia and microglia. There is limited understanding of how IL-6 communicates with different cells in the CNS and how the activation of individual gp130-coupled signal transduction pathways are regulated and mediate the actions of IL-6. Astroglia lack IL-6Ra but respond to IL-6/sIL-6Ra in a process termed trans-signaling. Microglia respond to IL-6 via both classical (IL-6/IL-6Ra) and trans-signaling. We found that IL-6 actions in the brain involve specific molecular and cellular responses that are dependent on trans-signaling. Further highlighting the importance of trans-signaling in mediating many of the CNS effects of IL-6 is the finding that inhibiting trans-signaling reduces the severity of neuroinflammatory disease induced by astrocyte-targeted IL-6 production. Downstream of gp130, the signaling intensity and kinetics of activation of the JAK/STAT and SHP2/MAPK pathways in response to IL-6 trans-signaling is similar in astrocytes and microglial cells. While gp130-JAK/STAT signaling drives increased expression of many STAT1 and STAT3-dependent genes, few genes are regulated in response to gp130-SHP2/MAPK signaling whose primary function is negative regulation of the gp130-JAK/STAT pathway via SOCS3. The role of gp130 signaling in the glial response to neuronal injury was determined in the facial nerve axotomy model (FNA) using mice with knock-in mutations that selectively disable either the JAK/STAT or the SHP2/MAPK pathway. These studies showed: (i) both pathways contribute to the early activation and accumulation of microglia in the injured facial nerve nucleus (FNN), and (ii) astrocyte GFAP expression is STAT3-dependent being markedly reduced in the absence of gp-130-JAK/STAT3 signaling. However, normal levels of glutamine synthetase and S100β in the brain and FNN of gp130-JAK/STAT3 incompetent mice suggest that astrocyte development is not compromised nor crucially dependent on this pathway. In summary, the genetic dissection of IL-6 communication and gp130 signal transduction provides new insights into key mechanistic processes that control the functional plasticity of astroglia and microglia. vivo approaches. Myasthenia gravis (MG) is an antibody-mediated disease with considerable clinical and serological heterogeneity. Patients can be separated into several groups according to age at onset, HLA association, thymic pathology, and serology. Early onset MG (EOMG) is defined as b40 years at onset, is mainly females, with a high incidence of HLA-B8 DR3. Late onset MG (LOMG) is defined as N40 years at onset, and is increasingly recognised with a peak incidence in patients over the age of 70 years. The remaining 10% of patients have thymomas. Overall, the total numbers of LOMG exceed EOMG by about 3:1 with more males than females. In MG there are acetylcholine receptor (AChR) antibodies in 80%, muscle specific kinase (MuSK) antibodies in up to 8% and "low-affinity" AChR antibodies in about 5%. IgG from patients with these different antibodies can passively transfer disease to mice, indicating their pathogenicity, and active immunisation is also effective. The MuSK patients often have severe bulbar symptoms and the reasons for this are not yet clear but the antibodies may act both pre and postsynaptically. The frequency of MuSK antibodies appears to differ with latitude suggesting an environmental influence. In the Lambert Eaton myasthenic syndrome there are antibodies to the voltage-gated calcium channel (VGCC); and in acquired neuromyotonia antibodies to voltage-gated potassium (VGKC) channels.
Over the last few years is has been found that antibodies to proteins complexed with VGKCs are associated with central nervous system dysfunction. Morvan's syndrome includes peripheral nerve hyperexcitability, autonomic dysfunction and central involvement. Recent evidence shows that many of the antibodies in these patients recognise Caspr2, a protein that is tighly complexed with VGKCs in neuronal tissues, and is often associated with thymoma. Antibodies to another VGKC-complex protein Lgi1 are found in patients who have only central involvement with memory loss and seizures.
Identifying novel anti-glycolipid antibody targets using combinatorial glycoarrays Willison Hugh ⁎
Glycolipids act as plasma membrane receptors for a wide range of antibodies, lectins and microbes. It has long been recognised that the local topography of glycolipids in the plasma membrane is critical to these recognition events, although the biological basis for this has been relatively under-investigated. Within the last five years, emerging evidence indicates that heterodimeric clusters of different glycolipids can form highly distinct and specific epitopes for antibody binding in human autoimmune neuropathy sera. These ganglioside and glycolipid complexes are comprised of a wide range of lipid partners, and can either dramatically enhance or equally well inhibit the binding of neuropathyassociated antibodies. This presentation will describe glycoarray methodology that allows for the rapid identification of the diversity of complexes that have been so far demonstrated. In living peripheral nerve plasma membranes, complex formation can be demonstrated that either enhances or inhibits antibody binding, and the subsequent pathophysiological events, this being demonstrated for GM1 in complex with GD1a. Potential mechanisms underlying these effects are considered, although much remains to be investigated and explained. However, the implications for neurological autoimmunity are widespread, ranging from glycoarray design through to the immunopathological consequences of glycolipid complex organisation in neural plasma membranes.
Actually approved disease modifying drugs (DMDs) for relapsing-remitting multiple sclerosis include recombinant interferon (IFN-beta) and glatiramer acetate (GA). All these immunomodulatory treatments have been shown to reduce the frequency and severity of relapses, as well as reducing progression of neurological disability. However all DMDs are administered parenterally and need frequent injections which may be inconvenient and uncomfortable for patients. In addition, not all patients respond adequately and common side effects associated with these therapies may reduce adherence. The development of drugs with easier administration, such as oral agents, would further promote adherence, increase patient satisfaction and thereby improve efficacy. Two phase III clinical trials CLARITY and TRANSFORM have provided promising results for Cladribine and fingolimod respectively.
The results of the CLARITY study show that annual short-course treatment with both doses (3.5 mg/kg and 5.25 mg/kg) of Cladribine Tablets led to a significant reduction in the rate of clinical relapses, disability progression and brain lesions, as well as a significant increase in the proportion of patients who remained relapse-free. Overall, the frequencies of adverse events in both Cladribine treatment groups were comparable to those observed in the placebo group dose.
The results of TRANSFORM study, also show a significant reduction in annualized relapses rate and MRI activity. During this study, two fatal viral infections occurred. Moreover, the FREEDOMS study demonstrated a significant reduction of disease activity of both doses of Fingolimod against Interferon beta 1a (Avonex). The safety profile of the drug open some concern for the risk herpes infections and cardiovascular problems.
Other oral drugs in earlier phase of the development include BG12, Teriflunomide and Laquinimod For all these three drugs, a preliminary efficacy emerged from Phase II studies and phase III studies are ongoing.
Interferon beta (IFNb) treatment is beneficial for a majority of Relapsing-Remitting Multiple Sclerosis (RRMS) patients. However, a significant proportion of patients show poor clinical response. It has been shown that baseline IFN-response activity serves a role as biomarker to predict the clinical responsive to IFNb. Here, we hypothesized that genetic variation within the IFN-pathway, in particular the IRF5 gene, is associated with differential IFN-response gene activity and clinical response upon IFNb treatment.
A test group of 30 and a validation group of 261 patients with RRMS who started with IFNb therapy were genotyped for IRF5 rs2004640 and rs4728142 polymorphisms. IFN-response gene activity in whole blood was determined at baseline and after the start of therapy by qPCR in the test group. Treatment response to IFNb was measured by the absence/occurrence of new T2 lesions on magnetic resonance imaging (MRI) in the test group and by the time to first relapse during IFNb treatment in the validation group.
In accordance with previous studies, poor pharmacological response (low or absent induction IFN-response gene activity) was observed in patients with elevated IFN-response gene activity at baseline. A poor pharmacological response to IFNb was associated with the IRF5 rs2004640 TT and rs4728142 AA genotypes (p = 0.0006 and p = 0.0023). Accordingly, all patients (100%) with either the rs2004640 TT genotype or the rs4728142 AA genotype developed new T2 lesions during IFN-β treatment. Moreover, the number of annualized T2 lesions was higher in patients with the rs2004640 TT genotype (p = 0.020). The clinical relevance of the rs2004640 TT genotype was further demonstrated by its association with a shorter time to first relapse compared to the other genotypes in an independent group (p = 0.037).
Our findings indicate an association of IRF5 rs2004640 with the pharmacological and clinical response to IFNb in RRMS. Patients homozygous for the rs2004640 T-allele have a poor clinical response represented by more frequent development of new T2 lesions and shorter time to first relapse during IFNb treatment. The identification of genetic factors that predispose to the clinical response to IFNb treatment in RRMS is an important clinical finding, especially with the current availability of alternative drugs. If confirmed in future prospective studies, the determination of IRF5 genotypes could provide additional information for treatment decisions in RRMS patients.
Thompson Sara ⁎ , Jones Joanne, Robertson Vicki, Compston Alastair, Coles Alasdair
University of Cambridge, Cambridge, United Kingdom BAFF (B cell Activating Factor) is essential for B-cell survival and function and binds to three receptors BR-3 (BAFF-receptor), TACI (calcium modulator and cyclophilin ligand interactor) and BCMA (Bcell maturation antigen), BR-3 being the main receptor. APRIL (a proliferation-inducing ligand), also has a role in survival of plasmablasts and like BAFF, is a member of the TNFalpha superfamily of genes, but only binds TACI and BCMA not BR3.
The aim of this study was to observe changes in these B cell survival markers and whether they correlated inversely with B cell death.
In a longitudinal study on early relapsing remitting multiple sclerosis (MS) patients, B cell survival/differentiation factors, proliferation and B cell death were measured monthly over 7 visits and their disease activity measured by EDSS (expanded disability status scale) and MRI (magnetic resonance imaging).
Results demonstrate that BAFF, APRIL and TACI mRNA are increased in untreated multiple sclerosis patients compared to healthy controls whereas cell death is lower. No differences are seen between active and inactive patients.
These preliminary data show an increase in B cell survival markers with a corresponding decrease in B cell death, suggesting the possibility of exaggerated survival of potential auto-reactive B cells in these patients. This could, therefore, have implications on the pathogenicity of MS.
In CIS, sFasL correlated inversely with FA measurements of corpus callosum splenium (p= 0.01, r = −0.643) and IL-2 levels correlated inversely with FA measurements of corpus callosum rostrum (p= 0.008, r = −0.883). In RRMS, sFasL correlated inversely with FA measurements of caudate nucleus (p = 0.005, r = − 0.494) and positively with that of centrum semiovale (p= 0.010, r = 0.457). In SPMS, MIF correlated significantly with ADC values of centrum semiovale (p= 0.006, r = 0.616) and IL-2 with ADC values of capsula interna (p= 0.006, r = 0.831). In PPMS, TNF-a correlated inversely with FA levels of corpus callosum genu (p= 0.009, r = −0.579) and positively with ADC levels of corpus callosum genu (p = 0.003, r = 0.640), truncus (p= 0.002, r = 0.653), optic radiation (p= 0.008, r = 0.591) and thalamus (p= 0.008, r = 0.591). Levels of IFN-g also correlated positively with ADC values in PPMS (p= 0.001, r = 0.682).
Correlations between immunologic and DTI findings in different MS subtypes are consistent with evolution of pathophysiology over disease course. It seems that some of immune molecules and DTI parameters evaluated by us might be utilized as biomarkers in MS.
Chanvillard Coralie ⁎ ,2 , Swaminathan Bhairavi 1 , Alloza Iraide 1 , Vandenbroeck Koen 1 , Infante-Duarte Carmen 2 1 Neurogenomiks, Ikerbasque and Universidad del País Vasco (UPV/EHU), Leioa, Spain; 2 Experimental Neuroimmunology, Experimental and Clinical Research Center, Charité Universitätmedizin, Berlin, Germany CX3CR1 is the only known receptor for fractalkine (CX3CL1), a chemokine that exists in soluble and surface-bound form and mediates both chemotaxis and adhesion of leucocytes. Our previous data from gene expression and flow cytometry studies demonstrated a significantly lower expression of CX3CR1 on natural killer (NK) cells in MS patients compared with healthy individuals. This suggested that disease activity might be related to the expression of CX3CR1. To explore this hypothesis we examined genetic variants of CX3CR1 and their possible association with MS.
A clinically well-defined Spanish cohort of 574 MS patients and 563 healthy controls was genotyped for single nucleotide polymorphisms (SNPs) in the CX3CR1 gene, using the 7500HT Fast Real-Time PCR system. We examined the SNPs rs3732379 and rs3732378, leading respectively to amino-acid substitutions V249I and T280M. The odds ratio of these SNPs with MS susceptibility was calculated with the Cochran-Armitage test using the PLINK software. The Haploview software was used to determine the haplotypes ratios and their correlated chi-squared test and p-value. Following the analysis of the whole dataset, we are currently in the process of stratifying data based on MS clinical progression (primary-progressive versus relapsing-remitting MS), as well as gender or geography.
Analyses of screening results and their correlations with disease susceptibility, clinical phenotype, gender, or ethnicity allow us to determine whether these genetic variants of CX3CR1 might represent a biological marker of MS.
Detection and quantification of microRNA expression in human peripheral blood microvesicles from multiple sclerosis patients treated with interferon-beta-1b Thamilarasan Madhan ⁎ , Koczan Dirk, Hermann Goertsches Robert, Hecker Michael, Thiesen Hans-Juergen, Zettl Uwe Klaus University of Rostock, Rostock, Germany
The purpose of this study was to investigate the presence of various microRNA species and their quantity in peripheral blood microvesicles of multiple sclerosis (MS) patients treated with interferon-beta-1b. We aim to find out the role of these microRNAs present in the microvesicles in relation to prognosis of disease course.
Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. MV contain various bioactive molecules and may (i) directly stimulate cells as a kind of 'signaling complex', and (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g. mitochondria) between cells.
Employing Applied Biosystems low density real-time micro fluidic cards, eight MS patients starting subcutaneous interferon-beta-1b therapy and three healthy controls were monitored at four different time points (baseline as well as after 48, 96 and 168 hours). Peripheral blood from the MS patients and healthy controls was subjected to differential centrifugation for the isolation of MV. The mirVana kit was used to isolate microRNAs from the MV. Putative microRNA targets were obtained from the MicroCosm database.
The real-time PCR low density microRNA array is capable of detecting 673 different microRNAs. Of those, we detected 260 in the blood-derived MV. In MS patients treated with interferon-beta, there was an up-and downregulation of about 30 different microRNAs, e.g. hsa-miR-501-3p, hsa-miR-148a and hsa-miR-16-1. These microRNAs are potentially involved in the regulation of various processes: among the predicted microRNA targets were cell adhesion molecules such as ESAM (hsa-miR-501-3p) as well as inflammatory cytokines, e.g. CCL1 (hsa-miR-501-3p) and TNF (hsa-miR-148a). However, further experiments need to be carried out to validate these results.
To conclude, we could show differential microRNA expression dynamics in peripheral blood MV of MS patients and healthy controls. More efforts are needed to associate these data with (i) gene regulatory targets in immune cells, and (ii) clinical data including disease severity and progression and therapeutic outcomes, which is both currently underway. It was described that Prenatal Stress (PS) stress can influence the behavior of the offspring. The aim of this work was investigate alterations in behavior and neurotrophins expression in adults animals subjected to PS. For this purpose, pregnant mice were individually restrained 2 hour a day, since gestational day 14, until delivery. Stressed offspring mice (n = 10) were tested at 2-months of age together with control matched mice (n = 10). Animals of each group (n = 5) were submitted to chronic stress. Results shown that PS did not induce significant alteration in the performance in the open field and in alternation task. A decrease in BNDF levels by Real-Time PCR was observed in hippocampus. Moreover BNDF and NT3 levels were decrease in lymphocytes. On the other hand these animals showed a poorer performance in the alternation test after exposure to chronic stress respect to normal animals. In addition an important decrease in BNDF levels in Clinical manifestations of intravascular large B-cell lymphoma (IVL) are varied and related to organ dysfunction caused by the occlusion of small vessels. IVL shows cognitive dysfunction and spinal cord disorder as CNS manifestations, and therefore mimics MS. It is necessary to pay attention to IVL in the neuroimmunological field. Particularly in the past, the diagnosis was usually only recognized at autopsy. Therefore, we need biomarkers for early diagnosis.
B cell attracting factor (BCA)-1 is a CXC chemokine. Recently, it is proved immunepathologically that lymphoma B cells produce BCA-1. We clarified the utility of CSF BCA-1 as a biomarker in the early clinical stage of IVL.
The study included 3 patients with pathologically definite IVL, who showed differential clinical types (following below). In the control group, various neurological disorders including MS were selected. We measured the level of BCA-1 in serum and CSF by ELISA. Case 1 is 53 years old man. He showed amnesia from three months, and then cerebellar ataxia from one month before hospitalization. During the deterioration course, he had hemophagocytic syndrome which was suggestive of Asian Variant IVL. Case 2 is 68 years old man. He showed ataxic gait from four months, and then dementia from one month before hospitalization. He was finally diagnosed classical type IVL by immunehistological examination of brain biopsy specimens. Case 3 is 54 years old man. He showed paraplegia from four months. Subsequently, he had pollakisuria from two months before hospitalization. Thoracic spine MRI revealed thoracic cord involvement. We found increased CSF BCA-1 level in the early clinical stage. The CSF levels of BCA-1in IVL were, respectively, 1638 pg/mL (Case 1), 737 pg/ mL (Case 2), and 370.2 pg/mL (Case 3), which significantly elevated. In addition, CSF BCA-1 in case 1 reflected disease activity in IVL, compared with other parameters. In contrast, CSF levels of BCA-1 in MS, NMO, CIDP, and OND were 27.3 ± 26.3 pg/mL, 100.3 ± 108.9 pg/ mL, b7.8 pg/mL, and b7.8 pg/mL, respectively. No significant differences in the serum levels of BCA-1 were found between each group. We also did an immunopathological study. Specimens obtained from Case2 brain biopsy showed BCA-1 autocrine by malignant B cells in the vessels.
We recognized the significant rise of the CSF BCA-1 level in the IVL cases. CSF BCA-1 could be a diagnostic marker for IVL in the early clinical stage. Myxovirus A (MxA) is a protein encoded by the MX1 gene with potent antiviral activity. MxA has proven to be a sensitive measure of the biological response to interferon-beta (IFNb) treatment and its activity is significantly reduced during the development of neutralizing antibodies (NABs). Nevertheless, the use of MxA as a biological marker for IFNb treatment in multiple sclerosis (MS) has been criticized for the lack of evidence of its role on disease pathogenesis and clinical response to IFNb.
Gene expression microarrays (Affymetrix Human Genome U133 Plus 2.0) were performed in peripheral blood mononuclear cells from 8 MS patients at baseline and after 3, 12 and 24 months of IFNb treatment. Four patients were negative for NABs and 4 patients developed NABs at 12 and/or 24 months. Changes in gene expression induced by IFNb over time in NAB positive and NAB negative patients were analyzed.
Nine genes followed a pattern in gene expression over time similar to the MX1 and were selected for further experiments. These genes were significantly induced by IFNb treatment and their expression was greatly reduced by the presence of NABs. Changes in gene expression of the selected genes were validated by real time PCR. In vitro experiments to characterize their specific induction by type I (IFNb and IFN-alfa) but not type II (IFN-gamma) IFNs are currently in progress. Selected genes are potential new biomarkers of IFNb bioactivity that may have functional roles in MS and in the clinical response to IFNb. 1 IDIBAPS, Hospital Clinic Department of Neurosciences, Barcelona, Spain; 2
Neuroinflammation and myelin degradation that accompany such debilitating brain diseases as multiple sclerosis are notoriously hard to study in humans due to the logistical obstacle of in vivo imaging of central nervous tissue. The human retina, which in past years has been more and more accessible via new imaging technologies, is understood to be a "window to the brain" in that its molecular environment mimics that of brain tissue during pathological events. For this reason, we have used non-resonant near-infrared Raman spectroscopy to image several key molecules implicated in neuronal inflammation. We have isolated the Raman spectra of NADH and FAD, both Krebb's cycle metabolites and markers of cellular energy production; iNOS, a protein responsible for the creation of the cellular free radical nitric oxide; Lactate, a source of neuronal energy and a reporter of glucose metabolism in neurons; Cytochrome C, a protein implicated in mitochondrial function and triggering the apoptotic cascade; L-glutamate, a major factor in excitotoxicity; N-Acetyl-Aspartate, a neurotransmitter used as a marker of neuronal degradation; and AB-Crystallin a molecular chaperone that has shown to be upregulated in the early stages of MS and is thought to be a myelin antigen. Moreover, we have obtained the Raman spectra of neural tissue using organotypic cultures of murine retina. We are currently measuring the levels of such metabolites in an in vitro model of neuroinflammation using retinal organotypic cell cultures challenged with LPS.
Hsu Nai-Jen ⁎ , Randall Philippa, Keeton Roanne, Sebesho Boipelo, Allie Nasiema, Kellaway Lauriston, Jacobs Muazzam
Pulmonary tuberculosis (TB) is the primary infection of TB, caused by the gram positive bacilli Mycobacterium tuberculosis (MTB), but the most predominant extra-pulmonary form of TB is TB Meningitis (TBM). Relatively little is known about the pathogenesis, the mechanism underlying the neurological complication, and the immunological responses during TBM. Although it is known that microglial cells take up MTB, such cells have a close relationship with neurons, may induce neuronal damage. Interestingly, another intracellular pathogen, Listeria monocytogenes has been shown in vitro to internalise in neurons. The interaction of MTB directly with neuronal cells remains largely unexplored.
An in vitro infection model was adopted to characterise the immunology of TBM in the nervous system. The primary neuron and microglia cultures were cultivated from the hippocampus and cortex of C57Bl/6 17 days old embryos. The primary cultures and neural celllines, included HT22 (neuronal), Neuro2A (neuronal) and BV2 (microglial), were infected with differing concentrations of laboratory MTB (H37Rv or H37Rv-GFP). At different time points of infection, the cells were subjected to a Ziehl Neelson or an immunofluorescent stain then analyzed using microscopy. A direct association between neuronal cells and the H37Rv at all multiplicity of infection (MOI) has been observed. To verify whether the bacilli are not only associated with the neurons but are also "internalised" by the neurons, confocal microscopy was employed. The GFP-expressing bacilli were clearly found inside the neuronal cytoplasm labelled by phalloidin marker.
In this study we show that neurons can act as target cells for MTB, which is comparable to what is seen in microglia. Visualisation of neurons actually showing uptake of MTB introduces a new dimension in studying the characteristics of TBM infection. Such neuronal responses may account for the neurological morbidity seen in patients suffering from TBM. Subacute sclerosing panencephalitis (SSPE) is a rare, persistent slow virus infection of the central nervous system (CNS) caused by measles virus (MV) that affects children several years after natural measles infection. Defective host immune response caused by MV has been proposed as a possible mechanism for the disease development.
To investigate the regulatory activities of T cells in SSPE patients, we have stimulated isolated CD4+ T cells from 12 SSPE patients, from 13 patients with other inflammatory diseases (ICON) and from 12 healthy children (CON) with anti-CD28, anti-CD28+MV peptide pool (MVp) and anti-CD3+CD28. IL-10, IL-2, IFN-g, IL-12p40, IL-12p70 and granzyme B (GrB) were measured in culture supernatants with multiple cytokine bead assays.
Spontaneous IL-10 secretion levels of CD4+ T cells and upon stimulation with anti-CD28+MVp were significantly higher in SSPE patients than in ICON (p = 0.038 and p = 0.007, respectively). IL-12p40 levels stimulated with anti-CD28+MVp were also significantly up-regulated compared to CON (p = 0.017). IL-2 secretion with costimulation alone (anti-CD28) or with TCR stimulation (anti-CD3) was lower in SSPE patients than in CON (p = 0.017 and p = 0.05). We observed no difference in IFN-g, IL-12p70 and GrB levels between groups.
Higher basal levels of IL-10 and higher response to MVp implicate the induction of IL-10 by MV in SSPE to suppress the immune response. Higher secretion of regulatory subunit of IL-12, namely IL-12p40, by anti-CD28+MVp stimulation in SSPE patients may also contribute to the suppressive effects of the virus. In accordance, decreased IL-2 levels of T cells also associate with the suppression of T cells in SSPE. 1 University of Ferrara, Ferrara, Italy; 2 Arcispedale S. Anna, Ferrara, Italy
Although Epstein-Barr virus (EBV) has recently received increasingly attention as a potential causative infectious agent in Multiple Sclerosis (MS), the significance of EBV-specific humoral immune response in MS still remains to be clarified. The aim of our study was to investigate CSF and serum levels and the presence of an intrathecal synthesis of anti-EBV IgG in MS and controls.
We measured by ELISA technique cerebrospinal fluid (CSF) and serum levels of anti-EBV IgG in 100 relapsing-remitting (RR) MS patients, grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity, in 109 patients with other inflammatory neurological disorders (OIND) and in 87 patients with non-inflammatory neurological disorders (NIND). Anti-EBV nuclear antigen-1 (EBNA-1) and anti viral capsid antigen (VCA) IgG levels were expressed as arbitrary units and quantitative intrathecal synthesis of anti-EBNA-1 and anti-VCA IgG was determined by Antibody Index (AI). The presence of EBV-specific CSF oligoclonal IgG bands (OCB) was assessed in MS patients by antigen-specific immunoblotting. CSF concentrations were higher in OIND than in MS (p b 0.0001) and NIND (p b 0.01) for anti-VCA IgG, and in MS than in NIND (p b 0.01) and in OIND than in NIND (p b 0.05) for anti-EBNA-1 IgG. Serum levels of anti-EBNA-1 IgG were more elevated in MS than in OIND and NIND (p b 0.0001). Serum titers of anti-EBNA-1 IgG were inversely (p b 0.001) correlated with EDSS. An intrathecal IgG production of anti-VCA and anti-EBNA-1 IgG, as indicated by Antibody Index, was present only in a small proportion of MS and controls (range from 1.1 to 6.4%). EBV-specific CSF-restricted OCB were detected in 25/100 (25%) MS patients. Serum levels of anti-VCA IgG were greater in MS patients without than in those with EBV-specific CSF OCB.
These findings argue against a direct pathogenetic role of EBVtargeted humoral immune response in MS. However, an intrathecal release of EBV-specific oligoclonal IgG can occur in a subset of patients with MS in whom an EBV brain persistent infection may act as a cofactor in the development of the disease. Accordingly, serum anti-EBNA-1 IgG response seems to mark MS patients compared to other inflammatory and non inflammatory conditions. Work supported by FISM (2008-R-12) and by Programma di ricerca Regione-Università 2007 -2009 Fine specificity of the B cell response to EBNA-1: A potential link between multiple sclerosis and infectious mononucleosis Mechelli Rosella ⁎ ,1 , Anderson Jourdan 2 , Annibali Viviana 1 , Cannoni Stefania 1 , Vittori Danila 1 , Coarelli Giulia 1 , Aloisi Francesca 3 , Ristori Giovanni 1 , James Judith 2 , Salvetti Marco 1 1 Sapienza University, Rome, Italy; 2 Oklahoma Medical Research Foundation, Oklahoma City, United States; 3 Istituto Superiore di Sanità, Rome, Italy
Together with epidemiological results, serological data on the humoral response to Epstein-Barr virus (EBV) in multiple sclerosis (MS) are the mainstay of the association between virus and disease. At variance with work characterizing disease-associated T cell epitopes of EBV antigens, published work within humoral EBV responses in MS have focuses on the relative quantity of antibody to select antigens and not on humoral epitopes. In order to provide insights to molecular mechanisms that may link EBV and MS, we evaluated the fine specificity of the B cell response to EBNA-1 in disease discordant monozygotic (MZ) twins, characterized for their humoral response to various pathogens that have been associated with the risk of MS.
In 20 MZ twin pairs discordant for MS we investigated the serology for EBV, human herpesvirus-6 (HHV-6), human cytomegalovirus (CMV), measles virus (MV), varicella zoster (VZV), herpes simplex (HSV [1] [2] and Bordetella pertussis (Bp) by means of commercially available assays. In 9 of these twin pairs plus 3 healthy MZ twin pairs, 3 non-twin MS patients and 2 healthy subjects, sera were evaluated for antibodies to all unique, maximally overlapping octapeptides of Epstein-Barr nuclear antigen-1 (EBNA-1). No seroprevalence differences against HHV-6, CMV, MV, VZV, HSV1-2, Bp) were noted between affected and unaffected twins. Seven healthy twins were EBV seronegative, but mixed results were obtained when these twin pairs were re-tested with other kits in different laboratories. The fine specificity experiments showed that patients and controls produced antibodies that recognized the glycine-alanine-rich portion of EBNA-1, as expected in EBV positive individuals. However, MS patients consistently responded to an additional sequence near the middle of the EBNA-1 protein, a region that is not antigenic in healthy EBV seropositive individuals. Surprisingly, this pattern is similar to that observed in patients with IM and was independent from the response to the other microbes as well as from a history of IM or smoking.
Furthermore the identified epitopes falls within a sequence that has been described as a target of the B cells response in MS patients [1] .
These results reflect the persistence of an IM-like humoral immune response to EBV in MS, supporting the epidemiological data on an increased risk of MS for individuals with a clinical history of IM and suggesting an unsatisfactory control of EBV infection in MS. [1] Sundström P, Nyström M, Ruuth K, Lundgren E. Antibodies to specific EBNA-1 domains and HLA DRB1*1501 interact as risk factors for multiple sclerosis. J Neuroimmunol. 2009 Oct 30;215 (1) (2) :102-7.
Genetic locus on chromosome 4 composed of immune-related genes regulate Herpes simplex-1 encephalitis in rat recombinant inbred strains Abdelmagid Nada ⁎ ,1 , Bereczky-Veress Biborka 1 , Bergström Tomas 2 , Sköldenberg Birgit 1 , Hubner Norbert 3 , Olsson Tomas 1 , Pravenec Michal 4 , Diez Margarita 1 1 Karolinska Institutet, Stockholm, Sweden; 2 Göteborg University, Göteborg, Sweden; 3 Max-Delbruck-Center for Molecular Medicine, Berlin, Germany; 4 Czech Academy of Sciences, Prague, Czech Republic
Herpes simplex encephalitis (HSE) is a rare disease with high mortality and significant morbidity among survivors. We have previously shown that susceptibility to HSE was host-strain dependent, as severe, lethal HSE developed after injection of human Herpes simplex type 1 virus (HSV-1) into the whiskers area of Dark Agouti (DA) rats. In contrast, HSV-1 did not penetrate into the trigeminal ganglia of Piebald Virol Glaxo (PVG) rats, which remained completely asymptomatic.
The aim of this study is to identify genetic regulation of HSE susceptibility using recombinant inbred rat strains.
Studying susceptibility to HSE in other inbred rat strains, we identified that the spontaneously hypertensive rats (SHR) are susceptible to HSE, while Brown Norway (BN) rats are completely resistant. However, in contrary to the resistant PVG in which HSV-1 did not penetrate to the CNS, in the clinically resistant BN rats the virus entered both the trigeminal ganglia and the brain stem. Immunohistochemistry revealed lower virus spread in the whiskers area and trigeminal ganglia of the susceptible SHR compared to resistant BN rats; although in the brain stem, the virus spread was more pronounced in the susceptible SHR rats.
To identify the gene(s) responsible for the susceptibility to HSE in these rat strains, we infected 29 recombinant inbred lines (RIL) between SHR and BN. Two RIL were completely resistant, seven lines showed an intermediate phenotype while twenty lines were susceptible. Linkage analysis of the HXB/BXH RIL strains revealed a significant quantitative trait locus (QTL) near the end of rat chromosome 4 (160-174 Mb) LRS 16.19 (LOD score 3.51) associated with incidence, weight loss, onset and survival after the infection. This QTL is comprised of several immune related genes. In addition, several suggestive QTLs in chromosome 1, 4, 9 and 10 were identified.
Identification of the gene(s) underlying these QTLs may reveal pathways regulating HSE development and possible new targets for therapeutic intervention.
Available data support a role of a virus in MS pathogenesis. These findings constitute a pathology very similar to that of virus-induced demyelination and have renewed research interest in HHV, particularly in the neurotropic viruses herpes simplex virus varicella zoster virus (VZV) and HHV type 6A.
This project is an attempt to highlight HSV as a probable contributing factor in MS pathogenesis. In order to pinpoint towards a probable association we have tried to correlate serum antibody titers for HSV-1, -2 with Multiple Sclerosis Functional Composite (MSFC) scale scores in MS patients and controls.
The MS group of our study consisted of 35 cases, 26 (74%) women and 9 (26%) men, an analogy quite similar to the analogy of gender in the MS population. Concerning the disease type, there were 24 (68.6%) cases of RR, 8 (22.9%) of SP and 3 (8.6%) of CIS. The mean age of the participants was 40 years (SD = 9.47, min = 23, max = 57) and the mean disease duration was 9.83 years (SD = 5.50, min = 1, max = 22) . Of the MS group, 12 (33.3%) participants were on systematic medication (mainly IF β-1a).
We used the Pearson's chi-square test, corrected by Fischer's exact test as in all cases there was one degree of freedom.
After that we performed a binomial sequential logistic regression analysis so as to discover whether the capability of the MSFC scale to differentiate MS patients from healthy controls, was further enhanced by the presence of HSV-2.
The IgG HSV 2 was significantly more frequently present in the MS (57.1%, 20 participants) versus the control group (17.1%, 6 participants), 2 (1, n = 70) = 11.99, p = 0.001.
Correct classification on the basis of the MSFC score alone was 68.6% for the control group and 57.1% for the MS one; the overall correct classification rate was 62.9%. With the addition of the second predictor the overall correct classification rate improved to 80%, reflecting success rates of classification of 85.7% and 74.3% for the control and the MS group respectively.
Our results add to the current knowledge despite the limitation of the small number of patients and controls examined and the wide range of the confidence intervals of the odds ratio. Prevalence of HSV-2 antibodies seems to be higher in MS population when compared with controls. Furthermore HSV-2 serum titers seem to enhance the predicting power of MSFC as an evaluation tool of Multiple Sclerosis natural history where higher MSFC scores favor better functionality.
In vivo HIV-1 Vpr molecular diversity: Domain-and residue-dependent neuroimmunity and neurodegeneration Na Hong 1 , Acharjee Shaona 1 , Jones Gareth 2 , Vivithanaporn Pornpun 1 , Noorbakhsh Farshid 1 , Barsby Nicola 1 , Maingat Ferdinand 1 , Ballanyi Klaus 1 , Pardo Carlos 3 , Cohen Eric A. 4 , Power Christopher ⁎ ,1 1 University of Alberta, Edmonton, Canada; 2 University of Calgary, Calgary, Canada; 3 Johns Hopkins University, Baltimore, United States; 4 University of Montreal, Montreal, Canada Viral molecular diversity and tissue-specific inflammation are the defining properties of human immunodeficiency virus (HIV)-1's biology and pathogenicity. Neuroinflammation accompanied by cytotoxic viral gene expression represent the chief effector mechanisms for the development of HIV-associated neurovirulence. Previous studies indicate the HIV-1 viral protein R (Vpr) is highly neuropathogenic in vitro and in vivo. Herein, we investigated the molecular diversity within HIV-1 Vpr and its relationship to neuroinflammation and ensuing neurovirulence.
The diversity of the HIV-1 accessory gene, Vpr, was examined in RNA sequences derived from brain and blood of HIV/AIDS patients with or without HIV-associated dementia (HAD). Specific mutations were identified at amino acid residue 77 within brain-derived Vpr sequences, which distinguished patients with HAD (77Q) from non-demented (ND) HIV/AIDS patients (77R) (pb 0.05). HIV-1 clones encoding 77R-ND stimulated higher IFN-alpha, MX1 and BST-2 transcript levels in human astrocytes relative to the 77Q-HAD encoding virus (pb 0.05). Likewise, Vpr-tranfected myeloid cells also expressed increased levels of IFNalpha and MX1 (pb 0.05). Vpr peptides (amino acids 70-96) containing the 77Q-HAD or 77R-ND motifs induced cytosolic calcium fluxes when exposed to human neurons. Human glia exposed to the 77R-ND peptide showed higher transcript levels of IFN-alpha, MX1, PRKRA and BST-2 relative to 77Q-HAD peptide (pb 0.05). The Vpr 77R-ND containing peptide was also more neurotoxic in a concentration-dependent manner when exposed to human neurons (p b 0.05). Stereotaxic implantation of full length Vpr, 77Q-HAD or 77R-ND peptides into the basal ganglia of mice revealed full length Vpr and the 77R-ND peptide caused greater neurobehavioral abnormalities associated with neuronal loss and microglial activation, compared with 77Q-HAD peptideimplanted animals (pb 0.05).
These observations underscore the potent neuropathogenic properties of Vpr but also imply that viral diversity might modulate innate neuroimmunity and neurodegeneration in unique manner to permit persistent viral gene expression and the development of neurovirulence. EBV is associated with an increased risk to develop multiple sclerosis (MS), but the mechanism underlying this association is not understood. We have investigated whether infusion of autologous Blymphoblastoid cells (B-LCL) induced with the EBV-related herpesvirus papio, which had been pulsed with relevant peptides for induction of autoimmune neuroinflammation can initiate a central nervous system targeting autoimmune response in rhesus monkeys.
Three groups of 5 animals were included; each group received three intravenous infusions of B-LCL (days 0, 28 and 56) that were either pulsed with the encephalitogenic self-peptide myelin/oligodendrocyte glycoprotein (MOG) 34-56 (group A), a mimicry peptide of the major capsid protein of cytomegalovirus (CMVmcp981-1003; group B) or citrullinated group C) . Groups A and B received on day 98 a single immunization with MOG34-56 in mineral oil (IFA). Group C monkeys were euthanized at day 98 just prior to day 98.
The infused B-LCL induced PBMC proliferation against all three peptides on top of a proliferative response against the B-LCL. The proliferation declined after termination of the B-LCL infusion and could not be sustained by the immunization with MOG34-56 in IFA. Proliferating PBMC in groups A and B were exclusively CD8+ T-cells, whereas in group C expansion of both CD4+ and CD8+ T-cells was observed. Anti-MOG antibodies were formed, mostly after the boost. Using immunohistochemical staining we could detect in the brains of group C monkeys perivascular infiltrates of mainly CD3+ and CD68+ cells together with clusters of CD3+ and CD20+ cells in the meninges. Such structures were less prominent in groups A and B. Clinical signs of autoimmune encephalitis (EAE) other than weight loss were not observed.
Infusion of gamma1 herpes virus infected autologous B-LCL pulsed with MOG (related) peptide, in particular cMOG34-56, induces a CNS targeting autoimmune reaction causing early pathological signs of MS.
Neuronal-astroglial interactions with Toxoplasma gondii cysts in the brain Sandra Halonen ⁎ , Melzer Tyler, Cranston Harlan, Pitts Betsey
Toxoplasma gondii is a protozoan parasite that is prevalent in humans and results in a chronic infection characterized by cysts located predominantly in the central nervous system. In immunosuppressed hosts, such as AIDS patients, the infection can reactivate from cysts in the brain resulting in severe and potentially fatal encephalitis. Recent studies also suggest the chronic infection may be a cause of cryptogenic epilepsy and T. gondii infection has been suggested to be associated with schizophrenia and psychosis, suggesting the chronic infection may have neuropathological effects in immunocompetant hosts. An understanding of the cyst stage of the parasite in the brain is of importance to chronic Toxoplasma infection in the immunocompetant host. Neurons are known to harbor cysts but in vitro studies suggest astrocytes can also foster development of the cysts. In this study we have addressed the question of whether neurons and astrocyte both serve as host cells for T. gondii cysts in a chronic infection in mouse brain.
Mice were infected with T. gondii and the intracellular localization of the cysts analyzed during a chronic infection (1, 2 and 6 m months post-infection). Brains of infected mice were fixed, cryosectioned, and stained with the lectin, FITC-Dolichos biflorans, to identify Toxoplasma cysts, antibodies to MAP2 and GFAP, to identify neurons and astrocytes respectively, and analyzed via confocal microscopy.
Cysts were found to occur almost exclusively in neurons throughout the chronic infection. Astrocyte interactions with neuronal-cysts were frequently observed. Astrocytes are known to associate with neuronal synapses with bidirectional communication between neurons and astrocytes know to occur, and it is thought that disruption of mechanisms involved in astrocyte-neuron signaling may result or be involved in brain diseases. The astrocyte associations with Toxoplasma cysts in neurons observed in this study, suggest a mechanism for cysts to affect glial cell physiology in chronic Toxoplasma infection in immunocompetant hosts.
14 Previous infection with murine gamma herpesvirus 68 modifies the course of EAE in C57Bl/6 mice
The University of British Columbia, Vancouver, Canada
The etiology of MS is not known, however epidemiological data and geographic patterns indicate that MS is triggered by an environmental factor in genetically susceptible individuals. A number of recent findings have identified Epstein-Barr virus (EBV) as a putative environmental trigger of MS but the mechanisms by which this virus causes MS remains elusive. Inconsistent EBV detection in the brain of MS patients indicates that EBV could be triggering disease without directly infecting the CNS. As EBV infection is restricted to humans, we used murine gamma herpesvirus 68 (MgHV), the murine homolog to EBV, to examine how MgHV infection could aid in the enhancement of an autoimmune reaction against the CNS.
C57Bl/6 mice were infected with MgHV and five weeks later EAE was induced. Mice previously infected with MgHV developed EAE earlier and with a more severe disease course showing both signs of paralysis and other neurological symptoms such as ataxia. Mice previously infected with another virus (LCMV) showed an EAE course comparable to uninfected EAE mice, confirming that increased EAE severity was a phenomenon specific for MgHV infection. Intriguingly MgHV infected mice showed T cell infiltrations both in the spinal cord and the brain parenchyma, whereas T cells were absent from the brain parenchyma of uninfected EAE mice. In CD4 T cells, IL-17 production after MOG restimulation in vitro was detected in uninfected EAE mice but was absent in T cells from MgHV EAE mice. FACS analysis on T cell activation markers showed an increased CD4 and CD8 T cell activation in all MgHV infected mice without correlation with paralysis development. Serum cytokines and chemokines like IFN-g, TNF-a, CXCL-9, CCL-4 and CCL-5 were significantly increased in MgHV infected mice at different time points post EAE induction. However these differences did not correlate with T cell infiltrations in the brain.
To date, these results showed that previous infection with MgHV enhances and modifies EAE symptoms. The absence of IL-17 production in infected mice further suggests that the disease might be triggered by different and more pathogenic T cell subsets than typical EAE. More experiments are planned to analyze the composition of the infiltrate in brains and spinal cords as well as to assess the role of innate immunity in the development of EAE in MgHV infected mice.
Rare and minute reactivation of JC virus in natalizumab long-term treated multiple sclerosis patients: A cross sectional multicenter study retardation, looking for the probable role of CMV infection in psychomotor development as an immunological mechanism involved in this process. Methods.32 children from 6 to 36 month of age with neurological disabilities of unknown cause and 50 controls sex/agematched were analyzed. CMV-specific IgG was determinate with enzyme-linked immunosorbent assay in serum/cerebrospinal fluid paired samples and the AI was quantified. Specific anti-CMVIgG was detected in 13 patients of control group and 20 patients suffering psychomotor retardation, since pathologic AI was also found in 15 patients with psychomotor retardation. Only, one patient from control group was positive to AI. Matched polymerase chain reaction (PCR) on filter paper used for biochemical screening that was kept from the neonatal period and AI data was available in 8 patients. PCR was simultaneously positive or negative in seven cases and only one patient belonged to a control group showing PCR positive with AI negative. The main clinical manifestations of the 15 patients with congenital infection were seizure (70%), chorioretinitis(28%), hepatosplenomegaly (20%) microcephaly and macrocephaly (20%). Conclusion: IgG-CMVAI should be considered as an important diagnostic tool to determinate if CMV infection is involved in patients suffer from psychomotor congenital impairment. Trypanosoma cruzi is a parasite protozoan able to colonize the central nervous system (CNS) and induce meningoencephalitis restricted to the acute phase of infection. Regardless the severe chronic myocarditis observed in one-third of the immunocompetent patients, the CNS inflammation and parasitism self-resolves in the chronic phase. However, in immunosuppressed patients the CNS is the major site of infection reactivation during the chronic infection leading to severe and, frequently, fatal meningoencephalitis. However, it remains unknown whether or not there are cognitive and behaviour alterations in T. cruzi infection. Several aspects of the CNS abnormalities detected in chagasic patients are replicated in T. cruzi-infected C3H/He mice. In this model the parasite infects glial cells, such as astrocyte and microglia, and provokes severe acute CD8-enriched meningoencephalitis paralleling parasitemia peak. Further, parasites are scarce and meningoencephalitis is absent in the chronic infection. In contrast, in C57BL/6 mice T. cruzi parasitism in the CNS self-resolves in the absence of inflammation. Herein, we analyzed cognitive deficits (learning and memory) and behaviour alterations (anxiety and depression) adopting infected mice that are resistant (C57BL/6) or susceptible (C3H/He) to T. cruzi-elicited meningoencephalitis. Several tests including open field (locomotor/anxiety), inhibitory avoidance shock and object recognition (memory) as well as tail suspension (depression) were performed. In contrast to susceptible mice, the resistant ones showed reduction in the numbers of crossings and the numbers of rearings during T. cruzi infection, indicating alterations in locomotor/exploratory activities. Neither difference between non-infected and infected nor between resistant and susceptible mice was demonstrated in the step-down latency of inhibitory avoidance shock test. However, T. cruzi-infected animals of both lineages presented similar time lapsing exploring familiar and novel objects, indicating an inability to form memory of this task. Interestingly, the tail Activin A (Act A), a member of the TGF-beta superfamily and multifunctional cytokine, is involved in the immune system and inflammatory response and has neuroprotective properties. Among other organs Act A can be found in the central nervous system and is elevated in the cerebrospinal fluid of patients with meningitis. Act A is released by microglial cells upon stimulation with bacterial TLR-agonists and has been shown to modulate the release of NO and cytokines by microglial cells. We investigated whether Act A influences the phagocytosis of Escherichia coli K1, which can cause meningitis, by microglial cells.
Primary mouse microglial cells were stimulated with different concentrations of Act A overnight followed by co-stimulation with agonists of TLR2 [P3C (Tripalmitoyl-S-glycerl-cysteine)], TLR4 [LPS (lipopolysaccaride)] and TLR9 [CpG(oligonucleotides containing unmethylated cytosine-guanosin motifs] for 24 hours. Then, microglial cells were challenged with 5 × 10 6 CFU/well E. coli K1 for 90 min at 37°C, 5% CO 2 . After bacterial exposure, extracellular bacteria were killed by treatment with Gentamicin 100 μg/ml for 60 minutes. The cells were lysed with distilled water. The number of the intracellular bacteria was determined by quantitative plating of serial 10-fold dilutions. Mann-Whitney-U-test, followed by Bonferroni's multiple comparisons test, was used to analyse the results.
Pre-treatment with Act A did not influence the phagocytosis rate of unstimulated microglial cells. In concentrations of 0.01 nM, 0.1 nM, and 1 nM, pre-treatment with Act A further increased the phagocytosis rate of microglial cells activated by agonists of TLR2, TLR4, and TLR9. The highest effects were achieved by pre-treatment with 1 nM Act A, which led to a 7-fold increase of the phagocytosis rate of microlgial cells stimulated with 0.1 μg/ml P3C (p = 0.0012), and a 3fold increase of the phagocytosis rate of microglial cells stimulated with 0.01 μg/ml LPS (p = 0.0174) and 1 μg/ml CpG (p = 0.0042). In higher concentrations (1000 nM) Act A seems to have an inhibitory effect on phagocytosis.
Pre-treatment with Act A increases the phagocytosis of E. coli K1 by murine microglia cells activated by agonists of the principal TLRs involved in the recognition of bacteria. Our findings provide further evidence for a role of Act A in the defense of bacterial infections and suggest that Act A has a stimulating effect on phagocytosis during CNS infections.
Acute stress may activate microglial cells in the brain via modulating adrenal corticosterone Sugama Shuei ⁎ ,1 , Takenouchi Takato 2 , Hiroshi Kitani 3 , Hashimoto Makoto 4 1 Nippon Medical School, Tokyo, Japan; 2 National Institute of Agrobiological Sciences, Tsukuba, Japan; 3 National Institute of Agrobiological Sciences, Tsukuba, Japan; 4 Tokyo Metropolitan Institute for Neuroscience; Tokyo; Japan Microglia is reported to be involved in various neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Our previous studies demonstrated that stressful experiences could induce morphological microglial activation. Yet, the mechanism of stress-induced microglial activation remains to be elucidated. In the present study, we investigated the mechanism by which acute stress induces microglial activation in rodent brains.
Adrenalectomized rats (ADX), sham-operated rats (SHM) and ADX plus corticosterone-administered rats (ADX+ CORT) were exposed to 2 h period of restraint combined with water immersion stress prior to brain analysis with immunohistochemistry with OX-42, OX-6, and with real time PCR quantification. Under control conditions, no morphological difference of OX-42 immunoreactive microglia was observed between sham-operated (SHM), ADX, and ADX plus corticosterone (ADX+ CORT) rats. Exposure to acute stress induced morphological microglial activation most significantly in the ADX, followed by ADX+ CORT, and SHM rats. In addition, exposures to acute stress did not induce MHC class II positive microglia cells in SHM rats, while it substantially induced HMC class II-positive activated microglial cells in the hippocampus in ADX rats. The number of activated microglia was slightly decreased by administration of CORT. Furthermore, a single session of acute stress did not increase iNOS in the brain in SHM rats. However, in the ADX rats acute stress significantly induced increased expressions of iNOS messages, whereas it was significantly decreased in ADX + CORT. In cultured microglial cells (MG6), glucocorticoids administration significantly suppressed the Several lines of evidence indicate that anti-aquaporin-4 antibody (AQP4-Ab) not only serves as a disease marker but also plays a pivotal role in the pathogenesis of Neuromyelitis optica (NMO). Animal models have recently provided evidence of the pathogenicity of AQP4-Ab in vivo. AQP4-Ab-positive IgG reproduces pathological changes strikingly similar to those of NMO patients when passively transferred to rats with experimental autoimmune encephalomyelitis (EAE). Although these observations suggest the pathogenicity of AQP4-Ab in NMO, it still remains unclear whether the antibody is a disease-modifying factor or the primary cause of the disease. In the present study, we examined whether AQP4-Ab can induce astrocytic cytotoxicity in the absence of CNS antigenspecific T cells.
After being treated with complete Freund's adjuvant (CFA) alone, eight-week-old rats were given daily injections of purified IgG collected from NMO or control patients for four consecutive days. Immunohistological examinations revealed remarkable swelling of astrocytes and perivascular loss of GFAP-and AQP4immunoreactivities (IRs) only in the NMO group. The active lesions in the NMO group were accompanied by perivascular deposition of immunoglobulin and active complement. To further examine the pivotal role of complement in the AQP4-Ab-mediated astrocytic cytotoxicity, the recipient rats were pre-treated with cobra venom factor to inhibit the complement cascade. In addition, predominant infiltration of granulocytes was observed in active lesions of the NMO group, accompanied by remarkable production of IL-8.
In the present study, we showed that when rats are pre-treated with complete Freund's adjuvant (CFA), AQP4-Ab induces its pathogenic effect in the absence of CNS antigen-specific T cells. These findings strongly support the primary role of AQP4-Ab in the pathogenesis of NMO, and suggest that danger signals provided by nonspecific inflammation can be a trigger for those who harbor the antibody to develop NMO. expression observed after LPS administration, we focused on possible gender differences for these enzymes during the acute immediate neuroinflammatory process observed during septic shock.
We have analyzed the influence of an intra peritoneal LPS injection (10 mg/kg) on the distribution pattern and expression levels of the PDE4B3 mRNA splicing variant in both male and female mice brain. Characterization of the cell populations involved in the PDE alterations was established by double in situ hybridization histochemistry and immunohistochemistry. We observed that PDE4B3 mRNA levels showed clear changes in females 24h post-injection, whereas, in male the altered expression was less evident and peaked 8h after treatment. Furthermore, we found that this downregulation was reflected in a lower percentage of Olgs expressing PDE4B3 mRNA.
Knowledge about PDE4B mRNAs expression in mouse brain in both sexes and the alterations provoked by LPS administration might help us to clarify sex-related differences in the susceptibility to autoimmune diseases. Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes, with neuroprotective properties in ischemic injury. To investigate the question of whether OPN could act as an opsonin in a disease model of the brain, we investigated the postischemic expression and localization of OPN mRNA and protein in a rat model of ischemic stroke. In addition, we characterized the subcellular localization of OPN protein, in particular in relation to the phagocytosis in the ischemic core, using ultrastructural techniques.
The middle cerebral artery in rats was occluded for 1 h by the intraluminal thread method and then recirculated. The ischemic core can be assessed by Fluoro-Jade staining and loss of cresylviolet-stained cells and microtubule-associated protein-2 immunoreactivity. Induction of OPN mRNA occurred in activated microglia/ macrophages in the ischemic core at day one after reperfusion, and this was sustained up to at least day 28. By three days after reperfusion, OPN expression was localized in the majority of Iba1+/ ED1+ cells, which were large, round, ameboid-like brain macrophages. OPN immunohistochemistry showed that homogeneously labeled profiles predominated in the ischemic core, where patchlike areas were devoid of OPN immunoreactivity. In addition, the concomitant induction of OPN and CD44 hyaluronic acid receptor, which has been identified as a receptor for osteopontin, was observed in the ischemic core. Ultrastructural immunohistochemistry showed that OPN labeling was associated with the membrane of neuronal fragments of various sizes, which were scattered in the ischemic core. In addition, intense labeling was observed over the Golgi complex and the periphery of the engulfed neuronal fragments within the macrophages.
Our data indicated that the increased immunoreactivity for OPN and CD44, which shared overlapping expression patterns in the ischemic core, occurred on the surface of neuronal fragments, and that OPN was also localized over the Golgi complex and neuronal fragments internalized by macrophages, suggesting that OPN secreted by macrophages may act as an opsonin, thereby facilitating phagocytosis by microglia/macrophages in the ischemic core in a rat model of stroke.
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2010-0000333) .
Ben-Gurion University of the Negev, Be'er-Sheva, Israel
Microglia are central nervous system (CNS) leukocytes, comprising up to 20% of the non-neuronal cell population. Arising from the hematopoietic system, they colonize the CNS during development. In the adult CNS, microglia provide the first line of defense against pathogens, however a growing body of evidence points to their unique role in maintaining homeostasis of the neuronal network, with a crucial impact on neurodegenerative processes. The cellular and molecular mechanisms by which the microglial phenotypes are gained, maintained and regulated in the healthy and diseased brain are albeit poorly understood. Herein, we established an ex-vivo system where lin-CD117+ mouse hematopoietic precursor cells and BM dendritic cells (BMDCs) are cultured on organotypic hippocampal slice cultures (OHSCs) and examined for the dynamics of their differentiation to microglia-like cells.
Our study demonstrates that when placed on the cellular milieu of the brain, both CD117+ and DCs adopt the microglia ramified morphology in a process that lasts approximately seven days. While CX3CR1, a microglial chemokine receptor involved in communication with neurons, is only slightly induced on day 2 of the culture, it is abundantly expressed by the majority of the differentiating cells on day 7, accompanied by increased expression of Iba-1, a microglial calcium-binding protein that is involved in cell mobility and phagocytosis. Concomitantly with the adoption of microglia-like features, the DC markers CD11c, MHC class II and CD86 were reduced, indicating the attenuation of the DC phenotype.
We assume that this differentiation process was imposed by a unique complex net of signals provided by the cellular milieu of the CNS. Our ex-vivo system may thus serve as a useful tool for studying how the array of signals provided by the CNS milieu induces functional changes in developing microglia, thus shedding light on the function of these cells in the healthy and diseased brain.
Comparative analysis of gene expression profiles of microglia from demyelinative lesions in osmotic demyelination syndrome rat model ation, are associated with local inflammation or immune cell infiltration mediated by cytokines. The direct effect of cytokines on oligodendrocyte survival and function is largely unknown.
We developed an ex vivo model of functional myelination utilizing organotypic cerebellar slice cultures. We studied myelination in real time by generating slice cultures from mice expressing green fluorescent protein (GFP) under the proteolipid protein (PLP) promoter. GFP was expressed by oligodendrocyte lineage cells including mature cell bodies, myelin processes, and oligodendrocyte progenitor cells. Slice culture viability was assessed by propidium iodide uptake by dead cells, and viable cultures maintained robust GFP expression for several weeks. Myelin tracts and individual oligodendrocytes were morphologically identifiable allowing for qualitative and quantitative analysis by microscopy and fluorescent signal patterns. We studied the direct effect of immune molecules on myelination including pro-inflammatory Th1 and Th17 cytokines (IFN-g, TNF-a, IL-17, IL-23, and GMCSF) and anti-inflammatory Th2 cytokines . Individual cytokines and cytokines in specific combinations readily induced cell death in slice cultures. Myelination was directly enhanced or inhibited by specific cytokines in slice cultures independent of overall cell viability. We next assayed the direct effects of cytokines on myelin de-and regeneration following a demyelinating insult. Demyelination indicated by a loss of GFP signal was induced in slice cultures by treatment with lysolecithin. De novo myelin formation was subsequently observed and returned to baseline levels over several days. Individual cytokines and cytokines in specific combinations were observed to inhibit or enhance myelin regeneration following the demyelinating insult.
Cerebellar slice cultures are an effective means to rapidly and objectively assay oligodendrocyte survival and functional myelination in real time with treatments. We demonstrated that cytokines which modulate the inflammatory microenvironment in the CNS directly enhance or inhibit myelin degeneration and de novo formation.
Delayed and opposite effects of kinins on astrocyte prostaglandin synthesis
Ben-Gurion University, Beer-Sheva, Israel
It has been shown that kinins and their receptors are over expressed in the brain under pathophysiological conditions such as inflammation. However, much less is known about the possible role of kinins, and especially bradykinin in brain inflammation. Although kinins are thought to have immediate effects, peptides may also exert longer and protein synthesis dependent actions. To evaluate this possibility, we assessed the regulation of prostaglandin E2 synthesis after 15 h bradykinin or Lys-des-Arg(9)-bradykinin B1 receptor agonist treatment in rat neonatal astrocytes.
Bradykinin, dose dependently stimulated basal and lipopolysaccharide (LPS)-induced prostaglandin E2 production, whereas exposure of astrocytes to the B1 receptor agonist decreased both basal and LPS-induced prostaglandin E2 release in a dose-dependent manner, as measured by radioimmunoassay (RIA). These kinin effects on PGE2 synthesis were completely abrogated by actinomycin-D and cycloheximide, suggesting de novo synthesis of proteins. Bradykinin also increased cyclooxygenase-2 (COX-2) protein levels about 2-fold, while the B1 receptor agonist decreased COX-2 protein expression. There was no change in COX-1 protein levels after treatment with either of the kinins.
Our data suggest a delayed feedback regulatory mechanism of kinins on astrocyte inflammation, whereby astrocyte prostaglandin synthesis is initially enhanced by bradykinin (B2) and eventually blocked by kinin breakdown product, acting on B1 receptors. At least part of this presumed feedback loop could be mediated by de novo protein synthesis of This research was supported by the Israel Science Foundation (grant No. 349/07).
Distinct alterations of glial gap junction proteins in normal appearing multiple sclerosis brain Sargiannidou Irene ⁎ ,1 , Markoullis Kyriaki 2 , Hadjisavvas Andreas 3 , Reynolds Richard 4 , Kleopa Kleopas 5 1 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; 2 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; 3 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; 4 Imperial College Faculty of Medicine, London, United Kingdom; 5 The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
Gap junction (GJ) deficient mice as well as patients with mutations in oligodendrocytic (Cx32 and Cx47) or astrocytic (Cx43) GJ proteins present with different degrees of transient or chronic CNS demyelination. Although glial GJs are essential for the integrity of CNS myelin, their role in multiple sclerosis (MS) pathology remains unknown. In this study, we examined the expression of glial GJ proteins in postmortem human brain tissue from MS patients and controls kindly provided by the UK MS Tissue Bank. Unfixed brain samples from 8 MS and 5 non-MS control brains were analyzed. Sections were characterized by immunostaining with antibodies to myelin oligodendrocyte glycoprotein (MOG) and microglia marker Iba-1. Sections included normal appearing cortex (NACx), normal appearing white matter (NAWM), and demyelinating lesions in the white matter and cortex of the brains. On average 15 images of NAWM and NACx from 5 different brain areas per case were evaluated in this study for microglia load to access the degree of inflammation. Expression of Cx32, Cx43 and Cx47 was analyzed by immunoblot and real time PCR. Results were controlled for age. Surprisingly, the expression of Cx32, which is the main GJ protein in large myelinated fibers was increased in MS NAWM compared to controls both by immunoblot and by real time PCR. In contrast, the expression of Cx43, which is the major astrocytic partner in oligodendrocyte-astrocyte GJs was markedly reduced in MS NAWM compared to controls. Cx47, which is expressed in cell bodies and proximal processes of oligodendrocytes was not significantly altered in this group.
This study provides evidence of significant alterations in the expression of glial GJ proteins in MS NAWM. Furthermore, glial GJ proteins are differentially involved and may play distinct roles in the inflammatory environment even without demyelination. Dysfunction of the GJ network in NAWM may contribute to the chronic progression in MS pathology. This project is funded by the Cyprus Research Promotion Foundation (Grant ACCESS/0308/11) and Cyprus Telethon. In the injured central nervous system (CNS), astrocytes appear as a key component of reactive gliosis. Reactive astrocytosis is defined as a hypertrophy and often hyperplasia of these cells that is associated with changes in gene expression like an increased synthesis of and cytokines. The Notch pathway is implicated in many aspects of the CNS development and functions. More recently, the Notch pathway has been identified to be involved in inflammatory events of the CNS. To address the role of Jagged1 in the modulation of an IFN-gamma induced inflammation in astrocytes we used a siRNA mediated silencing of the Notch ligand Jagged1 (siRNAJ1).
Primary astrocyte cultures, treated with siRNAJ1, were activated with IFN-g and RNA extraction were performed 1 hour and 3 hours after cell activation. In this study, we used Affymetrix Rat Gene 1.0 ST Arrays (27,000 genes) to examine alterations in gene expression of astrocytes treated with siRNAJ1. DNA microarray data filtration by intensity signal, x-fold change and p-value, revealed around 3900 genes varying for each comparison: ctr / siRNAJ1 and IFN-g / IFN-g + siRNAJ1. Several clusters of genes involved in various cellular processes such as immune response, cell signaling, cell adhesion and many others were altered by the inhibition of the Notch pathway. Previous results showing a modulation of the JAK/ STAT pathway could be confirmed in the microarray study. The microarray analysis allowed us to identify several genes and pathways (ex: IGF-1-, EGF-, and chemokine-signaling), induced by IFNgamma and implicated in inflammation, which are down regulated by the inhibition of Jagged1. Moreover, Real-Time PCR/microarray comparison of results, for several target genes identified, revealed an excellent correlation. The downregulation observed for several genes, including members of the JAK/STAT/SOCS pathways, was confirmed by Real-Time PCR. These results could indicate possible crosstalks between these signaling pathways and the Notch pathway.
Overall, these microarray studies highlighted the importance of the Notch pathway as could be observed by the very important gene number variation induced by the treatment with the siRNAJ1 on astrocytes and confirmed that Jagged1 downregulation induces a decrease of expression in elements characteristic for inflammation. Further analysis should give us the means to identify possible links between those major pathways. Multiple sclerosis (MS) is the most common neurodegenerative disease of young adults. Hallmark of MS is an autoimmune attack on oligodendrocytes, the myelinating cells of the central nervous system. This results in demyelination and ultimately causes neuronal degeneration. Remyelination, as one therapeutic strategy to ameliorate disease progress and restore neuronal function, requires the differentiation of oligodendrocyte progenitor cells (OPCs), which are present in and around the MS-lesions. Surprisingly, anti-TNFa therapy, known to down regulate TNFR1 mediated inflammation and supposed to reduce the autoimmune attack on oligodendrocytes, causes an exacerbation of MS-symptoms. This may at least partially be due to impaired repair mechanisms involving TNFR2 activation, since remyelination is inhibited in cuprizone-treated TNF-receptor 2 (TNFR2) deficient mice.
For the selective activation of human TNFR2 we designed a humanTNFR2-specific single chain TNF, which was oligomerized by the trimerization domain of tenascin C thereby functionally mimicking membrane-bound TNF. To determine a potential beneficial activity of selective TNFR2 activation of oligodendrocytes precursor cells (OPC), we prepared primary cultures from transgenic mice expressing the human TNFR2 (tgE1335 mice). Interestingly, human TNFR2 is predominantly expressed by OPCs, but not during later stages of oligodendrocyte development. We therefore analyzed the response to humanTNFR2 activation exclusively on OPCs. Our results indicate that TNFR2 activation protects OPCs from apoptosis induced by oxidative stress.
This finding is in accordance with a proposed neuroprotective role of TNFR2 signalling and support future in vivo studies on the remyelinating function of TNC-scTNFR2 in appropriate mouse models of multiple sclerosis. Clinical and Experimental Neuroimmunology, Department of Neurology, Heinrich-Heine-University, Düsseldorf, Germany
Matrix metalloproteinases (MMPs) represent a group of enzymes mediating deleterious effects in the inflamed central and peripheral nervous system; recent evidence, however, indicates that these proteinases are also crucially involved in myelin repair. Estrogen receptors (ERs) and non-genomic receptors (mERs) have been implicated to mediate neuroprotective and anti-inflammatory pathways in the central nervous system, whereas their function and the influence of its ligands in the PNS have not been sufficiently studied. Aim of this study was to elucidate the role of ERs in Schwann cells (SCs) in regulating the expression of MMPs.
Dorsal root ganglia (DRGs) were isolated from E15 rat pups and cultivated; this ex vivo model is convenient to study myelin formation and the differentiation and proliferation of glial cells in a physiological environment. Furthermore, purified SCs from neonatal rats were prepared. In the DRGs as well as in the SCs, ERβ expression was detected on protein as well as on RNA-level, whereas ERa was not detectable in SCs on protein level. Membraneimpermeable conjugate estradiol-BSA exhibited a significant modifying influence on gene expression: thus, the existence of non-genomic estrogen signalling pathways in Schwann cells is assumable. In addition, SCs and DRGs were treated with selective ER ligands. Myelination was assessed by Western Blot analysis and Sudan Black staining. Expression and activity of matrix metalloproteinases (MMPs) and other glia associated genes were measured by rtPCR and zymography. Stimulation with ERβ specific agonists led to a modification of pathways concerning proliferation and differentiation such as myelin synthesis and MMP-2 expression. MMP-9 increase after lipopolysaccharide stimulation was modifiable as well. The deprivation of intrinsic estrogen production was performed by aromatase inhibition with formestane and revealed oppositional effects.
The investigation revealed that genomic ERβ as well as nongenomic acting ERs are functionally expressed on SCs. Differential stimulation of those influences basic glia cell pathways including MMP activity and myelination. Whether these presumably neuroprotective and myelin-forming properties can be used in the treatment of disorders of the peripheral nervous system is currently being studied.
Gamma-secretase is important for microglia activity in Alzheimer's disease Farfara Dorit ⁎ , Trudler Dorit, Segev-Amzaleg Niva, Galron Ronit, Frenkel Dan
Tel-Aviv University, Tel-Aviv, Israel
The cleavage of amyloid precursor protein (APP) by gammasecretase plays an important role in the pathogenesis of Alzheimer's disease (AD). Thus, gamma-secretase is a target for the development of AD therapeutics and specific inhibitors are currently in clinical trials. Nevertheless, in this work we aim to investigate the role of gamma-secretase in mediating microglia activity towards clearing brain Alzheimer's amyloid deposits.
We show that gamma-secretase inhibitors impair microglia activity as seen in genes expression, protein levels and migration ability which resulted in a reduction of soluble beta-amyloid (Abeta) 1-42 phagocytosis. Moreover, adult microglia isolated from presenilin 2 knock out mice (PS2−/−) that bear a specific depletion in the gamma-secretase catalytic site, have impairment in clearing insoluble A-beta from AD mouse brain as compare to microglia from WT mice.
In this work, we suggest for the first time, a role of gamma-secretase in AD in regulating microglia activity that is important for clearing amyloid deposits. Thus, the investigation of gamma-secretase mediated cellular pathways in microglia may provide useful therapeutic intervention targets for AD and other neurodegenerative diseases. IL-6 has been demonstrated to have an important role in the development and malignant progression of astrocytomas by promoting angiogenesis, cell proliferation and resistance to apoptosis and radiation. Control of expression levels of cytokines such as IL-6 is thus essential for retaining cell homeostasis and preventing malignant transformation. To guarantee their transient expression, all cytokines contain AU-rich elements (ARE's) in the 3′ untranslated regions (3′ UTR) of their mRNA's. These elements confer instability, ensuring rapid mRNA degradation after transcription. IL-6 mRNA contains six ARE's. In line with this, IL-6 has been described to have a short half life in several cell systems, varying from 30 to 50 minutes. Several extracellular stimuli have been described to have a stabilizing effect on IL-6 mRNA, among others IL-1 beta , IL-17 , TNF and IL-6 itself. It is has been proven in several systems that the kinase p38 is involved in IL-6 mRNA stabilization after IL-1 induction. On the other hand, Protein Kinase C (PKC) activation has been associated with mRNA stabilization of other mRNAs such as GAP-43, COX-2, IL-1 and p21.
During the last decade more insight has been gained into the physiological consequences of IL-6 upregulation in gliomas, however the molecular mechanisms involved remain largely unclear.
Here we focus on the molecular mechanisms leading to excessive IL-6 production in a human astrocytoma cell line, 1321N1 cells. Previously, we have shown that the synergistic IL-6 production after combined beta-adrenergic and TNF-receptor triggering in 1321N1 cells is due to a transcriptional enhancer mechanism. Here, we describe that excessive IL-6 production after triple induction with isoproterenol and the pro-inflammatory cytokines TNF and IL-1 beta is due to an additional molecular mechanism, involving IL-6 mRNA stabilization by IL-1 beta. Surprisingly, this effect was PKC-but not p38-dependent and did not involve HuR translocation to the cytoplasm. The involved PKC isoform was of the novel subtype family, as based on our pharmacological inhibitor experiments.
In conclusion, the mRNA stabilizing effect of IL-1 beta in human astrocytoma cells is able to induce a very potent synergistic IL-6 expression which might have important physiological consequences.
Immunochemical changes of the neural cells in ischemic rat retinas evoked by an ischemia/reperfusion injury and by venous cauterization Shin JiMan ⁎ , Lee Jong-Hyun, Chun Myung-Hoon, Oh Su-Ja
For understanding the pathophysiology of the glaucomatous neural degeneration, a variety of experimentally evoked ischemic models were used. The present study was aimed to investigate whether the attitudes of the neural cells differentially appear in ischemic retinas evoked by ischemia/reperfusion injury (IR) or evoked by venous cauterization (VC).
IR was made by elevation of the intraocular pressure (IOP). IOP was raised by maintaining the pressure with 120 mm Hg for 1 hour via a needle cannulation of the anterior chamber and reperfusion using Sprague-Dawley rats. VC was made by an electric cauterization on three episcleral veins. Attitudes of neuronal cells were analyzed by calbindin, nNOS and osteopontin (OPN) immunohistochemistry, and those of glial cells by Griffonia simplicifolia Isolectin (GSI) B4 and GFAP immunohistochemistry.
Calbindin was expressed in the horizontal cells, amacrine cells and ganglion cells at normal state. In IR retina, calbindin expression is slightly down-regulated in the ganglion cells than those in normal and in VC. nNOS was expressed in the amacrine and displaced amacrine cells, and rarely in a population of the bipolar cells at normal. nNOS expressed bipolar cells are slightly increased in cell number in VC retina. OPN was expressed in a population of the ganglion cells at normal. OPN expression is slightly increased in the earlier stage of VC. GSIB4 labeled microglial cells appeared in the inner plexiform layer (IPL) near the blood vessels in addition to the endothelial cells at normal. Microglial cells were more populated in the IPL of the earlier IR retinas. GFAP expression was in the astrocytes in the nerve fiber layer and the ganglion cell layer at normal. GFAP expression in IR retinas appeared gradually from the proximal to the distal in the radial processes of Mueller glial cells, and that in VC was similar.
These findings demonstrate that there is no large specific change in neuronal cell attitudes, but remarkable glial cell activation in the rat retina in response to IR injury or VC. Microglial activation appears more vigorously in IR retina.
In vitro differentiation of lineage-negative bone marrow cells and monocyte into microglia-like cells Microglia are believed to be originated from hematopoietic cells which invaded into the central nervous system (CNS) during development. Even in adulthood, hematopoietic-derived cells have been reported to develop into resident perivascular macrophages and microglia. Although it has been reported that only Ly-6ChighCCR2+ monocytes can invade and differentiate into microglia, the detailed mechanisms of differentiation and transformation of microglial cells are not fully understood.
We demonstrate that murine microglial cells show 2 morphological forms in vitro, small round cells expressing CD11b, Iba1, triggering receptor expressing on myeloid cells-2 (TREM2), and weakly expressing major histocompatibility complex (MHC) class II and large flat cells expressing only CD11b and Iba1. Moreover, lineagenegative bone marrow (LN) cells cultured with primary mixed glial culture cells could differentiate into the small round microglia-like cells despite the absence of CCR2 and Gr-1 expression. The LN cells derived-small round cells and microglia were similar in terms of their cell surface molecules and the proliferative capacity. Furthermore, we show monocyte also differentiated into the small round microglia-like cells when cultured with primary mixed glial culture. Analysis by confocal microscopy revealed that the LN cell derived-small round cells were positioned above the GFAP-positive astrocytes.
The cell-cell contact with glial cells, especially astrocytes, may be necessary to maintain the small round shape of the immature cells expressing TREM2.
Induction of vascular endothelial growth factor receptor-3 mRNA in glial cells following focal cerebral ischemia in rats Recent studies reported that induction of vascular endothelial growth factor receptor (VEGFR)-3, a receptor for VEGF-C and VEGF-D is implicated to be likely in pathophysiological conditions, including neuronal damage and glial reaction in response to cerebral ischemia. However, it remains unclear whether VEGFR-3 is involved in the pathophysiology of stroke. To identify whether VEGFR-3 is involved in pathophysiology of stroke, we investigated the spatiotemporal regulation of VEGFR-3 mRNA after transient focal cerebral ischemia.
A rat focal cerebral ischemia-reperfusion model induced by middle cerebral artery occlusion was used to investigate the spatiotemporal regulation of VEGFR-3 mRNA by in situ hybridization and RT-PCR. Moreover, double-and triple-labeling techniques were used to identify the phenotypes of cells expressing VEGFR-3. To identify the origin of VEGFR-3-expressing brain macrophages, we also examined the cellular localization of VEGFR-3 mRNA in organotypic hippocampal slice cultures subjected to ischemia-like oxygen-glucose deprivation. Most of the increase in VEGFR-3 expression in the ischemic core could be attributed to brain macrophages, whereas VEGFR-3 in the periinfarct region was predominantly expressed in reactive astrocytes. In the ischemic core, VEGFR-3 was induced in almost all brain macrophages, which were large, round, ameboid-like brain macrophages. By contrast, only a small proportion of microglial cells in the peri-infarct region expressed the receptor, and most VEGFR-3 and Iba1 doublelabeled cells were highly activated stellate microglial cells. A subpopulation of VEGFR-3-expressing brain macrophages was positive for NG2 proteoglycan and showed proliferative activity. In addition, in vitro model of stroke revealed no significant induction of VEGFR-3 in activated microglial cells, indicating that infiltrating exogenous macrophages expressed VEGFR-3 after focal ischemia.
These data demonstrated that VEGFR-3 was induced in reactive astrocytes and possibly infiltrating blood-borne macrophages after focal ischemia, suggesting that VEGFR-3 may be involved in the glial reaction and possibly in the recruitment of monocytic macrophages during ischemic insults. This work was supported by Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs (Grant number: A08-4288-A22023-08N1-00010A) and by a scholarship from Seoul Science Fellowship, Seoul City (Shin, Y-J).
Injury to peripheral gustatory nerve initiates a central immune response in the brainstem Bartel Dianna L. ⁎ , Finger Thomas E.
The chorda tympani nerve (CT) is purely sensorycell bodies lie in the geniculate ganglion with peripheral processes innervating lingual taste buds and central processes extending into the nucleus of the solitary tract (nTS). The CT is prone to injury during various inner ear surgeries resulting in destruction of the peripheral processes; the ganglion cells and their central projections remain intact. Though the nerve successfully reinnervates taste buds within a few weeks of damage, taste disturbances can persist for months. These perceptual disturbances may indicate central nervous system (CNS) alterations since the periphery appears near normal after reinnervation. Trauma to mixed-function or pure motor nerves involves retrograde changes to motoneurons in the CNS, neuronal cell death, and inflammatory responses involving resident immune cells, microglia. We sought to test whether microglia respond in the nTS in a system where no neuronal degeneration occurs in the CNS despite the peripheral damage.
In adult mice the CT was cut unilaterally in the middle ear; animals survived for 1-30 days (6 times, n = 4) . Microglia were detected in the nTS using the Iba1 antibody. Microglia on the injured side had an activated morphology with short thick processes and 'ragged' nuclei, while those on the uninjured side remained in the "resting" state and highly ramified. Already at 2 days post lesion more microglia were evident on the lesioned side. By 5 days post injury, microglia numbers on the injured side were 300% of control levels but returned to normal by 30 days (no changes in numbers on the intact side). Origins for the additional microglia were assessed. Chimeric bone-marrow grafted animals show no evidence of inmigration from the periphery after nerve injury. Ki-67 staining for mitotic cells showed that by 1 day post lesion, microglia within the nTS on the lesioned side began to proliferate. All Ki-67+ cells are also Iba1+ indicating that mature microglia proliferate to generate the additional cells. Mature microglia from nearby regions of the brain also seem to migrate into the area of CT termination since microglial numbers decrease in the region ventral to CT fibers 2-3 days post injury.
In summary, damage to a pure sensory nerve evokes rapid changes in the microglia near the terminal zone of the nerve although the nerve fibers themselves remain intact. This suggests that microglia detect signals from damaged, but non-degenerating sensory terminals. Liver X receptors (LXRs) are ligand activated nuclear receptors that play an important role in the control of cellular and whole-body cholesterol homeostasis. Both LXR isoforms (LXR-a and LXR-β) are expressed in the central nervous system (CNS) and are involved in the regulation of brain cholesterol metabolism. However, their presence in oligodendrocytes (OLGs) and their function in OLG cholesterol homeostasis remain largely unknown. High cholesterol levels in OLGs are essential for myelin membrane growth during maturation of the CNS. In order to gain insight into cholesterol homeostasis in OLGs, expression levels of the LXRs and their target genes were investigated in neonatal or mature rat OLG cultures using quantitative real time PCR.
The LXRs, as well as the LXR target genes (ApoE, ABCA1, ABCG1, ABCG4 and LDLR) were detected in both primary OLG cell types. Treatment of primary neonatal rat OLGs with the LXR agonist T0901317 (1 and 10μM) induces the expression of several established LXR target genes, including ApoE, ABCA1, ABCG1, ABCG4 and LXR-a itself. Furthermore, treatment of the OLGs with T0901317 resulted in an enhanced cholesterol efflux in the presence of Apolipoprotein AI or high density lipoprotein particles. LXR activation also induces morphological changes.
These data show that LXRs and their target genes are present in OLGs and regulate their cholesterol homeostasis.
Minocycline forces microglia in an "early activation stage", independently of their activation history Brône Bert ⁎ , Dries Eef, Janssen Daniel, Rigo Jean-Michel
Microglia is considered as the resident macrophages of the central nervous system. It is generally accepted that microglial cells shift between different activated phenotypes in response to various stimuli. Depending on the stimulus, microglia can execute a proinflammatory or anti-inflammatory action in terms of e.g. nitric oxide or cytokine production. We investigated the microglial phenotypes using an electrophysiological approach. Previously it has been postulated that resting microglia have a linear current/voltage (I/V) relationship and that they up-regulate inward rectifier potassium (KIR) currents in an early activated state. During strong activation, the functional expression of delayed rectifier (KDR) potassium currents is increased in addition to the KIR current. The aim of this study was to discriminate different activation stages (pro-and anti-inflammatory) in terms of the electrophysiological expression profile of microglia.
The I/V-relationship of primary cultured rat microglia was measured using the whole-cell patch-clamp technique. The I/Vrelationship was compared in control, lipopolysaccharide-stimulated (LPS, 100 ng/ml) and minocycline-conditioned cells. In control conditions, 46% of the microglia showed a linear I/V-relationship comparable to what was previously described for resting microglia. Fifty percent of the control cells increased the expression of KIR currents (− 120 ± 26 pA at − 120 mV, n = 14) typical for early activated microglia. The presence of a KIR (− 59 ± 35 pA at −120 mV) and a large KDR (733 ± 210 pA at + 20 mV, n = 4) current in LPS-stimulated cells were in agreement with previously published results.
Minocyline is an antibiotic and anti-inflammatory drug that decreases the NO production in microglia. Therefore, we applied 400 μg/ml minocycline to LPS-stimulated cells in order to bring the microglia in a resting condition. As expected, minocycline-treatment resulted in a significant decrease of the KDR current in LPS-treated microglia (− 148 ± 70pA, n = 7). Interestingly, minocycline increased the KIR current in untreated ( −123 ± 33pA, n = 4) as well as in LPStreated microglia (− 302 ± 77 pA, n = 7).
These results suggest that, if the potassium channel expression profile is considered as an activation marker in microglia, minocycline forces microglia in their "early activated state" phenotype, independently of their activation history. Microglia are CNS specific immune cells and serve as pathologic sensors responding with a complex activation process to brain damage or dysfunction. Microglia express various subtypes of nucleotide (P2X, P2Y) and adenosine (A1, A2A and A3) receptors, which control ionic conductances, membrane potential, phagocytosis, migration, gene transcription, the production of inflammatory mediators and cell survival. We have now studied microglial phagocytic activity in two in situ models, in slices from adult (postnatal day 70 to 140) and in slices from early postnatal (day 6 to 9) mice. In the first model, we studied ramified microglial cells, in the second motile, ameboid microglia.
To monitor the phagocytic activity, we applied fluorescent latex beads to acute brain slices. Koizumi et al. (2007) has previously shown that UDP facilitated phagocytosis in microglia mediated by P2Y6 receptor activation. In agreement with this, we found that stimulation with both UTP and UDP increased phagocytic activity of ameboid microglial cells. In contrast, in the acute slices from adult mice, UDP/ UTP stimulation caused a decrease of phagocytic activity in ramified microglia. CD39, or NTPDase-1, is a microglia-specific ectoenzyme, which hydrolyzes ATP, released from damaged tissue, to ADP and AMP, whereas UTP is preferentially hydrolyzed to UDP (Zimmermann, 2006) . We found that basal phagocytic activity of ameboid and adult ramified microglia from CD39−/− mice is higher as compared to the wild type. UTP or UDP stimulation did not influence significantly the phagocytic activity in CD39−/− microglia.
In a second approach, we studied the motility of microglial processes in response to a laser lesion. To visualize microglial motility in brain slices we used mice expressing EGFP under microglia-specific CX3CR promoter. The laser lesion caused the movement of microglia processes towards the lesion within a time span of 60 min, presumably mediated by ATP released from damaged cells (Davalos et al., 2005) . To study the importance of CD39, we used CD39−/− animals cross-bred with mice expressing EGFP under microgliaspecific CX3CR promoter. In the brain slices from CD39−/− animals, the response of the processes to be attracted to a laser lesion was either very slow or completely absent.
We conclude, that microglia respond differently to the purinergic stimuli, depending on the developmental (ameboid versus ramified) and / or activation stage (CD39 WT versus KO).
Redlich Sandra ⁎ ,1 , Catharina Diesselberg 1 , Ribes Sandra 1 , Ebert Sandra 1 , Nau Roland 2 1 University of Göttingen, Department of Neurology, Göttingen, Germany; 2 Evangelisches Krankenhaus Göttingen-Weende, Department of Geriatrics, Göttingen, Germany
Microglial cells play a crucial role in the inflammatory response and can phagocytize and kill invading pathogens. Microglia express Toll-like receptors (TLR) that recognize pathogen-associated molecular patterns (PAMPs). Lipopolysaccharide (LPS) from gram-negative bacteria activates TLR4, and Tripalmitoyl-S-glycerl-cysteine (P3C) activates TLR1/2. We investigated whether LPS and P3C influence the phagocytosis of Cryptococcus neoformans yeast, which can cause meningitis in immunocompromised patients, by microglial cells in vitro.
Primary mouse microglial cells were stimulated with agonists of TLR1/2 (P3C) and TLR4 (LPS) for 24 hours. After stimulation, microglial cells were washed and infected with 5 × 10 6 CFU/well Cryptococcus neoformans for 120 min. Extracellular Cryptococci were killed by incubation in culture medium (DMEM) containing 2.5 μg/ml Amphotericin B for 60 minutes. Microglial cells were lysed with distilled water, and the number of intracellular Cryptococci was determined by quantitative plating of serial 10-fold dilutions on Sabouraud agar plates. Data are presented as means + SD. ANOVA followed by Bonferroni's multiple comparisons test was used to compare groups.
Unstimulated microglial cells were able to ingest a low number of Cryptococci (100.0 + 47.5%). Pre-treatment of microglial cells with 0.1 μg/ml LPS significantly increased the phagocytosis rate approximately 9-fold (932.3 + 571.1%; p b 0.001). Pre-treatment with 0.1 μg/ ml P3C also led to a 9-fold increase of the phagocytosis rate (902.4 + 543.0%; p b 0.01).
Pre-stimulation with LPS and P3C increases the phagocytosis of Cryptococcus neoformans by murine microglia cells in vitro. Our results suggest, that microglial activation by TLR agonists might prove beneficial for the treatment of Cryptococcus neoformans meningitis in immunocompromised patients.
Productions and functions of interleukin-33 in the central nervous system
Kawanokuchi Jun ⁎ , Yasuoka Satoko, Doi Yukiko, Liang Jianfeng, Noda Mariko, Parajuli Bijay, Sonobe Yoshifumi, Takeuchi Hideyuki, Mizuno Tetsuya, Suzumura Akio RIEM, Nagoya University, Nagoya, Japan
Interleukin-33 (IL-33), recently identified functional ligand for ST2, is a novel member of the IL-1 cytokine family. IL-33/ST2 signaling has been described as a negative regulator of Toll-like receptor-IL-1 receptor signaling, but it also functions as an important effector molecule of Th2 responses. Recent studies have shown that pathogenassociated molecular patterns significantly increase IL-33 mRNA and protein expression in the central nervous system (CNS). However, the function of IL-33 in the CNS is still uncertain. Thus we investigated the expression and the function of IL-33 in the CNS.
We examine the expressions and functions of IL-33 in the CNS by means of RT-PCR, Western blotting, ELISA, FACS and BrdU cell proliferation assay. Murine brain endothelial cell-line bEND.5 cells and astrocytes express IL-33 mRNA and protein but microglia do not. Lipopolysaccharide (LPS) enhances IL-33 expression in astrocytes. Tumor necrosis factor-alpha (TNF-alpha) suppresses IL-33 expression in bEnd.5 cells. Astrocytes and microglia express ST2L, a transmembrane isoform of IL-33 receptors and sST2, a secreted isoform of IL-33 receptors. IL-33 induces microglial proliferation and phagocytosis. IL-33 induces production of proinflammatory cytokines and chemokines in microglia. However the supernatants of IL-33 stimulated microglia do not induce neurotoxicity. IL-33 also induces the production of antiinflammatory cytokines, anti-oxidant molecules and neurotrophic factors in microglia. In the presence of interferon-gamma (IFNgamma), IL-33 enhances the mRNA expression of antigen presenting cell (APC) related molecules and the production of NO in microglia. In the presence of LPS and IFN-gamma microglia stimulated with IL-33 are induced to express IL-12 family cytokines.
IL-33 is a multifunctional cytokine secreted by astrocytes and endothelial cells that acts on microglia through ST2L and exerts diverse biological effects in the CNS.
Regulation of progranulin expression in human microglia and astrocytes: Implications for neuroinflammatory diseases Suh Hyeon-Sook ⁎ , Choi Namjong, Krause Daniela, Lee Sunhee Albert Einstein College of Medicine, NY, United States Progranulin (PGRN) is a relatively newly discovered microglial protein with diverse functions described in the periphery. Haploinsufficiency resulting from mutations of PGRN causes frontotemporal lobar degeneration (FTLD). Although neurons and microglia (and astrocytes, less frequently) are known to express PGRN, little is known about the regulation and function of PGRN in the CNS.
In primary cultures of human fetal microglia and astrocytes, we studied the expression of PGRN using standard ELISA and Western blot analysis. Microglia released nanogram levels of PGRN which increased further following stimulation with Th2 cytokines (IL-4 or IL-13) but not with Th1 cytokines (IFN ± IL-1) or toll-like receptor ligands (poly IC or LPS). LPS or poly IC often suppressed PGRN in microglia. In astrocytes, lower nanogram levels of PGRN were released and their responses to inflammatory stimuli were different from microglia, with IFN/IL-1 and poly IC being stimulatory. Interestingly, astrocyte expression of PGRN closely resembled that of secretory leukocyte protease inhibitor (SLPI), a known inhibitor of progranulin cleavage to inflammatory granulin.
These results suggest that PGRN is a protein involved in immunoregulatory function in the brain. The immune profile suggests that PGRN expression denotes an alternative (M2) microglial activation rather than classical (M1) activation.
Serotonin modulates microglial phagocytosis and motility Tessmann Grietje ⁎ , Matyash Vitali, Färber Katrin, Kettenmann Helmut Cellular Neuroscience, MDC, Berlin, Germany Microglia, the brain macrophages, represent the immune cells of the brain. During any insult they transform from a ramified to an ameboid cell, can migrate to the site of injury and can release both neuroprotective and proinflammatory substances. It was shown that microglial cells express neurotransmitter receptors for GABA, norepinephrine and dopamine whose activation modulates microglial functions (Kettenmann and Pocock, 2007) . Here we studied the impact of serotonin on microglial phagocytosis and motility.
To quantify phagocytosis activity, we determined the uptake of serum coated latex beads, in cultured microglia from newborn NMRI animals, in acute brain slice preparations from 6-9 day old mice and from 8 weeks old adult NMRI mice. Application of 1 μM, 10 μM, 100 μM and 1 mM serotonin reduced significantly phagocytosis activity in primary microglia to 88% ±9%, 82%±8%, 81% ±9% and 85% ±10% respectively (SEM, n=5 ). In comparison we tested also acutely isolated brain slices as an in situ model to study microglia in their tissue environment. 1 mM Serotonin stimulation resulted in a decrease of phagocytosis activity to 76%±7% (SEM, n=5) in slices taken from p6-9 old animals whereas in slices of adult mice we did not observe any significant change in phagocytosis activity (n =6). In a second set of experiments we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion. We determined and quantified the movement of processes to the lesion in acute slices from CX3CR1-EGFP mice. Concentrations of 100 μM and 1 mM serotonin caused an increase in the response of microglial processes toward the laser lesion. Stimulation with 10 μM serotonin did not show any significant effect.
These data indicate that microglial cells express functional serotonin receptors which control microglial executive functions.
The glial response to Interleukin-17
Rodgers Jane ⁎ , Miller Stephen
Multiple sclerosis (MS) is a T-cell mediated autoimmune, demyelinating disease in the central nervous system (CNS). Myelin-specific Th17 cells have been characterized as major contributors to MS pathology by infiltrating into the CNS and upon reactivation, inducing local inflammation that results in myelin destruction. Activated Th17 cells produce interluekin-17 (IL-17), which signals through the IL-17 receptor complex consisting of at least two IL-17RA subunits.
To characterize how the Th17-mediated inflammation affects brain resident cells such as glia, IL-17RA expression was first quantified in astrocytes, microglia, oligodendrocytes, and progenitor cells. Glia expressed IL-17RA and upregulated it during disease. Mixed glia cultures and purified OPCs were stimulated with IL-17 and their cytokine production, proliferation, and maturation were studied. Interleukin-6 and chemokines monocyte chemoattractant protein 1 (MCP-1) and keratinocyte chemoattractant (KC) were produced in a dose and time-dependent manner.
Oligodendrocyte progenitor cells (OPCs) are the major source of remyelinating cells, thus it is especially necessary to understand how they are affected by IL-17. Understanding how IL-17 affects glia may lead to effective therapies for enhancing remyelination.
The role of 3-hydroxyanthranilic acid, a tryptophan metabolite, as a neuroprotective agent in neuroinflammation Krause Daniela ⁎ ,1 , Suh Hyeon-Sook 2 , Choi Namjong 2 , Zhao Meng-Liang 2 , Lee Sunhee 2 1 Ludwig-Maximilians University, Munich, Germany; 2 Albert-Einstein College of Medicine, New York City, United States Indoleamine 2,3 dioxygenase (IDO) is an IFN-inducible, ratelimiting enzyme in the kynurenine pathway (KP) of tryptophan metabolism. Activation of the KP leads to the generation of various kynurenine metabolites depending on the cell type. In the CNS, the kynurenic acid arm of the KP is present predominantly in astrocytes, whereas the 3-hydroxykynurenine (3-HK) arm, which leads to the generation of 3-hydroxyanthranilic acid and quinolinic acid, is present exclusively in microglia. In addition to quinolinic acid (an NMDA receptor agonist), 3-HK and 3-HAA are also shown to be neurotoxins owing to their pro-oxidant activities. Based on these and other observations, the KP enzymes (kynurenine monooxygenase: KMO, for example) are being actively targeted for drug development for neurodegenerative diseases such as Huntington's disease. However, 3-HK and 3-HAA have also been shown to inhibit nitric oxide production and to ameliorate experimental autoimmune encephalitis, suggesting a neuroprotective activity.
We investigated the role of 3-HAA in modulating gene expression and neurotoxicity in primary cultures of human fetal brain cells. Cultures were stimulated with IL-1 ± IFN or toll-like receptor (TLR) ligands in the presence or absence of 3-HAA and results were analyzed by microarray, western blot, ELISA and vital dye exclusion test. We found that 3-HAA suppresses glial cytokine and chemokine expression and also reduces cytokine-induced neuronal death. Unexpectedly, 3-HAA was also found to be highly effective in the induction of hemoxygenase-1 (HO-1), an antioxidant cytoprotective enzyme. Furthermore, HO-1 was expressed by astrocytes and not by microglia in human CNS cultures. Cytokines or TLR ligands had negligible effects in inducing HO-1 as compared to 3-HAA.
Our results demonstrate novel anti-inflammatory and neuroprotective activities of microglial kynurenines. They also suggest that inhibitors of certain KP enzymes might potentiate neuronal damage by changing the CNS inflammatory and redox balance. Oligodendrocytes form one of the most highly specialized cellular structures in the body, the myelin sheath, which allows the electrical insulation of nerve fibbers. So, defects in myelin insulation can lead to several central nervous system disorders. Interestingly, previous studies showed that newborns with increased total serum bilirubin levels display white matter abnormalities, suggesting that unconjugated bilirubin (UCB) may compromise myelination. Hence, we aimed to investigate whether and how UCB determines the demise of oligodendrocyte precursor cells (OPCs) .
Mixed glial cultures were obtained from cerebral cortex of neonatal rat pups. At 10 days in vitro (DIV) OPCs were isolated by shaking after dislodging of microglia. Enriched OPCs cultures were achieved using specific proliferating factors. After 6 DIV cells were treated with 50 μM UCB plus 100 μM of human serum albumin for 4 and 8 h. The purity of OPC cultures was evaluated by immunocytochemistry using antibodies directed to OPCs (A2B5 and O4) and astrocytes (GFAP). Cell viability was assessed by LDH leakage, caspase-3 activity using specific substrate cleavage, mitochondrial abnormalities using MitoTracker Red (500 nM) and endoplasmic reticulum (ER) stress by the activity of calcium-dependent calpain using a specific substrate BOC-LM-CMAC (10 μM).
Approximately 80% of our enriched OPCs cultures were A2B5/O4 positive with less than 3% GFAP positive, and the remaining oligodendrocytes in varying stages of differentiation. Exposure of OPCs to UCB led first to a mitochondrial dysfunction at 4 h (~0.8-fold, p b 0.01) followed by ER stress determined by an increase of calpain activation at 8 h (~1.6fold, p b 0.01) and of caspase-2 and -12 activation (preliminary data). These sequential events triggered cell death by rupture of plasma membrane, with increased LDH release (N1.5-fold, p b 0.05), as well as by apoptosis with enhanced caspase-3 activity (N1.6-fold, p b 0.05) and nuclear fragmentation by TUNEL labelling (preliminary data).
Our results suggest that UCB exerts cytotoxicity in OPCs leading to a cascade of programmed intracellular events, including early signals of mitochondrial dysfunction and activation of calpains that ulti-mately result in death by necrosis and apoptosis. Targeting OPCs dysfunction by UCB may lead to new therapeutic strategies aimed at preventing abnormal myelination following neonatal jaundice.
White matter reactive astrocytes express nuclear estrogen receptor alpha (ESR1) in experimental autoimmune encephalomyelitis and multiple sclerosis Giraud Sebastien N. 1 The mechanism of action of estrogens as modulators of inflammation and neuroprotection in neurodegenerative disorders is a matter of great debate. Whereas an active astrocytic involvement in the physiopathology of neurodegenerative or neuroinflammatory disorders has now emerged, the glial expression pattern of estrogen receptors (ER) in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) remains undefined. We found that nuclear ERalpha is expressed by reactive astrocytes in the white matter cord during chronic EAE in mice, and that estradiol treatment after EAE onset alleviated ongoing EAE symptoms and was associated in the spinal cord white matter with a reduction of astroglial reactivity, leukocytic infiltration and axonal loss.
In order to investigate the astrocytic expression of ERalpha in MS, archival paraffin sections from frontal cortex of secondary progressive MS patients and control subjects were used for double immunocytochemistry. ERalpha was hardly detected in the white matter tracts of control subjects, or around blood vessels where increased GFAP staining was observed in control subject 1 (Wegener case). In the grey matter, moderate ERalpha immunoreactivy in astrocytic fibers could be observed in layer I, particularly for astrocytes contacting dilated blood vessels in control subject 1. Otherwise, cortical astrocytes with several processes and in close proximity to blood vessels were not stained for ERalpha in control and MS subjects. In the normal appearing white matter of MS patients, astrocytes expressed relatively low levels of extranuclear ERalpha compared to those in the white matter at the rim of chronic plaques (identified by the lack of Sudan black staining). Reactive astrocytes in the demyelinating white matter (as assessed by lower Sudan black staining and CD68 immunoreactivity on adjacent sections) exhibited nuclear ERalpha staining whereas ERalpha was not evidenced in chronic plaques though dense GFAP immunoreactive fibers were detected.
Herein, we show for the first time that ERalpha is expressed by reactive astrocytes in MS white matter, with a nuclear staining that could be only observed in demyelinating lesions. These data support white matter astroglia as an important direct target for estrogen antiinflammatory actions in MS. To study of interaction of immune and nervous systems actually on the purpose of disclosing of pathogenesis and search of new approaches to treatment. Aim: To study of clinical features and laboratory criteria of immunological indicators.
Clinical research on 39 patients at the age of 20-44 years is carried out. Out of them men are 27 (69.2%), women 12 (30.8%). Statistical data processing was spent with application of parametrical and nonparametric methods.
Statistically significant clinical signs of the favorable forecast were: presence of positive dynamics, mild expressiveness of the disorder of sensitivity and a pain syndrome. During the treatment the minimum residual phenomena were observed in 28 (71.8%) patients, severe in 11 (28.2%). Results of the immunological researches have shown authentic decrease in migratory activity of leukocytes of peripheral blood by 32%.
The decrease in the production of helper lymphokin, the factor oppressing migration of leukocytes − 0.71 + 0.02 is following criterion of a decrease in functional activity of T-helper. The increase in active serum lymphokin 1.42 + 0.03 in 24 (61.5%) patients was the criterion of the disorder of an immune homeostasis. Increase of the level of the general lymphocytes and T lymphocytes in 15 (38.5%) patients was criterion of the severe course of the disease.
Revealed prognostic criteria promote differential diagnostics and a correct choice of medical tactics.
Do polymorphisms at the CD14 promotor gene and soluble CD14 levels effect biological response to IFNβ therapy in multiple sclerosis? CD14 is a potential marker for macrophage activity; may modulate LPS-triggered apoptosis and regulate T and B lymphocyte function. Elevated serum levels of sCD14 have been found to be associated with inflammation. sCD14 has been reported acting as a negative regulator of cell activation for human T-cells. It was reported that a C to T exchange at position −159 in the promotor region of the CD14 gene (5q31) leads to higher sCD14 levels. Additionally carriers of the TT genotype showed an increased CD14 expression.
The aim of this study was to evaluate the CD14 polymorphisms and sCD14 levels in our multiple sclerosis population and to assess whether sCD14 levels and CD14 promotor gene polymorphisms may reflect any therapeutical effect.
250 multiple sclerosis and 183 controls were included in the study. MS patients were grouped according to their therapeutical response to IFNβ. Individuals either with relapse rates of more than 1/year, or 1point of EDSS deterioration/year were described as evidence for lack of therapeutic response. Three different banding patterns were obtained; CC, TT and CT.
Serum levels of sCD14 by ELISA were also measured for both 77 patients before immune-modulating, immunosuppressive or steroid treatment and for 67 healthy subjects.
In our study sCD14 levels were not statistically different between controls and patients. However, in both controls and patient groups, females had increased sCD14 levels compared to males (p = 0.001p = 0.013).
139 patients and 98 controls were genotyped as CT; 71 patients vs. 49 controls were TT, and 40 patients and 34 controls were CC genotype. There were no differences in genotype frequencies between the controls and the MS patients (p = 0,611).
The distribution of the C dominant genotype (CC+CT) (p = 0,709) and T dominant genotype (TT+CT) (p = 0,321) frequency was similar in both groups.
The clinical response for IFNb was not different in C dominant and T dominant genotypes (p = 0.709-p = 0.321). In the T dominant patient subgroup, sCD14 levels were significantly lower than the CC genotype ( p = 0.013).
Our results point out that sCD14 may not be a reliable marker for evaluating biological response to IFNβ therapy. Secondly in a large scale patient group genotype differences for CD14 did not prove to influence therapeutical response to IFNβ.
A detrimental role for EMMPRIN in multiple sclerosis and experimental autoimmune encephalomyelitis Agrawal Smriti ⁎ , Silva Claudia, Fan Yan, Yong V. Wee
Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a member of the immunoglobulin (Ig) super family. It is an upstream inducer of matrix metalloproteinases (MMPs) that are known to be detrimental in multiple sclerosis (MS), as they result in CNS infiltration of inflammatory cells and myelin destruction. In this study, we investigate a role for EMMPRIN in MS and target its function in murine experimental autoimmune encephalomyelitis (EAE).
Using immunofluoresence staining, Western blotting and flow cytometry of mouse EAE and control tissue, we found an upregulation in EMMPRIN levels with worsening EAE disease severity. A marked increase in EMMPRIN levels was observed in both CNS infiltrating leukocytes and CNS resident cells. Interestingly, EMM-PRIN levels were observed to be similarly upregulated in MS plaque areas compared to adjacent white matter in human CNS samples. Functionally, treatment of EAE-afflicted mice with a blocking anti-EMMPRIN antibody decreased CNS parenchymal infiltration of leukocytes and ameliorates EAE clinical severity. Finally, we demonstrated that the reduction in EAE disease severity in anti-EMMPRIN treated mice is associated with reduced MMP proteolytic activity at the glia limitans, the final barrier before infiltration into the CNS parenchyma.
Our results are the first to emphasize an early detrimental role for EMMPRIN in EAE and MS, through a mechanism that involves MMP activity. EMMPRIN is a novel therapeutic target in MS to ameliorate neuroinflammation and pathology. Most of the present strategies for the treatment of Multiple Sclerosis (MS) are based on the modulation of immune response, but there is a lack of needed treatments to slow disease progression through inhibition of inflammatory and degenerative pathways within the CNS that result in demyelination, axonal damage and neuronal degeneration. Activated microglia plays a key role in the course of inflammatory demyelinating pathologies of central nervous system. Progressive forms of MS are intimately associated with the presence of reactive microglia and it is postulated that downregulating microglial/macrophage activation may delay or halt disease progression.
There is a growing evidence that the modulation of microglial potassium channels is as a good approach to regulate its activation. In the present study we demonstrate that pharmacological regulation of glial inward rectifier potassium channel is effective to improve clinical and histological parameters in Experimental Autoimmune Encephalomyelitis (EAE) induced-mice. Daily oral administration of NT-KC-003 reduces the clinical score of EAE mice, decreases microglia activation, white matter demyelinization and the area of cell infiltration and inhibits inflammatory response when compared to control animals that received vehicle. Tissue integrity and neuronal viability on the spinal cord is also better preserved in treated mice.
Taken together, our results indicate that the inhibition of microglia-mediated neuroinflammation through the glial inward rectifier potassium channel regulation is a prospective strategy for treating CNS demyelinating disorders such as MS that could be also combinable with inmmunomodulatory therapies.
A novel strategy against microglia-induced neurodegeneration Suzumura Akio ⁎ , Takeuchi Hideyuki, Mizoguchi Hiroyuki, Doi Yukiko, Noda Mariko, Sonobe Yoshifumi, Jin Shijie, Mizuno Tetsuya Nagoya University, Nagoya, Japan Pathophysiology of neuronal degeneration still remains to be elucidated. Glutamate released by activated microglia induces exitoneurotoxicity and likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). In this study, we examined whether pharmacological blockade of gap junction hemichannel inhibited glutamate release from activated microglia and significantly ameliorated symptoms of mouse models of neurodegenerative disorders.
We have synthesized several compounds to effectively block gapjunction on microglia, using carbenoxolon as a lead compound. By means of in vitro screening, we got several compounds which effectively suppressed glutamate release by microglia. One of the compound (INI-0602) significantly ameliorated clinical symptoms of mouse models of ALS and AD without detectable toxicity.
The compound may provide a novel strategy against a variety of neurodegenerative disorders.
ABR-215031 suppresses experimental autoimmune neuritis in Lewis rats via inhibition of T cell activity Zhu Wei ⁎ ,1 , Mix Eilhard ⁎ ,3 , Nennesmo Inger ⁎ ,2 , Zhu Jie ⁎ ,2 1 Jilin University, Changchun, China; 2 Karolinska Institute, Stockholm, Sweden; 3 University of Rostock, Rostock, Germany
The therapeutic effects of ABR-215031, which is a new immunoregulator have been evaluated in experimental autoimmune neuritis (EAN), a CD4+ T cell-mediated animal model of Guillain-Barré syndrome in man. In the present study we investigated the effects of ABR-215031 on T cell-mediated autoimmunity of EAN in Lewis rats induced by inoculation with peripheral nerve myelin P0 protein peptide 180-199 together with complete Freund's adjuvant (CFA).
Our data indicate that in two different therapeutic regimens of active EAN and passive transfer EAN, ABR-215031 administered daily orally effectively ameliorated and inhibited the clinical and pathological signs of EAN. The suppression of EAN was associated with marked decrease of inflammatory cell infiltration into the peripheral nervous system (PNS), and an insufficiency of autoreactive T cells, as reflected by inhibited P0 peptide-specific mononuclear cell proliferation, decreased in CD3-, CD4-positive T cell subpopulation and substantially increased the number of macrophages and stimulates NO release, thus increased apoptosis of autoreactive T cells. We conclude that the insufficiency of autoreactive T cells induced by ABR-215031 in the PNS and lymphoid organs may contribute to the absence of inflammatory cells in the EAN lesions and ameliorate the disease.
These effects indicate that ABR-215031 has a strong ability to inhibition of T cell response in EAN and may have therapeutic potential in human GBS. We have previously shown that phospholipase A2s (PLA2s) play a role in the onset, progression or remission phases of experimental autoimmune encephalomyelitis (EAE), a widely used animal model of multiple sclerosis. PLA2s also regulate the production of prostaglandins via cyclooxygenase-1 and 2. Prostaglandins mediate either proinflammatory or protective effects depending on the type of receptors to which they bind. In the present study, we assessed changes in expression of prostaglandin E2 synthases (mPGES-1, cPGES) and prostaglandin receptors (EP1-4) in relapsing-remitting EAE (RR-EAE).
RR-EAE was induced in C57/BL6 mice by subcutaneous injection of MOG30-35 in complete Freund adjuvant, along with pertussis toxin. Spinal cords were obtained at onset, peak and remission stages of EAE for RT-PCR, Western blotting, FACS and double immunofluorescence labelling. The mRNA of mPGES-1 was upregulated at the onset and peak stages of EAE, followed by a decline to naive levels at remission, while no changes were seen in the mRNA of cPGES. The PGE2 receptors also exhibited marked changes in mRNA expression throughout the course of EAE. EP1, EP2, and EP4 mRNA levels were elevated at the onset of EAE and increased further at the peak stage, followed by decline to normal levels at the remission phase. No changes were seen in EP3 mRNA levels. Immunobloting revealed no change in EP1 at the protein level but increased expression of EP2 and EP3 receptors while EP4 protein expression decreased at onset, and was restored to normal levels at peak and remission. FACS analysis revealed that the EP2 and EP3 receptors were predominantly expressed in CD11b+/Gr1+ monocytes/neutrophils (~18%) and CD11b+/Gr1− macrophages/ microglia (27-29%). A small population of T and B lymphocytes also expressed these receptors. Furthermore, mPGES-1 synthases were also expressed primarily in CD11b+/Gr1+ and CD11b+/Gr1− leukocytes. Double immunofluorescence labelling of EAE spinal cord tissue sections showed that in addition to infiltrating immune cells, EP2 and EP4 were also expressed in astrocytes near the EAE lesions.
These data show that alterations occur in expression of the prostaglandin synthases and PGE2 receptors during the course of EAE in infiltrating immune cells and CNS resident cells, suggesting that these receptor-mediated pathways may modulate the inflammatory response during EAE. Recent evidence indicates dysregulated expression of neuroimmune factors in the central nervous system (CNS) can contribute to the neural impairment associated with a variety of CNS conditions, including infection, injury, aging, drug abuse and neurodegenerative or psychiatric disorders. However, the role of specific neuroimmune factors in normal and abnormal CNS function is limited. In this study we investigated the CNS actions of two neuroimmune factors, the chemokine CCL2 (CC chemokine ligand 2, previously known as monocyte chemoattractant protein-1 or MCP-1) and the cytokine interleukin-6 (IL-6). Both neuroimmune factors are expressed in the normal CNS, in aging, and in CNS disorders. To identify CNS actions of CCL2 and IL-6 under conditions of chronic exposure, we have measured synaptic transmission and short-term plasticity in acutely isolated hippocampal slices from adult transgenic mice that exhibit elevated CNS expression of CCL2 (Huang et al., J. Neurosci. 22, 2002) or IL-6 (Campbell et. al., PNAS 90, 1993) and their non-transgenic littermates controls.
Extracellular field potential electrophysiological recordings at the Schaffer collateral-CA1 synapse showed a significant reduction in the magnitude of synaptic responses in hippocampal slices from the CCL2 transgenic mice compared with slices from non-transgenic littermate controls. In contrast, synaptic transmission at the Schaffer collateral-CA1 synapse of IL-6 transgenic mice showed a significant increase in the magnitude of synaptic responses compared with slices from non-transgenic littermate controls. Short-term synaptic plasticity (post-tetanic potentiation, PTP; and short-term potentiation, STP) was enhanced in hippocampal slices from CCL2 mice but not in hippocampal slices from the IL-6 transgenic mice. Long-term synaptic plasticity (long-term potentiation, LTP) was not altered in the CCL2 or IL-6 hippocampal slices compared with the respective non-transgenic littermate control slices. Western blot analysis of hippocampus from the CCL2 and IL-6 mice show increased levels of the astrocyte protein GFAP but no change in level of neuronal specific enolase, a neuronal marker, or cdllb, a microglia marker.
These results show that neuroimmune factors known to be chronically produced at elevated levels within the CNS in certain disorders can significantly alter neuronal function and show that different neuroimmune factors have distinct actions on CNS synaptic function. (Supported by MH083723, AA019261)
Are multifocal motor neuropathy patients underdiagnosed?
Epidemiological studies of amyotrophic lateral sclerosis (ALS) have been reported, but those of multifocal motor neuropathy (MMN) have remained unknown.
We reviewed clinical and demographic details of MMN/ALS patients from 2004 through 2009 in major neurological centers in Japan.
The diagnosis of MMN was made on the basis of revised EFNS/PNS criteria. We diagnosed ALS using the El Escorial criteria except for "possible" ALS. We surveyed the gender, the age at diagnosis, and the results of anti-ganglionic antibodies. The study included 17 patients with MMN and 390 patients with ALS. The median number of patients with MMN was 2.1 per center (range, 0-9 per center) and was influenced by the method used to detect mild or occult conduction blocks in each hospital. The median onset age of MMN was 50.5 years (range, 17-79 years) and that of ALS was 65.1 years (range, 33-87 years). Females accounted for 29.5% of MMN patients and 40.0% of ALS patients. Serum anti-ganglionic antibodies were detected in 41.2% of the MMN patients.
The ratio of MMN:ALS patients was 1:22.9. The large variation in the number of patients with MMN among centers suggests that the diagnostic sensitivity depends on the method used to detect conduction blocks.
Axonal mitochondrial dysfunction in an in vitro model of neuroinflammation Axonal degeneration, Wallerian degeneration as well as dyingback degeneration, are active phenomena characterized by highly regulated new protein synthesis. Mechanisms controlling axonal dye are still poorly understood and further investigation must be undertaken to establish the sequence of molecular events involved in axonal degeneration.
We built a model of neuroinflammation by using organotypic cultures of cerebellum challenged with LPS for 1 to 168 hours. This model reproduces all the molecular events that take part in a neuroinflammatory and neurodegenerative disease like MS: microglial activation, demyelination and axonal damage. In particular, we observe by ELISA assay an increase of TNF-alpha and IL1-beta after 3 and 24 hours of LPS stimulation respectively. Additionally, we observe a decrease of MBP and CNPase, and the formation of multiple axonal spheroids (suggesting axonal transport impairment) after 24 hours of LPS stimulation. We also detect an increase of reactive oxygen species (ROS) and iNOS after 12 and 24 hours of LPS stimulation respectively. Morphological analysis using electron microscopy shows an altered mitochondrial morphology with enlarged mitochondria all along the axons. We are currently assessing a loss of mitochondrial membrane potential by different probes like JC-1 and Mitotracker.
Neuroinflammation promotes axonal dysfunction, oxidative stress and mitochondrial morphological changes in axons, pointing that energetic failure participates in axonal damage in this model of MS.
Characterization of plasmacytoid dendritic cells in multiple sclerosis Nakano Akiko ⁎ , Ifergan Igal, Kebir Hania, Prat Alexandre
Multiple sclerosis (MS) is an immune-mediated disorder of the central nervous system (CNS) characterized by multifocal areas of leukocyte infiltration, demyelination and axonal damage. Typically, demyelination is associated with an accumulation of CD4+ T lymphocytes and macrophages/dendritic cells (DCs) that arise from migration of peripheral blood immune cells across the CNS microvascular endothelium. Moreover, MS lesion formation is thought to be mediated by autoreactive T lymphocytes directed against myelin peptide sequences. Therefore, trafficking of antigen presenting cells (APC) into the CNS is thought to contribute to lymphocyte reactivation within the CNS compartment.
Using an in vitro model of the human BBB, we showed that upon migration across BBB-endothelial cells (BBB-ECs), 60% of monocytes differentiate into CD123+ plasmacytoid DCs (pDCs). We showed that these cells carry the markers CD123high, HLA-DRhigh, CD14low-interm, CD11c low-interm, CD86high-interm, CD83neg, CD80neg, CD40lowinterm, DC-SIGNneg. Functional analysis of these pDCs indicated that CD4+ T lymphocytes grown in the presence of CD123+ pDCs had a tendency to produce lower levels of GM-CSF, TNF-a and IFN-g, but higher level of IL-4 when compared to those cultured with CD14+ monocytes. Also, using tissue from MS patients, we confirmed by immunohistochemistry the abundance of such perivascular CD123+ pDCs within acute MS lesions, closely associated with microvascular BBB-ECs.
These results suggest that this novel population of BBB-associated pDCs play an anti-inflammatory role in the CNS and are present within MS lesions.
Correlation of MMP-2 and MMP-9 with pro-inflammatory cytokines in Guillain-Barré syndrome The role of matrix metalloproteinases (MMPs) and cytokines in the pathogenesis of Guillain-Barré syndrome (GBS) largely remains unknown. We studied the role of MMP-2, MMP-9, TNF-α and IL-1β in disease progression and recovery of patients with GBS.
Sixty five patients with GBS and 68 healthy controls were enrolled in the study. Serum levels of MMP-2, MMP-9, TNF-α and IL-1β were analyzed by ELISA and activities of MMPs were measured by zymography.
Expression of MMP-9, TNF-α and IL-1β was higher in progressive phase 200.06 ± 97.23 vs. 92.47 ± 34. 19, p b 0.001; 105.77 ± 68.06 vs. 17.16 ± 6.20, p b 0.001; 149.48 ± 89.53 vs. 63.49 ± 27.42 , p b 0.001) and lower in recovery phase 46.38 ± 21.36 vs. 92.47 ± 34. 19, p b 0.001; 14.34 ± 6.71 vs. 17.16 ± 6.20, p = 0.013; 56.31 ± 17.77 vs. 63.49 ± 27.42 , p = 0.077) of GBS than healthy controls. A positive correlation of MMP-2 with IL-1β (r= 0.254, p = 0.044) and MMP-9 with TNF-α (r= 0.25, p = 0.044) and IL-1β (r= 0.311, p = 0.012) was observed with progressive phase of GBS.
The study shows that up-regulation of MMP-9 along with proinflammatory cytokines in early course appears to be associated with immune-mediated disease progression due to inflammation in the PNS, while during later phase, down-regulation of MMP-9 and pro-inflammatory cytokines is implicated in the recovery from the disease.
Effects of IL-18 on human SH-SY5Y neuroblastoma cell line proteome Neuropathological changes in Alzheimer's disease (AD) brain include extracellular Amyloid-β (Aβ) plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated tau-protein. There are also signs of chronic inflammation. Interleukin-18 (IL-18) is an inflammatory cytokine largely produced in the brain by activated microglia. Our previous studies suggest that IL-18 may have an impact on tau and kinases related to tau phosphorylation. However, the link between IL-18 and AD pathogenesis is still poorly understood, and requires further studies. In this study, we wanted to study the effect of IL-18 on differentiated human SH-SY5Y neuron-like cell proteomes in vitro.
The impact of IL-18 on differentiated human SH-SY5Y neuron-like cell protein profiles were examined at different time-points (24, 48 and 72 h) using two-dimensional difference-gel-electrophoresis (2D-DIGE). Non-treated differentiated SH-SY5Y cells were used as controls. Proteins exhibiting changes were cut out from silver stained gels; in-gel digested with trypsin, and identified using mass spectrometry and database searches. The most significant protein changes were found after the treatment with IL-18 for 24 h but proteomes were also altered after the 48 h and 72 h treatments when compared to the untreated cells. The number of protein spots was the greatest at 48 h maybe indicating in enhanced modifications of proteins. Altogether 57 proteins exhibiting alterations were identified and they were related to e.g. cell proliferation and/or differentiation, inflammation, regulation of oxidation and cell signaling. Expression of some of the most interesting proteins will be examined with Western blots.
Our preliminary data suggest that IL-18 has a time-dependent effect on proteome profiles in SH-SY5Y cells. Some of the detected changes may be linked to up-or down-regulation in protein expression whereas some to altered modifications. We are currently examining protein changes further by immunoblotting.
Experimental autoimmune encephalomyelitis in the absence of lysophosphatidic acid receptor LPA1 Lysophosphatidic acid (LPA) is an endogenous phospholipid which is involved in many different cellular processes through specific Gprotein coupled receptors (LPA1-5). Works with viable Malaga variant maLPA1-null mice, lacking LPA1 receptor, demonstrated the requirement of this receptor for normal proliferation and differentiation of neural precursors, both in development and adult life. Their characterization has included myelinating glia which highly expresses LPA1 receptor during myelination, and pointed out that maLPA1-null mouse may be a suitable model for the study of demyelinating diseases, such as multiple sclerosis. The absence of LPA1 decreases the number of oligodendrocytes in adult and generates a myelin-deficient pattern.
Besides, LPA also acts on neuroinflammatory processes through LPA1, LPA2 and LPA3 receptors influencing interleukins secretion, blood brain barrier permeability, and microglia activation. Therefore, the role of LPA in a neuroinflammatory and demyelinating model, such as the experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 immunization, has been studied. The response has been evaluated in both normal and LPA1-null mice based on clinical and histological analysis. The results show that the presence of LPA1 receptor is necessary for a normal immune response.
Although both genotypes developed similar clinical courses displaying relapsing-remitting forms, not only did wild type mice show more severe symptoms than null animals but also the onset of disease was delayed in those animals lacking the receptor and they reflected a better recovery after the relapses. Mice with conditional SOCS3 deletion (SOCS3−/−) in myeloid lineage cells were generated. In this study we have evaluated the polarization of macrophages and microglia and progression of EAE in wild-type (WT) and SOCS3−/− mice. We found that bone marrowderived macrophages and microglia were stimulated to express higher levels of genes related to M1 polarization, such as IL-6, TNF-alpha, IL-12 and iNOS at the mRNA and protein levels, in SOCS3−/− cells compared to WT mice upon LPS, IFN-gamma or LPS plus IFN-gamma stimulation. Furthermore, SOCS3 deletion enhanced LPS and IFN-gamma-induced STAT1 and STAT3 activation, but had no significant effect on LPS-induced NF-B and MAPK activation. Experiments on the effect of SOCS3 on EAE development are under investigation; preliminary results suggest that SOCS3−/− mice have an earlier onset and more severe of EAE than WT mice. The function of macrophage/microglia SOCS3 on Th1, Th17 and Treg cell differentiation will be tested during the pathogenesis of EAE.
Our study suggests that SOCS3 is an important negative regulator of macrophage/microglia inflammatory responses, specifically in the EAE model. Spinal cord injury (SCI) is characterized by the mechanical disruption of the spinal cord tissue, ischemia and edema followed by the expression of cytokines and chemokines at the lesion site leading to the recruitment of peripheral blood leukocytes. These events result in cell death, denervation and paralysis. Oligodendrocytes are one of the most sensitive cells to traumatic injury in the CNS. Their loss leads to demyelination and axonal injury with concomitant loss of neurological functions. In a previous study using transgenic mice where NF-kB is specifically inhibited in astrocytes (GFAP-IkBadn, TG), we showed a reduction in the production of cytokines/ chemokines such as CCL2 and CXCL10 by astrocytes, a smaller lesion site, increased white matter preservation and improved locomotor function 8 weeks following SCI. Our goal is to gain insight into the molecular mechanisms leading to improved functional behavior and increased white matter preservation.
We performed a whole mouse genome microarray analysis on spinal cords from injured WT and TG mice at 3 days, 3 weeks, and 6 weeks following SCI. Surprisingly, we found the largest number of differentially regulated genes (994) at 6 weeks post-injury with genes involved in inflammatory/immune responses, chemotaxis, axonal growth, and cell death, all processes that may influence functional recovery. We also found a number of genes (SOX17, Tcf7L2, CNPase, Ki67) significantly up-regulated at 6 weeks, that suggest an increase in oligodendrogenesis in TG mice compared to WT mice. Moreover KEGG pathway analysis revealed that the Wnt signaling pathway, which is important for remyelination, was significantly activated in our TG mice compared to WT mice. We also found significant changes in RNA and protein levels of TLR2 and the number of CD11b+ cells suggesting an altered innate immune response in the TG mice.
We are currently testing the hypothesis that inhibition of astoglial NF-kB reduces and modifies the nature of the inflammatory response at 6 weeks post-injury and therefore may provide a more permissive environment for oligodendrocyte progenitor cells to develop into myelinating oligodendrocytes.
Imbalance in interleukin-18 and interleukin-18 binding protein in AD patients Reale Marcella ⁎ , Iarlori Carla, Bellante Veronica, Di Nicola Marta, Gambi Domenico University "G. d'Annunzio" Chieti -Pescara, Chieti, Italy
Interleukin-18 (IL-18), a member of IL-1 cytokine family, is a potent inducer of proinflammatory cytokines. IL-18 binding protein (IL-18BP) is a constitutively secreted protein able to bind IL-18 with high affinity, providing a potential mechanism whereby IL-18 activity is regulated. Our previous study confirmed that an imbalance of Th1 versus Th2 polarization in favor of Th1 cell subsets appears to be a key pathogenic mechanism in AD. The aim of this preliminary study, is to investigate if IL-18 and IL-18BP expression and production is may be involved in AD. 20 AD patients with clinical diagnosis, according to NINCDS-ADRDA criteria, and 10 HC subjects, matched for sex and age were enrolled. Interleukin-18 and IL-18BP plasma levels were assayed by a specific enzyme-linked immunosorbent assay kit according to the manufacturer's instructions. RT-PCR was performed to determine mRNA IL-18 and IL-18BP espression in PBMC.
In this study we show that IL-18 plasma levels in the AD group were significantly higher than those in the healthy controls, and IL-18BP levels were lower than those in the healthy controls (P b 0.01). An increase release of IL-18 and IL-18BP was observed in PBMC from AD patients compared to the control group. We found that there were significant increases in mRNA expression of IL-18, while IL-18BP expression was decreased in AD patients, as compared with those of healthy controls.
IL 18 is a potent upstream cytokine that is antagonized by IL-18BP, this leads to a vicious cycle that may represent an important contribution to the pathogenesis of AD. One interpretation suggested for the lower levels of IL-18BP in the plasma of AD patients and higher mRNA IL-18BP levels in PBMC from AD group when compared to HC, is that IL-18BP is bound by IL-18. Our study suggests that IL-18 in serum of AD patients promotes upregulation of Th1 cells and inhibits Th2 cells, which results in a greater imbalance of Th1/Th2 and aggravate patient illnesses.
Myelin-phagocytosing macrophages show immunomodulatory properties Bogie Jeroen ⁎ , Timmermans Silke, Stinissen Piet, Hellings Niels, Hendriks Jerome
Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) in which macrophages play a pivotal role. Nonetheless, it remains largely unknown which macrophage subpopulations are induced during CNS inflammation, what underlying cellular mechanisms govern the induction of these macrophage subsets and how they contribute to MS pathology. Initially, macrophages where thought to be merely detrimental in MS, as they internalize myelin and secrete toxic and pro-inflammatory mediators. However, recent evidence suggests that they may also have protective effects, especially following myelin phagocytosis.
In this study, we demonstrate that myelin-phagocytosing macrophages inhibit lymphocyte proliferation more pronounced than control macrophages. This inhibition was antigen independent and was mediated by an altered nitric oxide synthase/arginase balance. Additionally, we show that myelin internalization by macrophages induces the expression of liver X receptor (LXR) response genes. LXRs are nuclear receptors involved in cholesterol metabolism and inflammation. Prolonged LXR activation led to an induction of NO secretion by macrophages, possibly explaining the increase in NO production after myelin phagocytosis.
These observations suggest that myelin phagocytosis leads to an altered macrophage phenotype that modulates lymphocyte responses in MS. More research is needed to further elucidate this immunomodulatory role of myelin-phagocytosing macrophages and is essential to increase our understanding of neuroinflammatory responses in diseases like MS.
Neuropeptides as modulators of microglial function in Alzheimer's disease Fleisher-Berkovich Sigal ⁎ ,1 , Filipovich-Rimon Talia 1 , Ben-Shmuel Sarit 1 , Gera Lajos 2 , Kummer Markus 3 , Heneka Michael 3 1 Ben-Gurion University, Beer-Sheva, Israel; 2 University of Colorado, Denver, United States; 3 University of Bonn, Bonn, Germany
Microglial inflammation plays an integral role in the development of neurodegenerative disease. Although neuropeptides such as bradykinin (BK), somatostatin (SST) and endothelin (ET) are known to be important mediators of inflammation in the periphery, evidence of a similar function in the brain is scarce. The aim of the present study was to examine the effects of these neuropeptides on various aspects of microglial activity such as chemotaxis, phagocytosis of amyloid-β (Aβ) and release of proinflammatory mediators.
Using immunocytochemistry, we show expression of receptors for BK (B1, B2 subtypes), ET (ETA, ETB subtypes) and SST (SST 2, 3, 4 subtypes) in both microglial cell lines BV2 and N9. Our results also show that exposure of BV2 and N9, as well as primary neonatal rat microglial cells to BK or SST increased Aβ phagocytosis by the cells by more than two folds in a dose dependent manner. By contrast, endothelin decreased Aβ phagocytosis dose dependently. Pulse chase experiments confirmed that all neuropeptides increased the overall phagocytosis of Aβ but did not affect the Aβ degradation pathways. All neuropeptides increased chemotactic activity of microglia several folds. In addition, we show that Aβ-induced expression of genes encoding the proinflammatory molecules iNOS and COX-2 in microglia was inhibited by 50% by BK. ET also significantly decreased the Aβ-induced expression of monocyte chemoattractant protein 1 (MCP-1) and interleukin-6 (IL-6).
These results suggest that neuropeptides may play an important role in chemotaxis and the clearance of Aβ and modulate the brain response to neuroinflammatory processes such as the release of inflammatory factors from microglial cells. The neuropoietic cytokine oncostatin M (OSM) is expressed in multiple sclerosis (MS) lesions, but its effect on central nervous system (CNS) lesion development remains unknown. OSM limits neuronal death induced by excitotoxicity, but in non-neuronal disease models such as arthritis, it is associated with inflammation and tissue damage. This study was designed to elucidate the effect of local OSM expression on inflammatory CNS lesion development.
First, local expression of OSM was induced in the right striatum of healthy, adult C57Bl/6J mice by means of a stereotactic injection with lentiviral vectors encoding OSM. OSM disrupted the blood-brain barrier and induced a local inflammatory response, characterized by an upregulation of adhesion molecules, MHCII expression and infiltration of T-cells and macrophages in the otherwise healthy CNS. This indicates that OSM expression is sufficient to induce several aspects characteristic of inflammatory CNS lesions. Next, we investigated how OSM affects the underlying disease processes in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). When EAE was induced in mice with a local expression of OSM in the striatum, disease incidence and clinical symptoms were significantly reduced. In agreement with these findings, significantly less inflammatory lesions were detected in the spinal cord of OSM-treated mice. To explain how OSM can induce inflammatory lesions at the site of expression, while inhibiting development of EAE lesions, we hypothesized that the immune cells attracted to the site of OSM-expression may have a regulatory or immunosuppressive phenotype. Indeed, immunohistochemical staining revealed mannose receptor positive macrophages at the site of OSMexpression, a marker for alternatively activated macrophages. Moreover, OSM induced a shift in cytokine profile of EAE-animals as analysed by means of Q-PCR, while our in vitro studies show that OSM reduces NO-production by activated macrophages.
Our study demonstrates that OSM is an important regulator in the development of inflammatory CNS lesions.
Mitsugu Yamanaka ⁎ , Michael T. Heneka
Alzheimer's disease (AD) is characterized by chronic deposition of amyloid-beta (Abeta) and by a microglial-mediated inflammatory response. Peroxisome proliferator-activated receptor (PPAR) gamma agonists ameliorate glucose metabolism and suppress inflammation in type 2 diabetes patients. Recent evidence indicates that mild cognitive impairment in humans may be improved by treatment with PPAR gamma agonists. DSP-8658 is a selective PPAR alpha/gamma modulator currently under clinical development for the treatment of type 2 diabetes. In transactivation assays DSP-8658 was characterized by comparable EC50 values for human PPAR alpha (1.08 μM) and human PPAR gamma (1.01 μM), and partial activation of human PPAR gmma (76% compared to that of pioglitazone). In this study, we investigated the therapeutic potential of DSP-8658 for AD.
We analyzed Abeta phagocytosis using rat primary microglia and performed surface marker analysis of microglia in APP23 transgenic mice. DSP-8658 enhanced the uptake of Abeta in rat primary microglia. Of note, this effect was abolished by coincubation with inhibitors of transcription (Actinomycin D, 5 μM) or translation (cycloheximide, 10 μM), suggesting that the observed effect requires de novo protein synthesis. In vivo, DSP-8658 induced the expression of surface markers such as CD40, CD80 and CD86 in microglia of APP23 mice. PPARs heterodimerize with RXRs and subsequently bind to specific DNA responsive elements, thereby regulating the expression of target genes. Interestingly, the combination of DSP-8658 with several RXR agonists, including bexarotene and retinoic acid treatment showed synergistic enhancement on the uptake of Abeta by microglia in a concentration-dependent manner.
Together these data suggest that activation of PPAR alone or even more effectively in concert with RXR enhances microglial Aβ phagocytosis. In conclusion, DSP-8658 may serve as a therapeutic agent for AD treatment.
Prenatal immune challenge promotes Alzheimer's disease-like neuropathology in aged wild-type mice Krstic Dimitrije ⁎ , Doehner Jana, Breu Karin, Madhusudan Amrita, Knuesel Irene
Many age-associated degenerative diseases are associated with abnormal proteinaceous aggregates, often in the form of amyloid deposits. In Alzheimer's disease (AD), Amyloid-beta (A-beta) peptides are the major plaque components. Rare familial AD mutations, which all affect the proteolysis of amyloid precursor protein leading to increased production of aggregation-prone A-beta peptides, supports a central role of A-beta in AD pathophysiology. Growing experimental evidence suggest a critical role of soluble, prefibrillary oligomers A-beta species in triggering neuronal malfunction and cell death. However, it is currently unknown whether similar mechanisms also underlie aging-associated sporadic AD. We have reported previously that normal aging in rodents and primates is accompanied by loss of Reelin-expressing neurons and accumulation of Reelin-positive oligomeric aggregates in the hippocampal formation. These alterations correlate with cognitive impairments in mice. A prenatal immune challenge resulted in accelerated plaque deposition compared to controls, suggesting a role of early neurodevelopmental and immunological abnormalities for aging-related neuropathology.
Here, we examined 6 and 15 months-old wild type mice that were exposed in utero to the viral mimic, polyinosinic:polycytidylic acid (PolyI:C), using biochemical and immunohistochemical approaches, combined with immuno-electron microscopy to characterize the impact of a prenatal infection on A-beta and Tau pathology. We confirm the formation of ThioflavinS-positive and A-beta-immunoreactive extracellular deposits selectively in the hippocampal formation of PolyI:C-exposed mice. At 6 months, the area covered by Reelin-and Abeta-positive deposits was significantly enlarged as compared to controls. At 15 months, a further aggravation of the amyloid pathology associated with abnormal levels of phosphorylated Tau was observed following PolyI:C-exposure.
This suggests that an in utero infection favors AD-like neuropathology in normal wild type mice. Programmed cell death-1 (PD-1) is a co-inhibitory receptor expressed on activated immune cells. The interaction between PD-1 and its ligands (PD-L1 or PD-L2) suppresses immune responses. Experiments performed in animal models of multiple sclerosis (MS) have shown that PD-L1 or PD-L2 blockade leads to an earlier onset and increased disease severity. PD-1 and its ligands were observed on infiltrating immune and CNS cells in these models but which CNS cell types express these molecules have not been fully resolved, and data on human CNS are still incomplete.
OUR GOAL is to assess the expression of PD-L1 and PD-L2 in human CNS, and to establish whether these molecules could locally contribute to modulate immune responses.
We investigated whether PD-L1 and PD-L2 are expressed by human primary CNS cells (astrocytes, microglia, oligodendrocytes, neurons, brain endothelial cells (HBECs)) under basal and inflammatory conditions using cytokine treatments (IFN-gamma, TNF, IL-1beta). Astrocytes, microglia, oligodendrocytes, neurons, and HBECs expressed barely detectable PD-L1 and PD-L2 levels under basal conditions, at the mRNA (qPCR) and protein levels (flow cytometry, immunocytochemistry). However, inflammatory cytokine treatments increased the expression of PD-L1 and PD-L2 on these cells, especially PD-L1 on astrocytes and microglia, and PD-L2 on HBECs. Blocking PD-L1 using specific siRNA expression in astrocytes led to a significant enhanced alloreactive CD8 T cell responses (proliferation and cytokine production) showing the capacity of human glial cells to modulate CD8 T cell responses via PD-Ls. Moreover, the expression of PD-L1 was analyzed using immunohistochemistry in post-mortem brain tissues from MS and controls. Higher expression of PD-L1 was detected in MS brain sections compared to controls which colocalized with astrocyte or microglia specific cell markers. Finally, we detected PD-1 only on a subset of infiltrating CD8 T cells in human MS brain lesions.
Our results demonstrate that PD-L1 and PD-L2 although barely expressed by human brain cells both in vitro and in vivo, are significantly boosted in an inflamed environment like the one observed in MS lesions. Finally, CNS-provided PD-Ls molecules have the capacity to reduce human CD8 T cell responses, but numerous CNS infiltrating CD8 T cells do not express PD-1 and are thus insensitive to this attempt by the inflamed CNS to dampen their responses.
Role of Chlamydia pneumoniae antibodies in smokers with acute ischemic stroke: Indian study Bandaru VCS Srinivasarao ⁎
Background: Smoking is one of the major risk factors for cerebrovascular and cardiovascular diseases. Several reports have implicated Chlamydia pneumoniae (C. pneumoniae) as a causative factor for atherosclerosis and stroke. Limited data at present in India and no such data available on the association between C. pneumoniae infection and smoker with acute ischemic stroke. Objective: We investigate the role of C. pneumoniae antibodies in smokers with acute ischemic stroke. Methodology: This study was conducted at Nizam's Institute of Medical Sciences, a tertiary care hospital in south India; between January 2006 and December2008. Consecutive patients aged above 18 years with acute ischemic stroke who admitted within 72 hours of stroke onset were included. All patients underwent stroke work-up. Risk factors data were collected for all patients. We measured Creactive protein (CRP) levels and C. pneumoniae antibodies IgG and IgA by microimmunofluorescence technique (MIF) in all patients. Results: Out of 220 patients with acute ischemic stroke more than 18 age group smokers were 69( 31.4%) and nor-smoker were151 (68.6%). C. pneumoniae seropositivity found in 40(57.9%) smokers and 42 (27.8%) non-smokers (p b 0.0001). CRP was elevated in smokers compared to non-smokers (p b 0.0001). In smoker we did the multiple logistic regression test after adjustment for various risk factors like hypertension (odds 0.8; 95% CI: 0.44-1.71); diabetic mellitus (odds 1.3; 95% CI: 0.66-2.65); alcoholics (odds 1.3; 95% CI: 0.71-2.75; and C. pneumoniae seropositivity (odds 3.5; 95% CI: 2.15-7.15). After adjustment IgG antibodies (odds 8.5; 95% CI: 3.90-10.68) were more in smokers with stroke compared to IgA antibodies (odds 3.2; 95% CI: 1.28-8.06).
C. pneumoniae IgG antibodies were independently associated with acute ischemic stroke in smokers.
T cells specifically targeted to amyloid plaques enhance plaque clearance in a mouse model of Alzheimer's disease
Ben-Gurion university, Be'er-Sheva, Israel
Patients with Alzheimer's disease (AD) exhibit substantial accumulation of amyloid-beta (Abeta) plaques in the brain. Here, we examine whether Abeta vaccination can facilitate the migration of T lymphocytes to specifically target Abeta plaques and consequently enhance their removal.
Using a new mouse model of AD, we show that immunization with Abeta, but not with the encephalitogenic proteolipid protein (PLP), results in the accumulation of T cells at Abeta plaques in the brain. Although both Abeta-reactive and PLP-reactive T cells have a similar phenotype of Th1 cells secreting primarily IFN-gamma, the encephalitogenic T cells penetrated the spinal cord and caused experimental autoimmune encephalomyelitis (EAE), whereas Abeta T cells accumulated primarily at Abeta plaques in the brain but not the spinal cord and induced almost complete clearance of Abeta. Furthermore, while a single vaccination with Abeta resulted in upregulation of the phagocytic markers triggering receptors expressed on myeloid cells-2 (TREM2) and signal regulatory protein-beta1 (SIRPbeta1) in the brain, it caused downregulation of the proinflammatory cytokines TNF-alpha and IL-6.
We thus suggest that Abeta deposits in the hippocampus area prioritize the targeting of Abeta-reactive but not PLP-reactive T cells upon vaccination. The stimulation of Abeta-reactive T cells at sites of Abeta plaques resulted in IFN-gamma-induced chemotaxis of leukocytes and therapeutic clearance of Abeta.
TGF-β signalling in myeloid cells during experimental autoimmune encephalomyelitis is crucial for the entrance into the remission phase Trogrlic Ivana ⁎ , Malipiero Ursula, Ackermann Friederike, Suter Tobias, Fontana Adriano University Hospital Zurich, Zurich, Switzerland TGF-beta is a potent regulatory cytokine with diverse effects on hematopoietic cells and it has an important role in controlling autoimmunity. In T cell-mediated autoimmunity, mostly studied on the experimental autoimmune encephalomyelitis model (EAE), it has been shown that TGF-beta is increased during the remission phase. Treatment of mice with TGF-beta1 ameliorates EAE, whereas anti-TGF-beta antibodies lead to exacerbation of EAE.
Dendritic cells (DC) and macrophages (Mph) are known effector cells in EAE. Whether TGF-beta is also essential in the control of EAE, not only by acting on T cells but also by regulating the function of the innate immunity, remains to be established. In-vitro studies show TGF-beta to be a powerful Mph deactivator by inhibition of proinflammatory cytokines such as TNF-alpha and IL-1 beta. Furthermore, TGF-beta impairs the production of oxygen radical intermediates and the generation of nitric oxide (NO) by the inducible NO synthase.
In order to dissect the role of TGF-beta in the innate immune system and in autoimmunity, we use knockout mice with a targeted deletion of the TGFbRII gene only in activated phagocytes of myeloid origin (phag-TGFbRII−/−). We found that in the absence of TGFbRII signaling in myeloid cells, the chronic phase of EAE takes a more severe course and mice don't go into remission phase. Unexpectedly, the phenotype was not associated with more prominent expression of iNOS, TNF-alpha and IL-1beta in Mph, DCs or microglia derived from TGFbRII knockout mice, but rather with the accumulation of the myeloid cells in the CNS of the phag-TGFbRII−/− animals in the late phase EAE. We have found dendritic cells to be 2-3 fold increased in the CNS of the knockout animals. Analysis of surface markers like integrins, molecules involved in cell migration and extravasation, showed no difference in expression on the accumulating cells. Activation markers for these cells were also not differently expressed compared to littermate controls.
Present data strongly support the involvement of TGF-beta signalling in myeloid derived cells in the remission phase of the EAE. Currently, we are further investigating the influence of TGF-beta on cell survival or recruitment of inflammatory cells to the CNS.
The chemokine receptor CXCR3 is critical for the Alzheimer-like plaque formation in transgenic APP/PS1 mice Chemokines are a family of cytokines with structural similarities and chemoattractive properties. They are important molecules in orchestrating inflammatory processes and are able to modulate the course of neuroinflammatory and neurodegenerative diseases.
Descriptive studies in brain tissue from Alzheimer patients and the according animal models revealed high levels of the chemokine CXCL10 arguing for an important role of this chemokine in the pathogenesis of Alzheimer's disease (AD). The corresponding receptor is expressed by activated T cells but also by microglia and neurons. Recent functional studies addressing the role of CXCR3 in neurological diseases presented data arguing for a very complex role of this chemokine system in CNSdiseases, which can be both beneficial and detrimental.
Here we examined studied the impact of CXCR3 signalling on AD progression in transgenic mice that co-express the two human AD mutations for amyloid precursor protein (APPswe) and presenilin-1 (PS1E9). These mice were crossed with CXCR3-deficient mice.
Eight months old mice with the genotypes APP/PS1/CXCR3−/−, APP/PS1/CXCR3+/+, CXCR3−/− and wild-type mice were analysed for amyloid-beta; deposition, APP processing, and expression of inflammation-related genes. Furthermore microglial phagocytosis assay of CXCR3 deficient and wild type primary microglia were used to analyse the in vitro impact of CXCR3 on the microglial phagocytosis of amyloid-beta 1-42.
Immunohistochemical Abeta staining and ELISA detection of amyloid-beta in the brain revealed strongly reduced plaque burden and reduced amyloid-beta protein-levels in CXCR3 deficient APP/PS1 mice compared to APP/PS1/CXCR3+/+ mice. Morphological activation and periplaque accumulation of microglia was diminished in APP/ PS1/CXCR3−/− compared to APP/PS1/CXCR3+/+ mice. Moreover, CXCR3 deficiency leads to decreased RNA levels of proinflammatory cytokines like TNFalpha and the CXCR3 ligands in APP/PS1 brains. In vitro, the phagocytosis assay data indicated an increased uptake of FAM-labelled fibrillar amyloid-beta 1-42 for microglial cells isolated from APP/PS1/CXCR3−/− mice compared to controls.
In conclusion, CXCR3 is critically involved in the AD-like plaque formation of APP/PS1 transgenic mice. Our data suggest that the reduction of amyloid plaques in APP/PS1/CXCR3−/− mice is mediated by the modulation of microglial function due to CXCR3deficiency and argues for CXCR3 as a novel and promising therapeutic target in AD.
The contribution of p38alpha MAPK versus p38beta MAPK in the microglia cytokine response to LPS or beta-amyloid Adam Bachstetter ⁎ , Linda Van Eldik University of Kentucky, Lexington, United States Activated microglia are a major pathological feature of Alzheimer's disease (AD). Microglia activation is not necessarily detrimental, as there are many beneficial microglia responses, such as the sequestering and removal of protein aggregates. However, activated microglia are the primary source of proinflammatory cytokines in the brain. Two proinflammatory cytokines that are elevated in AD, and believed to contribute to the neuronal damage, are IL-1beta and TNFalpha. The Mitogen-Activated Protein Kinase (MAPK) p38 is one kinase pathway involved in the production of IL-1beta and TNF-alpha. The p38 family consists of at least four isoforms (p38alpha, beta, gamma, delta) encoded by separate genes, but the p38gamma and p38delta isoforms are not expressed at significant levels in the brain. Commercially available p38 inhibitors, which have been shown to inhibit IL-1beta and TNF-alpha biosynthesis, interact with both the p38alpha and p38beta isoforms, and the relative contributions of microglial p38alpha and p38beta to IL-1beta and TNF-alpha biosynthesis in response to various stimuli have not been addressed.
To address this question, we isolated microglia from p38alpha and p38beta knockout (KO) mice, and stimulated the microglia with two different activating stimuli: lipopolysaccharides (LPS) or beta-amy-loid1-42. Interestingly, we found quantitative differences in the microglia responses to LPS and beta-amyloid1-42 depending on the p38 isoform KO. Microglia from p38beta KO mice were responsive to LPS, but were nearly unresponsive to beta-amyloid1-42. A diminished but detectable response to both stimuli was seen in the p38alpha deficient microglia.
These results suggest that unique functions may be attributed to the p38alpha and p38beta isoforms in activated microglia, which might be exploited in the future by the development of new classes of CNS-active, isoform-selective p38 inhibitors.
The effect of medicinal plants on neuroinflammation in neurological diseases: A pragmatic randomized ethnopharmacological survey in Chapai-Nawabganj district of Bangladesh Mollik Md. Ariful Haque ⁎ Peoples Integrated Alliance, Dhaka, Bangladesh Epilepsy, malaria, and sexually transmitted diseases have been prevalent and even endemic in various parts of the world since ancient times. In recent years, attention has focused on these diseases because of the emergence of drug-resistant varieties of these diseases. As a result, it has become imperative to discover novel compounds to treat such diseases. Since medicinal plants form one of the best sources for obtaining pharmacologically active constituents, which can be used as remedy for diseases like epilepsy, malaria, and sexually transmitted diseases; the objective of this present study was to conduct an ethnopharmacological survey amongst the traditional health practitioners of Chapai-Nawabganj district, Bangladesh to obtain information on medicinal plants used by them as remedy for the above ailments. It is noteworthy in this regard that all the above-mentioned ailments are prevalent in Bangladesh, and the primarily rural population of the country relies on medicinal plants or plant parts prescribed by traditional health practitioners to treat the above ailments.
Interviews were conducted of traditional health practitioners with the help of a semi-structured questionnaire and medicinal plant samples were photographed and identified at the Bangladesh National Herbarium. The collected information showed that the following medicinal plants were used to treat epilepsy: Citrus maxima Merr., Bacopa monnieri (L.) Pennell, Datura stramonium L., Calotropis gigantea (L.) W.T.Aiton, Nigella sativa L., Bixa orellana L., Carica papaya L., Acorus calamus L., and Boerhavia repens L.. Anti-malarial medicinal plants included Justicia adhatoda L., Brassica napus L., Ocimum tenuiflorum L., Solanum nigrum L., Saccharum officinarum L., Piper nigrum L., Momordica charantia Descourt., Cocos nucifera L., and Citrus acida Roxb.. Medicinal plants used to treat sexually transmitted diseases included Borassus flabellifer L., Terminalia bellirica (Gaertn.) Roxb., Colocasia esculenta (L.) Schott, Melia azadirachta L., Achyranthes aspera L., Cicer arietinum L., Vitex negundo L., Curcuma longa L., Saraca indica L., Bombax ceiba L., Basella alba L., and Aconitum napellus L.
Since the rural patients of Bangladesh appeared to be generally satisfied with the treatment offered through these medicinal plants, it is important to conduct proper scientific studies towards discovery of compounds of interest in these medicinal plants, which can be used as safe and effective medicines.
The inhibitory neurotransmitters glycine and GABA ameliorate the disease course of experimental autoimmune encephalomyelitis Carmans Sofie ⁎ , Hendriks Jerome, Rigo Jean-Michel, Stinissen Piet, Hellings Niels Biomed, Hasselt University, Diepenbeek, Belgium
The inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA) have been shown to modulate peripheral immune cell responses. Such an immunomodulatory role can be important in neuroinflammatory diseases like multiple sclerosis (MS). We recently demonstrated that glycine modulates macrophage effector functions implicated in central nervous system (CNS) inflammation in vitro.
In this study, the in vitro effect of GABA on disease-contributing macrophage functions was explored. Furthermore, the role of glycine and GABA in the disease process of MS was further elucidated in its animal model experimental autoimmune encephalomyelitis (EAE).
Mouse peritoneal macrophages were pre-incubated with GABA (0-10 mM) for 24 hours, followed by lipopolysaccharide (10 ng/ml) stimulation for 6 hours. GABA reduced tumor necrosis factor-alpha production in a concentration-dependent manner. In contrast, interleukin-6 production was increased at low GABA concentrations (30-300 μM). The effects of GABA were mediated by GABA-A and GABA-B receptors, as their respective antagonists gabazine and saclofen reversed the GABA-evoked effects. In addition, the macrophage capacity to phagocytose myelin was significantly reduced after GABA treatment (100 μM-10 mM) for 6 and 24 hours. To elucidate the influence of GABA and glycine on neuroinflammation in vivo, chronic EAE was induced in C57Bl/6J mice and animals were then treated daily from day 3 onwards with glycine (150 mg/kg), or vigabatrin (250 mg/kg). Vigabatrin is a GABA-transaminase inhibitor resulting in an increase in GABA concentration. Glycine treatment delayed EAE onset and reduced the clinical symptoms, while vigabatrin treatment completely prevented EAE development.
These findings demonstrate that glycine and GABA have a beneficial influence on neuroinflammation, possibly by modulating macrophage function. Therefore, these inhibitory neurotransmitters and their metabolic pathways may set the basis for the development of new therapeutic agents for inflammatory diseases affecting the CNS.
The roles of gp91phox (NOX2) expressed in classical activated microglia after traumatic brain injury Kenji Dohi ⁎ ,1 , Hirokazu Ohtaki 1 , Kazue Satoh 1 , Tomoya Nakamachi 1 , Sachiko Yofu 1 , Kazuyuki Miyamoto 1 , Dandan Song 1 , Shohko Tsunawaki 2 , Seiji Shioda 1 , Tohru Aruga 1 1 Showa University, Tokyo, Japan; 2 National Research Institute for Child Health and Development, Tokyo, Japan Traumatic brain injury (TBI) is associated with reactive oxygen species (ROS), and one of the major enzymatic sources of superoxide anion production in the brain is NADPH oxidase. gp91phox (NOX2), one of the catalytic subunit of NADPH oxidase enzymes (NOXs), plays as a key enzyme in various neuronal diseases. We hypothesized that gp91phox generates superoxide anion is a major causative role in TBI.
Unilateral TBI was induced in gp91phox knockout (gp91phox−/−) and the wild-type (gp91phox +/+) mice by CCI. The expressions and roles of gp91phox after TBI were investigated with the immunoblotting and staining. The mice were determined in situ superoxide anion detection and the oxidative metabolite in brain. The activating phenotype in microglia which expressed gp91phox and gp22phox was determined in microglial cell line BV-2 in the presence of gp91phox increased 24 h and 48 h in ipsilateral hemisphere of wild-type mice after CCI, and expressed mainly in ameboid-shaped Iba-1-positive microglial cells, but weakly in astrocytes and neurons. gp91phox−/− mice had less contusion area and TUNEL positive cells than the wild-type one, and also suppressed superoxide radical (O2−) and peroxynitrite metabolites. In the presence of IFN-gamma, BV-2 cells increased iNOS and NO levels which indicated classical activated phenotype and drastically increased gp91phox and p22phox (another subunit of NADPH oxidase), but not or decreased in the presence of gp91phox containing NADPH oxidase expressed in classically activated like microglia promotes ROS formation and has an important role on brain damages after TBI. Modulations of gp91phoxcontaining NADPH oxidase and gp91phox-derived ROS may provide a new therapeutic strategy to traumatic brain injury.
Therapeutic effect of the alkyl-lysophospholipid edelfosine on immune cells and experimental autoimmune encephalomyelitis The cytostatic drug edelfosine is a synthetic analogue of lysophosphatidylcholine. Edelfosine is incorporated by cells and it acts on cellular membranes rather than DNA by selectively activating the cell death receptor Fas/CD95. Edelfosine is incorporated to effective intracellular concentrations by highly proliferating cells like activated immune cells and tumor cells. Our aim was to study the effect of edelfosine on the immune system in the context of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Additionally, we examined the impact of this drug on human T cell proliferation.
In order to analyze the anti-inflammatory properties of edelfosine we have used the relapsing-remitting mouse model of EAE. SJL mice were immunized with the proteolipid protein peptide 139-151 (PLP139-151). The effect of edelfosine on EAE disease course was evaluated by clinical and histological analysis. Daily treatment by edelfosine reduced the mean EAE score of 2.9 in control animals (PBS administration) to 1.7 (10 mg/kg edelfosine administration) at EAE acute phase. Spinal cord immunohistological analysis at the acute phase of EAE revealed a reduced CD45+ as well as CD3+ cellular infiltration if mice were treated with edelfosine. Edelfosine treatment also led to a dose-dependent activation of caspase 3 in CD4+ and CD8+ cells. We found a significant difference between 10 mg/kg edelfosine and PBS treatment independent of the cell type (CD4+ and CD8+ cells) or tissue (lymph nodes and spleen) examined. Lymph node cells of edelfosinetreated mice retained their proliferative function upon restimulation ex vivo. Re-activation of cells with PLP139-151 resulted in an edelfosine dose-dependent proliferative response. Edelfosine was also found to interfere in a dose-dependent manner with the activation and proliferation of MBP83-99 specific human T cells.
Our results imply that edelfosine may ameliorate EAE by acting on disease-specific immune cells, possibly via apoptosis induction. The results point to a dose-dependent interference of edelfosine with PLP139-151 specific immune cells in vivo as well as their proliferative capacity in restimulation experiments ex vivo. Further experiments to explore the therapeutic effects of edelfosine, when started at EAE disease onset, as well as its effects on formation of the immunological synapse are ongoing.
*Supported by the Gemeinnützige Hertie Stiftung.
A 30 years old libyan lady presented with left eye blurred vision 6 months ago which resolved within 1 week after oral steroid usage. Three months later she got bilateral lower limb numbness and parentheses followed by weakness, urinary retention and unsteadiness. Those symptoms resolved partially after usage of intravenous steroids. Two months later she got severe neuropathic pain and allodynia all over the body with bilateral lower limb numbness which resolved partially after usage of intravenous steroids and oral pregabalin. Clinically she has ataxic paraparesis with brisk reflexes all over the body and bilateral lower limb hyposthesia and allodynia up to the level of D4, with Lhermit sign. Investigations: Cervicodorsal spine MRI showed a single and continuous spinal cord lesion extending from D4 to D12 with another 2 small ovoid cervical spine lesions which brain MRI showed 5 white matter demyelinating lesions in the brain, 4 of them disappeared after steroids. CSF Aquaporin 4 antibodies were negative as well as the CSF oligoclonal bands, with normal CSF IgG index. Visual evoked potentials showed delayed p100 response on the left eye. Somatosensory evoked potentials were normal. Conclusion: The patient has relapsing-remitting neurologic disease with MRI brain lesions suggestive of MS, but she has single continuous and gadolinium enhancing dorsal spine lesion extending from D4 to D12. The presence of such long spinal cord lesion is atypical for MS and it is suggestive of Devic Disease in spite of negative CSF Aquaporin 4 antibodies.This case highlights the challenges in diagnosing demyelintating diseases, which has important implication regarding patient prognosis and therapy.
Diabetic ketoacidosis increases inflammatory transcription factor activation and adhesion molecule expression Close Taylor E. ⁎ , Bezzina Kathryn A., Patterson Eric K., Cepinskas Gediminas, Fraser Douglas D.
The University of Western Ontario, Lonond, ON, Canada Diabetic ketoacidosis (DKA) is a complication of type-1 diabetes that requires urgent recognition and treatment. During DKA therapy, children are at high risk for intracranial complications including: edema, stroke, and haemorrhage. The causes of these intracranial complications are unknown; however, blood brain barrier (BBB) compromise and neuroinflammation may be contributing factors. We have previously shown that there is a difference in the immune response systemically and in the cerebral cortex. We illustrated significantly higher levels of the cytokines interleukin (IL-6), IL-8 (KC), IL-10 and TGF-b1 in serum of DKA mice. However, in the cerebral cortex IL-10 decreased and TGF-b1 increased without corresponding changes to . The aim of this study was to further investigate the neuroinflammatory response. We hypothesized that the transcription factor nuclear factor-kB (NF-kB) is activated in endothelial cells of the BBB by DKA and that NF-kB subsequently increases expression of vascular cell adhesion molecule-1 (VCAM-1).
To investigate NF-kB activation and VCAM-1 expression during DKA, we used juvenile mice injected with either pancreatic beta-cell toxins; streptozocin and alloxan (DKA) or vehicle alone (control). Spleen and cerebral cortex samples were obtained from both control and DKA mice at various time points (12, 24, 48, and 72 hours) . Activated NF-kB p65 protein levels in spleen and cerebral cortex were analyzed using NF-kB p65 transcription factor assays. VCAM-1 mRNA expression in cerebral cortex was quantitatively analyzed using realtime PCR.
DKA was confirmed in mice by demonstrating significantly higher serum glucose, beta-OH-butyrate levels, and weight loss compared to controls. Analysis of cerebral cortex revealed significantly increased activated NF-kB p65 levels at 48 and 72 hours, while in spleen, there were no significant differences between control and DKA mice. An upregulation of cerebral cortex VCAM-1 mRNA was found at 48 hours, mirroring NF-kB p65 activation, but this finding was not yet statistically significant (n = 3).
These data implicate an inflammatory response in cerebral cortex during DKA, involving NF-kB p65 protein activation and VCAM-1 mRNA expression at 48 hours, without a corresponding response in the spleen. This observed neuroinflammatory response may contribute to the intracranial complications observed in some children during DKA. The resultant MOGTCR × Thy1CFP mice develop ON leading to neuronal loss that can be monitored longitudinally in "real-time" in the living animal, therefore offering a refinement of previous models of MS, such as "classical" EAE. This new model will be invaluable for the study of neuroprotective and repair strategies in autoimmune diseases and provides a direct experimental correlate for studies that could be undertaken in humans.
A C57BL/6 mouse expressing a T cell receptor (TCR) transgene specific for 35-55 residues of myelin oligodendrocyte glycoprotein (MOG), restricted by H-2Ab [Betteli et al. 2003. J. Exp. Med. 197 :1073] develops ON spontaneously (approximately 5%) or following induction with a combination of immune adjuvants (Pertussis toxin, MOG-specific Z12 monoclonal antibody), to give a co-ordinated 100% incidence of disease. Optic neuritis, which can develop in the absence of clinical or histological EAE, is associated with extensive demyelination and axonal loss in the optic nerve. This nerve loss can be quantified by loss of RGC in the retina. These animals were crossed with C57BL/6.Thy1 CFP mice, which express cyan fluorescent protein (CFP) under control of a Thy1 promoter that limits expression of CFP to the RGC in the eye [Feng G et al. 2000. Neuron 28:41] .
The resultant MOGTCR × Thy1CFP mice develop ON leading to neuronal loss that can be monitored longitudinally in "real-time" in the living animal, therefore offering a refinement of previous models of MS, such as "classical" EAE. This new model will be invaluable for the study of neuroprotective and repair strategies in autoimmune diseases and provides a direct experimental correlate for studies that could be undertaken in humans.
Central diabetes insipidus is associated with an infiltration of mainly T-cells in the pituitary gland, infundibulum, and possibly the hypothalamus. There, the destruction of vasopressin-expressing neurons leads to polydipsia and polyuria. The pathophysiology and the role of the T-cell subpopulations in this disorder are still unknown and could be studied in animal models.
To study the relative contribution of CD8 and CD4 T-cells and their cooperation in CNS autoimmunity, we have developed a mouse model in which hemagglutinin (HA) is expressed as a neo-self antigen in neurons (CamK-HA mice). Complementary HA TCR-transgenic mice are used as a source of HA-specific Tcells to address the individual or combined contribution of different T cell subpopulations in their potential to induce damage to neurons.
A knock-in mouse in which the HA cDNA is inserted in the Rosa26 locus with a floxed Stop cassette was crossed with the CamK-iCre mice, expressing the Cre recombinase under the control of the neuron-specific Calmodulin-Kinase IIa promoter.
After adoptive transfer of 30× 106 of in vitro activated HA-specific Tc1 cells in CamK-HA mice and controls, 41% of the CamK-HA mice died of general myelo-encephalitis, whereas all of the controls survived.
At the initial phase, the diseased CamK-HA mice showed a severe weight loss and neurological signs associating trembling and hypoactivity, without obvious paralysis. Histology at day 6 detected inflammation in spinal cord and brain, predominantly in the hypothalamus, brainstem and medulla oblongata. After this initial phase, the surviving mice recovered full mobility and normal weight. However, all the CamK-HA recipients developed from days 7 to 10 onwards signs of diabetes insipidus (polydipsia and polyuria). The high amount of urine (3-5 times more than the controls) could be reduced by injection of a vasopressin-analog, suggesting destruction of the hypothalamic vasopressin-producing neurons in these mice. Indeed, the virtual disappearance of vassopressin-expressing neurons was shown by histology.
This model should help generate novel information regarding the contribution of CD8 T-cells in neural tissue injury and their protection. As a first illustration of this potential, we developed a new model of diabetes insipidus induced by "autoreactive" cytotoxic CD8 T-cells, underlying the potential role of these T-cells in the pathophysiology of human idiopathic diabetes insipidus.
Characterization of experimental autoimmune neuritis models induced by P0 peptides and lipopeptides in Lewis rats Beaino Wissam, Trifilieff Elisabeth ⁎ Université de Strasbourg / CNRS, Strasbourg, France Thiopalmitoylation (i.e., the covalent attachment of palmitic acid via a thioester linkage to cysteine residues in the polypeptide backbone) is a common post-translational modification of proteins. In a previous study, we have shown that thiopalmitoylation of encephalitogenic T-cell epitopes of central nervous myelin proteolipid protein (PLP), as occur naturally in vivo, enhanced immune responses as well as the development and chronicity of experimental autoimmune encephalomyelitis (EAE), an animal model of the human inflammatory demyelinating disease, multiple sclerosis (MS) (1, 2) . These results suggest that the immune response induced by endogenous thioacylated peptides that are released during myelin breakdown may play a role in the development and chronicity of autoimmune inflammatory demyelinating diseases.
To confirm this hypothesis we have studied the effect of thiopalmitoylation on the immunogenic and neuritogenic properties of P0 protein, the major protein of peripheral nervous system (PNS) myelin. P0 protein is thiopalmitoylated at cysteine 153, and is a candidate autoantigen in AIDP (acute inflammatory demyelinating polyradiculoneuropathy) the most common subtype of Guillain-Barré syndrome (GBS), a human inflammatory demyelinating disease of the PNS. Valuable insights in the immunopathogenic mechanism of GBS has been gained from the animal model, experimental autoimmune neuritis (EAN), that can be induced in Lewis rats by injection of P0 protein or P0 peptides.
We have synthesized palmitoylated and non-palmitoylated P0 (180-199) and P0(152-171) peptides to study and compare their immunogenic and neuritogenic properties after injection in Lewis rats. P0(180-199) peptide, the immunodominant P0 neuritogenic epitope in Lewis rat, was used as the control peptide. The course of the disease was followed by clinical assessment and histopathology of the sciatic nerve.
Our preliminary results show that thiopalmitoylation has indeed important effect on the neuritogenic properties of both peptides and that a "CIDP-like relapsing model" can be induced with palm P0(180-199) while palm P0(152-171) seems to induce a more "AMAN-type" of disease (acute motor axonal neuropathy). The role of muscle-specific kinase (MuSK) antibodies (Ab) for the onset of myasthenia gravis (MG) has remained in dispute because the majority of MuSK Ab in the patients have shown to be incapable to activate complement, leading to the lysis of post-synaptic membrane at neuromuscular junction (NMJ). In order to reveal whether the activation of complement is indispensable for the pathogenicity of MG caused by MuSK Ab, complement (C5)-deficient mice were immunized with MuSK protein.
To investigate whether MG can be cause4 by MuSK-Abs without involvement of complement activation, we injected MuSK protein into C5-deficient mice twice at days 0 and days 14. The mouse strain has two bases "TA" deletion near the 5′ end of the C5 gene, which causes premature stop of the translation and C5 deficiency. This mutation cannot generate lytic membrane attack complex (MAC) by activation of all complement pathways. The mice demonstrated not only the characteristic features of electromyograpy observed in patients of MG, but also abnormal electrophysiological signs recorded in patients of MuSK MG. A single injection of neostigmine significantly decreased muscle weakness in only 2 of 6 MuSK-injected mice with severe muscle weakness. Reversal of CMAP amplitude decrements after neostigmine treatment was recorded at 3/s stimulation in 6 anesthetized mice within 9.2-24.5% (mean 16.6%) of the decrement. However, only one mouse could recover CMAP within 10% of decrement. Both pre-and postsynaptic structures of neuromuscular junctions (NMJs) were significantly degenerated in the mice. We examined the expression of AChE and Collagen Q (ColQ) at the postsynaptic membrane of NMJs in the mice. We found that the intensity of AChE expression at NMJs was significantly decreased compare with uninjected-control mice, and both ColQ and AChR expression were coordinately reduced at the NMJs.
We show the evidence that MuSK-Abs can cause MG without involvement of complement activation using mice strain which bears mutations in complement component C5.
Dominancy of encephalitogenic peptide itself directs sustainable regulation of a model of multiple sclerosis, by induction of 'armoured' Treg Lin Youwei ⁎ , Sachiko Miyake, Takashi Yamamura National Institute of Neuroscience, NCNP, Tokyo, Japan Immunization with a myelin peptide causes a variety of inflammatory reactions in the central nervous system, collectively referred to as experimental autoimmune encephalomyelitis (EAE). Though some models may represent relapsing-remitting disease (RR-EAE) resembling multiple sclerosis, others would follow monophasic disease (M-EAE). Surprisingly, no prior studies have addressed mechanistically why each EAE model would exhibit its own unique disease course, besides referring to genetic factors. Noting that immunizing SJL/J mice with overlapping myelin proteolipid protein (PLP) residues 136-150 and 139-151 could induce quite different diseases; M-EAE by PLP136-150 and RR-EAE by PLP139-151, we addressed if M-EAE and RR-EAE are differentially regulated in vivo.
From the phenomenon that M-EAE could never develop any relapse nor re-induction whereas RR-EAE could, we compared the kinetics of lymphoid cells from mice immunized with either peptide and found that highly potent regulatory T cells expressing CD69 and CD103 were induced and maintained in the draining lymph nodes of M-EAE after the appearance of encephalitogenic T cells. These CD69+CD103+ regulatory T cells were functionally more efficacious than other subsets of CD4+ CD25+ T cells as a reflection of showing more suppressive gene signature. They uniquely dominated by Foxp3+RORgt+ cells, expressed CTLA4, ICOS, GITR, FR4 and LAG3 at a much higher level and S1P-1 at a lower level, and produced IL-10, LAP and IL-17, enabling them to resist against continuously peripheral expansion of encephalitogenic T cells. Furthermore, elongation of N-termini of PLP136-150 and truncation of Cterminus of PLP139-151 were closely related to dominancy of peptide itself, and the more dominant peptide possessed the capacity to develop acute EAE, the less relapse and re-induction of EAE were occurred, correlating with the potency to induce activated regulatory T cells described above.
These results indicate that differential induction of the "efficacious" regulatory T cells by individual dominant self-peptide for 'tuned suppression' is relevant for pathogenesis leading to different clinical course and prognosis and also for treatment of autoimmune diseases. Tumor necrosis factor (TNF), a pleiotrophic cytokine involved in normal brain function, is associated with the development of inflammation following both traumatic injury and neurological diseases. Treatments aimed at eliminating excessive TNF are effective therapies for a number of inflammatory and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. However, the clinical use of TNF inhibitors is associated with an increased risk of infections. Genetic studies in mice have suggested that inflammation in disease models involves solTNF and that maintenance of innate immunity involves transmembrane TNF (tmTNF).
Therefore, we took advantage of a dominant-negative inhibitor of solTNF, XENP1595, that selectively inhibits solTNF and compared its effect to the more widely used IgG1 Fc-TNF receptor 2 fusion protein (Etanercept), an inhibitor of both solTNF and tmTNF, in a mouse model of spinal cord injury (SCI).
Behavioral testing of mice subjected to SCI and treated with either 1) XENP1595, 2) Etanercept or 3) vehicle, either directly to the traumatized spinal cord via micro-osmotic pumps (continuous delivery for 3 days) or subcutaneously every 3 days for 8 weeks, showed that mice treated with XENP1595 displayed no obvious improvement in functional outcome compared to Etanercept or vehicle. However, preliminary data of stereological estimations of lesion sizes show that mice treated with XENP1595 delivered directly to the spinal cord have smaller lesion sizes 7 days after SCI than mice treated with Etanercept or vehicle.
These preliminary data indicates that solTNF inhibition and maintenance of tmTNF might be neuroprotective following SCI.
Expression quantitative trait loci identifies candidate genes and regulating pathways in experimental autoimmune encephalomyelitis Thessén Hedreul Mélanie ⁎
Genome-wide association studies have successfully identified many risk genes regulating complex diseases in humans. Numerous quantitative trait loci (QTLs) that modulate disease have also been identified when performing genome-wide screening in experimental animal crosses. However, few have reached single-gene resolution and the knowledge of how disease risk genes truly regulate disease is incomplete. Advances in technology over the last couple of years have enabled mapping of gene expression levels (eQTLs) on a whole-genome scale. This is an important complement to traditional QTL mapping and availability of the full transcriptome even enables co-expression and network enrichment analysis of genetic risk variants, which can provide insights into disease pathogenesis.
The model we use for studying complex inflammatory responses is experimental autoimmune encephalomyelitis (EAE), the animal model of MS. In this study, we combined classical QTL with eQTL mapping to 1) facilitate candidate gene identification within our previously known EAE QTLs, as cis-eQTLs that reside within these regions constitute possible candidate genes, and to 2) define disease pathways that regulate neuroinflammation. A genome wide expression experiment (Affymetrix Rat Gene 1.0 ST Arrays) was performed on spleen sampled from 150 male rats from a (DAxPVG.1av1)DA backcross at day 35 post immunization of EAE.
We have now confirmed many of the EAE QTLs previously found in our group, mapped several cis-eQTLs and gained knowledge on how these are involved in regulating disease. Encephalitis lethargica (EL) affected a large number of people in the pandemic in the early 1900s (von Economo, 1930) . Whilst it is now considered a rare disorder it still occurs sporadically. Its frequency, presentation, disease course, treatment response and causes are unknown. Typically, EL presents as an acute illness in young adults with initial neuropsychiatric features, sleep disturbance and movement disorders. Histological and biochemical data suggest that autoimmune mechanisms play an important role in this disorder and recently serum anti-basal ganglia antibodies (ABGAs) have been detected in affected sporadic cases associated with evidence of recent streptococcal infection.ABGA are also associated with other neuropsychiatric disorders like Sydenham's chorea, PANDAS, Tourette syndrome and obsessive compulsive disorder. As ABGA are strongly associated with recent streptococcal infection, these disorders represent a potentially good model for the study of molecular mimicry and autoimmunity.
In the present study we have demonstrated the effect of immunopurified antibodies from EL patient plasma and recombinant enzymes (autoantigens) on neuronal cell function. Following the application of the purified autoantibodies to neuronal cells in culture a decrease in enolase activity, cell viability and metabolic activity has been observed, together with an increase in apoptosis. Furthermore, our preliminary data indicated that calcium influx via the L-type channel is significantly depressed by ABGA patient IgG, leading to neurite retraction and apoptosis. Thus we also aim to determine the effects of purified monospecific antibodies on KCl-evoked calcium responses as well as NMDA stimulated calcium responses. Single cell calcium (Ca2+) responses will be measured by fluorescence imaging. Post-streptococcal disorders are still poorly understood, largely due to the lack of suitable animal models. Therefore, we are developing an animal model of these disorders by passive transfer of EL patient IgGs or active immunization with group A streptococcus homogenate, human basal ganglia and recombinant proteins (autoantigens).
As auto-antibody mediated diseases respond to immunomodulatory therapy, identifying and defining the pathogenesis of these disorders is important so that patients can be appropriately treated. Establishing this group of disorders as a "true" autoimmune disease of the CNS and demonstrating that ABGA have functional effects will establish a whole new paradigm. Multiple sclerosis (MS) is an immune-mediated, demyelinating neurodegenerative disease of the central nervous system (CNS). Using experimental autoimmune encephalomyelitis (EAE) models of MS, we have previously demonstrated that the cannabinoid system can control: autoimmune, neurodegenerative and symptomatic aspects of disease, related to the actions of the two cannabinoid receptors (CB1 and CB2) so far described.
We identified that orphan G protein coupled receptor 55 (GPR55) as a novel lipid-receptor that is stimulated by lysophosphoinositol and importantly some cannabinoid ligands, suggesting that it may have some functional relationship with the cannabinoid system. Using quantitative polymerase chain reaction and in situ hybridization we show that GPR55 is weakly expressed in both the nervous and immune systems and as expected is absent in a novel Gpr55tm1Lexgene deficient knockout mouse, being developed by us.
The immunological phenotype in relation to the susceptibility of GPR55-deficient C57BL/6/129 and ABH mice to experimental autoimmune encephalomyelitis and the response to VSN16 [3-(5dimethylcarbamoyl-pent-1-enyl) -N-(2-hydroxy-1-methyl-ethyl)] a novel GPR55-selective, symptom modifying agent will be presented.
Herpes simplex virus type 1 (HSV-1) induced central nervous system (CNS) demyelination in mice Kastrukoff Lorne ⁎ , Lau Allen, Thomas Eva
Infection of the oral mucosa with a 'novel' strain of HSV-1 (lab strain 2) results in the development of demyelinating lesions throughout the brains of susceptible but not resistant mice. The lesions, characterized by demyelination, an inflammatory mononuclear cell infiltrate, and relative preservation of axons, develop in three phases over 6 months. The early phase lasts for~21 days postinfection (PI) and correlates with the transient spread of infectious virus throughout the brain; determined by viral titer studies.
To further characterize the early phase lesions, we performed histology, immunohistochemistry (IHC), in-situ hybridization, and solution phase PCR studies in inbred mice infected with HSV-1 via the oral mucosa.
Focal areas of viral antigen are identified up to 12 days PI in the brains of HSV-1 infected mice. The areas are restricted to the trigeminal root entry zone in resistant (C57BL/6) mice but develop sequentially throughout the brains in susceptible strains. HSV-1 infected cells in the focal areas have the morphological characteristics of neurons and glia. In susceptible strains, the number and size of demyelinating lesions in the brain correlate inversely with the resistance of the mouse strain to mortality (PL/J N A/JN SJL/JN BALB/c). Focal areas of viral antigen evolve into demyelinating lesions with the clearance of virus from these sites. Despite clearance of infectious virus, viral c-DNA positive cells remain in the areas of demyelination. Solution phase PCR studies identified viral DNA to be present in the brains of susceptible strains for at least 6 months.
In summary, specific strains of HSV-1 can induce CNS demyelination in susceptible strains of mice. In the early phase, virus replication occurs in focal areas in the brain while latent infection is established. Further, the development of demyelination correlates with the clearance of infectious but not latent virus from the focal areas of viral antigen. The presence of latent virus in the CNS beyond the early phase may play a role in the development of demyelination that occurs during the later phases.
HPA axis activation influences the susceptibility to develop Experimental Autoimmune Encephalomyelitis in a gender specific manner Harpaz Idan ⁎ Ben Gurion University, Beer Sheva, Israel Activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to stress increases the release of corticosteroids from the adrenal glands to maintain homeostasis. However, chronic stress prolongs the activity of the HPA axis and may interfere with its normal activity. This, in turn, may interfere with immune homeostasis and reduce the ability of the organism to cope with immune challenges. Given the different gender susceptibilities to experimental autoimmune encephalomyelitis (EAE), we investigated whether mice exposure to chronic stress affects the susceptibility to EAE in a gender-specific manner.
To this end, we exposed male and female mice to chronic stress for 24 days and then we induced EAE. Corticosteroids secretion was evaluated throughout the experiment. We show that, under both basal and chronic stress conditions, males were more susceptible to EAE as compared with females. This effect was associated with poor activation of the HPA axis both under basal and chronic stress in males as compared with females. In addition, while chronic stress induced anxiety-like behaviors, weight loss and reduced frequency of T cells in both genders, male mice showed an exacerbate form of the disease following exposure to chronic stress.
The data presented above demonstrate that HPA-axis activation in response to chronic stress is differentially regulated in males and females, a phenomenon which can directly impact on the regulation of autoimmunity and hence the susceptibility and progression of autoimmune diseases. Primary Sjøgren's syndrome (PSS) is a chronic autoimmune disease affecting exocrine glands. Fatigue is common and a major cause of disability. Sickness behaviour in animals is an adaptive response characterized by increased sleep, social withdrawal and loss of appetite and is mediated by pro-inflammatory cytokines such as interleukin (IL)-1 acting on the brain. This cytokine is also increased in humans with cancer related fatigue. Our group recently reported an association between activation of the IL-1 system and fatigue in PSS indicating that IL-1 might signal fatigue in several somatic diseases. The objective of the current study was to explore the effect of IL-1 inhibition on fatigue in PSS.
In a double-blind, placebo-controlled study 26 patients agreed to participate and written consent was received. During the four weeks of treatment the patients self-administered daily injections of either anakinraan IL-1 receptor antagonistor placebo (0.9% NaCl). Patients were examined at inclusion, week 0 (start), week 2, week 4 (end of treatment) and follow-up at week 5.
The patients were asked to rate their fatigue on a visual analogue scale (VAS). Baseline characteristics (age, disease duration, haematological and biochemical tests, and levels of fatigue) were similar in the anakinra-group (n = 13) and the placebo-group (n = 13).
Friedman test for repeated measures was used to test differences over time compared with baseline, and chi-square test was used to compare the number of patients who reached a 50% reduction in fatigueVAS in week 4 compared with inclusion.
There was a highly significant reduction in fatigueVAS during the study in the anakinra-group, not observed in the placebo-group (median fatigueVAS at inclusion: 65 (range 32-93), median week 0: 66 (22-89), median week 2: 60 (9-93), median week 4: 34 (5-91) vs median inclusion: 72 (47-97), median week 0: 69 (46-93), median week 2: 51 (21-80), median week 4: 60 (20-90), p = 0.009 vs p = 0.053). Significantly more patients had a 50% reduction in their fatigueVAS by week 4 in the anakinra-group (6/12) compared with the placebo-group (1/13, p = 0.03).
IL-1 inhibition reduces fatigue significantly in patients with PSS. This may be due to a decreased IL-1 signalling in the brain, as observed in animal studies. The results indicate that the IL-1 system is a biological factor associated with fatigue in humans.
Increased TRPM8 expression on rat brain in the pathogenesis of experimental autoimmune encephalomyelitis Yanjie Jia ⁎ , Jingjing Lu, Yonglin Jia, Lijun Jing
The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
The transient receptor potential (TRP) channels are a large family of proteins, which are involved in a wide range of processes ranging from sensing of thermal and chemical signals to reloading intracellular stores after responding to an extracellular stimulus. TRPM8 is conventionally reported as a cold-and menthol-sensing cation channel implicated in thermosensation. Here we reported that expression of TRPM8 might increase greatly on rat brain in the pathogenesis of experimental autoimmune encephalomyelitis (EAE).
In this study, we firstly established the Wistar rat models of EAE by footpad injection of guinea pig spinal cord homogenates and CFA. The expression of TRPM8 protein and mRNA on brain tissues, which were extracted from EAE rats at 1, 7, 14 and 21 days respectively, was measured with immunohistochemical stain, Western blot and RT-PCR. The correlation between the level of TRPM8 and symptoms of EAE was also investigated.
The results showed that various degrees of inflammation and demyelination of brains were found in the EAE group, after being immuned. Then we observed that TRPM8 protein and mRNA expression, were at very low level in the brain of the control group. After EAE was induced, the level of TRPM8 protein and mRNA expression on the brains increased gradually with the development of symptoms and brain pathology of EAE. On 14 days, the level of TRPM8 protein and mRNA expression on the brains was at significantly increased levels. At the same time, the clinical symptoms and pathology of EAE group reached the peak. Immunohistochemical stain demonstrated the expressions of TRPM8 protein were mainly localized on cell membrane and axon in the regions of lesion foci. On 21 days, the levels of TRPM8 protein and mRNA expression reduced gradually, which was in parallel with a gradual alleviation of parts of the clinical symptoms and pathological lesion of EAE group.
These findings indicate that TRPM8 may play some important roles in the pathogenesis of EAE.
Inhibition of autoimmunity is insufficient to stop secondary progressive, experimental autoimmune encephalomyelitis There has been poor translation of the use of immunosuppressive agents from animal models into the treatment of multiple sclerosis (MS). Although this may be because of differences in disease pathogenesis, the vast majority of studies in experimental autoimmune encephalomyelitis (EAE) models of MS examine prophylactic, treatment regimes. Whilst these may elucidate immune mechanisms, they are probably of limited relevance to the therapeutic application. This study aimed to investigate whether potent immunosuppression could inhibit long-established, progressive-autoimmune disease of the CNS in an animal MS model.
Chronic-relapsing EAE was induced in ABH mice using spinal cord antigens in complete Freund's adjuvant. FYY720 (fingolimod), which limits relapsing MS, was orally-administered at various times after disease induction and the influence on motor-function was assessed. Whilst prophylactic, FTY720-administration inhibited the development of disease, more importantly it inhibited subsequent relapsing-disease when used therapeutically during relapsing-EAE. This facilitated some recovery in motor-function suggestive of repair, once relapsing disease was halted. However, following the accumulation of neurologicaldeficits due to relapsing attacks, slow progressive-worsening in disability occurred. This was exposed by the elimination of relapsingautoimmunity by immunological tolerance induction, using transient T cell deletion and intravenous myelin administration. Months after disease induction, animals were randomised to vehicle or FTY720. As anticipated no further relapses occurred but there was a significant deterioration in motor function in both groups, suggesting that immunosuppression at this point was not inhibiting progressive EAE.
Early treatment with immunosuppressive agents may inhibit the generation of a neurodegenerative microenvironment, which is no longer responsive to potent immunosuppression. However, if treatment is initiated too late in these disease-processes, progressive neurological disease continues unabated. This suggests that immunosuppression alone will never be insufficient to control progressive neurodegeneration in both animals and in progressive MS.
Inhibition of neutrophil elastase attenuates TH17-induced neuro-inflammation Axtell Robert ⁎ , Steinman Lawrence
Neuromyelitis optica (NMO) is a neuro-inflammatory disease similar to relapsing-remitting multiple sclerosis (RRMS). However, NMO differs from RRMS in that the inflammatory lesions in NMO are more granulocytic and conventional RRMS treatments are not effective in NMO. The pathogenic mechanism of neutrophils in NMO is largely unknown. In this study we demonstrate that NMO can be modeled with experimental autoimmune encephalomyelitis (EAE) induce with TH17cells and this disease can be attenuated by targeting an enzyme found the granules of neutrophils, neutrophil elastase.
We found that compared to RRMS, NMO has increased serum levels of IL-8 and Gro-alpha, chemokines that recruit and activate neutrophils. In congruence with NMO, EAE induced with TH17 cells had increased levels of Gro-alpha and have a predominant neutrophil infiltrate compared to EAE induce with TH1 cells. In addition, inhibiting an enzyme found the granules of neutrophils, neutrophil elastase, significantly attenuated TH17-EAE and blocked the infiltration of neutrophils to the central nervous system.
Our data demonstrate that TH17 induced EAE has key features that resemble NMO. Furthermore, inhibiting neutrophil elastase is effective in treating TH17 EAE and could be a viable treatment option for NMO.
Magnetic resonance imaging analysis of brain lesions in a mouse model of progressive multiple sclerosis Levy Hilit ⁎ , Assaf Yaniv, Frenkel Dan Tel Aviv University, Tel Aviv, Israel
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by damage to the neuronal myelin sheath. The magnetic resonance imaging (MRI) is a noninvasive imaging tool for diagnosing and characterizing MS pathology. Experimental autoimmune encephalomyelitis (EAE) model is the most common animal model to study MS, nevertheless, in most EAE mouse models, the neurological damage mostly affects the spinal cord with limited damage to the brain that is not measurable by MRI. Our goal was to investigate the development of brain lesions in vivo in a model of chronic progressive MS using MRI analysis as done in human.
In this work we describe EAE course in NOD mice that results in both brain and spinal cord lesions. Post-gadolinium T1 MRI revealed increased blood-brain barrier (BBB) permeability in brain white matter specific to the corpus callosum, fimbria, and internal capsule as found in human. Diffusion-tensor imaging (DTI) analysis identified focal areas of abnormal anisotropy and diffusivities values. Immunohistological analysis indicated that these DTI/ BBB abnormalities are also characterized by demyelination and inflammation. Furthermore, we suggest that increased astrocyte toxicity in EAE induced NOD mice may be linked to brain lesion development.
We propose this EAE model as a suitable model for studying MS using MRI methods towards future drug development. The 22q11.2 deletion syndrome (22qDS) is the most common hemizygous microdeletion syndrome occurring in humans. Individuals with 22qDS show a variety of neuroanatomical/pathological defects. In particular, they suffer many brain anomalies, including reduced size of cerebellum, cerebral cortex (grey and white matter), polymicrogyria, and midline defects such as cavum septum pellucidum. Recently it has been shown that they have a reduction of microRNA (miRNA) dosage, possibly deriving from the absence of a Dgcr8 allele which is a part of the molecular machinery involved in miRNA biogenesis. However, the real contribution of miRNAs to the disease outcomes is far from coming out.
MiRNA contribution to forebrain development was tested by inactivating Diceri.e. a limiting enzyme for miRNAs biogenesis acting downstream Dgcr8at several time points of forebrain development. FoxG1Cre mediated Dicer inactivation, which occurs in E8.5 developing forebrain, leads to a substantial derangement of forebrain development and causes radial glial (RG) cell death that produces brain cyto-architecture alterations. However, their capabilities to progress in the cell cycle were not affected. In contrast, later Dicer inactivationi.e. mediated by GfapCre and occurring at E12.7resulted in less severe apoptotic phenotype. RG cell survival was preserved in both cortical and hippocampal anlage, but RG cell capabilities to progress in cell cycle were altered. Indeed, impaired dentate gyrus cells proliferation produced a dramatic shrinking of E18.5, P15 and P30 cKO dentate gyrus. Cortical RG cell proliferation is also impaired and starting from E16.5 a significant number of RG cells lose their phenotype acquiring those features of basal progenitor (BP) cells. The alteration of the balance between RG and BP cell populations leads to a reduction of adult neural precursor cells (aNPC) of the dorsal subventricular zone (SvZ).
Forebrain Dicer inactivation leads to different phenotypes depending on the time points in which such manipulation has been carried out. Cell survival is severely affected when we ablated Dicer before the onset of neurogenesis. Besides cell death, however, we did not find any alteration in cell cycle progression of RG cells. On the other hand, later Dicer inactivationi.e. after the onset of neurogenesisonly partially affects cell survival, but significantly alters cell cycle progression of dentate gyrus progenitors.
Neuronal PPARg-knockout causes obesity and peripheral insulin resistance in mice The peroxisome proliferator-activated receptors (PPARs) represent ligand-activated transcription factors that belong to a nuclear receptor superfamily. PPARg is present in most cell types where it mediates multimodal function, including control of glucose homeostasis, regulation of systemic insulin sensitivity, cell differentiation, and cholesterol metabolism. In the central nervous system PPARg is abundantly expressed in neurons where its function is less clear yet. This study aimed to investigate the role of neuronal PPARg in the regulation of body weight and glucose metabolism using neuronspecific PPARg-knockout (KO) mice.
Targeted deletion of PPARg in neurons was induced by using Cre-loxP technology and taking advantage of the NEX promoter to achieve neuronal specificity. Male PPARg KO and wildtype (wt) mice were fed a standard diet (CD) or high-fat diet (HFD) for a period of 4 months. Body weight, daily food and calorie intake were monitored. Each group underwent glucose tolerance (GTT) and insulin tolerance testing (ITT) after a 6 h-fast, and the impact of PPARg deficiency and high-fat feeding on insulin signalling and lipid metabolism was studied in the blood and peripheral organs. Hypothalamic RNA samples were subjected to whole genome array. The other brain hemisphere was used for histological analyses.
Maintained on unrestricted CD, PPARg KO mice showed significant body weight gain in comparison to wt mice and exhibited glucose intolerance and insulin resistance in GTT and ITT after 4 months. These findings were further exacerbated upon high-fat feeding. There was a significant increase in plasma total cholesterol in the CD-fed PPARg KO mice as compared to wt controls along with several other metabolic changes. Neuroanatomy of PPARg KO mice demonstrated a similar neuronal architecture and density as in controls and revealed that hippocampal and cortical neurons were morphologically intact. A microarray showed a 4.2-fold upregulation of the carboxypeptidase E gene (Cpe) in the hypothalamus of CD-fed PPARg KO mice as compared to CD-fed wt controls, whereas Cpe expression was 10.2fold downregulated in PPARg KO mice compared to controls under HFD conditions.
Our results suggest a key role for neuronal PPARg in the regulation of peripheral insulin sensitivity and body weight, which may be due to an influence on the expression of the obesity susceptibility gene Cpe in the hypothalamus. Additional studies are required to further elucidate these findings. Multiple sclerosis (MS) is an immune-mediated, demyelinating neurodegenerative disease of the central nervous system (CNS). Loss of myelin leads to the development of compensation mechanisms such as reorganisation of sodium channels along the axon. Whilst this may maintain signal transduction in the short-term, Na + loading may leave nerves vulnerable to the generation of toxic chemical/ionic imbalances caused by metabolic demands and subsequent death. This may be a mechanism of neurodegeneration that underlies progressive MS, which currently does not respond to immunosuppression. Most previous studies in the experimental autoimmune encephalomyelitis (EAE) model of MS suggest that Na + channel blockers are immunosuppressive in non-demyelinated EAE. However, we demonstrate that some Na + channel blockers such as carbamazipine and oxcarbazepine can exhibit marked neuroprotective-effects in the absence of overt immunosuppression of T cell-induced autoimmunity.
Spinal cord homogenate in complete Freunds adjuvant was used to induce EAE in ABH mice. Following the initial attack to establish tissue damage, a synchronized relapse was induced to trigger marked nerve damage. Bolus daily administration of Na + channel blockers, at doses equivalent to or lower than human use, did not induce immunosuppression and reduce the incidence immune attack or the maximum severity of clinical disease. However, the drugs promoted a better recovery in remission from the immune-attack as evidenced by: clinical scores, motor co-ordination assessed by RotaRod and total spinal cord nerve content assessed using a neurofilament ELISA. The neuroprotective effect was evident when treatment was initiated at onset and more importantly, days after the onset of paralytic disease.
These data indicate that there is a defined time-window following the development of attack during which neuroprotective agents work; we have called this the "inflammatory penumbra", which is analogous to the well defined ischaemic penumbra in stroke. The inflammatory penumbra appears to correlate with the period of active blood-brain barrier dysfunction, which lasts days in mice but weeks in MS. These results have therapeutic indications for neuroprotective strategies in MS; co-administration of Na + channel blockers with standard immunosuppressive agents, such as steroids during lesion formation and evolution may provide added benefit for limiting inflammatory-neurodegeneration in progressive disease. Multiple sclerosis (MS) is a neurodegenerative disease characterized by repeated demyelination events, accompanied by inflammation, gliosis and axonal loss. Remyelination occurs mostly at early stages of the disease, but as the disease progresses, remyelination fails. Thus, mechanisms behind demyelination relapse and remyelination failures are considered as key elements in the study of MS progression.
Systemic inflammation can affect central ongoing inflammatory processes by inducing cytokine expression within the central nervous system (CNS). As such, it is able to modulate various aspects of CNS function. Inflammation has been positively correlated with demyelinating processes, although under certain circumstances it can play a dual role in MS progression. Interleukin-1B (IL-1B) is present in active MS lesions and has been linked to demyelination relapses. However, its potential as a pro-remyelinating factor and in neuroprotection was also shown. Therefore, its precise role in MS progression needs to be further elucidated.
Our main hypothesis is that repeated central or systemic proinflammatory stimuli exacerbate previous demyelination due to a central demyelinating stimulus.
We have previously developed a chronic inflammatory rat model of demyelination, where chronic expression of IL-1B in the striatum causes reversible demyelination and blood-brain barrier breakdown. To test our hypothesis, we re-administered adenoviral vectors expressing IL-1B (AdIL-1B) or B-galactosidase (AdB-gal) as control in the striatum or i.v.
Animals which received a systemic injection of Ad-IL-1B 30 days after the first injection (remyelination phase) showed exacerbated inflammation and demyelination, along with microglial and astroglial activation. However, if the second stimulus was administered centrally a lower inflammation and demyelination was observed compared with animals receiving a unique pro-inflammatory stimulus.
In conclusion, peripheral IL-1B exacerbates the inflammatory and demyelinating effects of a previous central chronic IL-1B administration, while a repeated central IL-1B stimulus shows a diminished effect when comparing it with the first central episode. Our model shows that peripheral inflammation has a highly significant influence on central on-going inflammatory and demyelinating lesions. Inflammatory responses involve cAMP and pharmacological manipulation of cAMP levels by using specific phosphodiesterase (PDE) inhibitors provokes an anti-inflammatory response. Experimental autoimmune encephalomyelitis (EAE) is an animal model of the chronic inflammatory, neurodegenerative demyelinating disease multiple sclerosis (MS). Previously we demonstrated that in the brain and spinal cord of EAE rats there was a dramatic increase in the mRNA expression levels of the PDE4B isoenzyme, solely due to the splicing mRNA variant PDE4B2. This expression was found in the infiltrating T-cells and macrophage/microglia in microvessels and brain parenchyma.
We present our results on the alterations in the expression of the cAMP-specific PDE4 and of several inflammatory cytokine mRNAs in the brain and spinal cord of C57BL6 EAE mice model by neuroanatomical techniques (double in situ hybridization histochemistry and immunohistochemistry), and quantitative real time RT-PCR. We observed a PDE4B2 mRNA upregulation after the onset of the EAE model, being significant 30 days post-immunization. No changes on PDE4B3 mRNA splice variant could be detected. Double in situ hybridization and immunohistochemistry studies showed that cellular infiltrates in brain and spinal cord tissues of EAE mice are overexpressing PDE4B isoform. We found that PDE4B2 mRNA splice variant was overexpressed in T cells and macrophages/microglia in the inflammatory foci.
These findings support the important and distinctive role of different PDE4 isoforms during the neuroinflammatory response produced by EAE model and its importance as a therapeutic target for the treatment of some neuroinflammatory diseases using subtype-selective PDE4B inhibitors.
Rapid injury and steady recovery of the neuromuscular nerve terminal integrity after application of anti-ganglioside antibody in a murine model of Guillain-Barré syndrome Rupp Angie ⁎ , Willison Hugh J.
Circulating anti-ganglioside antibodies are considered to be important mediators of the disease in the human peripheral nerve disorder Guillain-Barré syndrome (GBS). Recent research conducted in a mouse model of GBS has revealed the neuromuscular junction (NMJ) as a potential site of anti-ganglioside antibody binding and complementmediated injury. The ganglioside composition of the neural and glial components of the NMJ determines which of these structures are injured. One explanation for both a rapid onset and recovery occasionally observed in humans suffering from motor axonal forms of GBS (AMAN) is that the distal motor nerve is capable of degenerating and functionally regenerating over a short period of time.
The ventral neck muscles of mice expressing cytosolic fluorescent proteins in their axons (CFP) and Schwann cells (GFP) were subjected to a single topical application of anti-GD1a ganglioside antibody followed by a source of complement. Changes to the NMJs of these muscles were documented both by in an ex vivo qualitative and quantitative imaging.
Within 30 min of the application of the source of complement, a rapid loss of CFP overlying the NMJ could be seen; the GFP signal remained stable and in control animals no changes to either GFP or CFP were observed. At 24 h after induction of the injury,~7% of the superficial sternohyoid NMJs exhibited CFP; the CFP-loss extended proximally until the axons formed little bundles. Recovery of CFP was monitored, with N99% of the NMJs exhibiting CFP by day 5. Auxiliary investigations revealed that the loss of CFP at the NMJ correlated with a loss of nerve terminal neurofilament. The return of CFP at the NMJ was accompanied by a return of neurofilament. The perisynaptic Schwann cells were not affected by this specific anti-ganglioside antibody mediated injury; they did, however, in some cases extend processes beyond the normal NMJ boundaries, which occasionally were accompanied by axonal sprouts.
The results describe above indicate that following an anti-GD1a ganglioside antibody-mediated attack a very rapid loss of nerve terminal integrity can be observed at the murine NMJ. This is followed by a steady recovery of the nerve terminal integrity over the next few days, which, on occasion, is accompanied by reactive changes in the perisynaptic Schwann cells. This data supports the notion that an equivalent mechanism may account for the rapid recovery seen in some clinical cases of AMAN.
Silencing of murine ADAM17 expression in vitro by a lentiviral vector-mediated RNA interference approach Sinagra Melanie ⁎ ,1 , Bunning Rowena 1 , Bolton Chris 2 , Azzouz Mimoun 3 , Woodroofe Nicola 1 1 Sheffield Hallam University, Sheffield, United Kingdom; 2 Queen Mary, University of London, London, United Kingdom; 3 University of Sheffield, Sheffield, United Kingdom ADAM17, also known as tumour necrosis factor-alpha (TNF-a) converting enzyme (TACE), is a membrane-bound enzyme belonging to the ADAM (a disintegrin and metalloproteinase) family. It was originally identified through its ability to cleave into a soluble form, TNF-a, a major immunomodulatory cytokine involved in central nervous system (CNS) inflammatory diseases, including multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). ADAM17 is also responsible for the shedding of several membrane-bound proteins involved in the pathogenesis of MS, including the TNF-a receptors, TNFR1 and TNFR2, cell adhesion molecules and fractalkine. This suggests that ADAM17 may play a key role in these inflammatory demyelinating disorders. This hypothesis is reinforced by the fact that ADAM17 is expressed in the CNS in MS, particularly in active plaques and in white matter (WM) blood vessel endothelial cells, astrocytes and activated microglia. Our group has also demonstrated that ADAM17 expression is up-regulated in inflammatory macrophages, endothelial and astrocytic cells in spinal cord WM of rats with EAE.
In this context, we investigated the putative role of ADAM17 in MS pathogenesis using a lentiviral (LV) vector mediated RNA interference (RNAi) approach.
We designed several short hairpin RNA sequences targeting various sites on mouse ADAM17 mRNA. We subsequently constructed the corresponding LV-ADAM17 lentiviruses containing those interfering RNAs in order to knockdown ADAM17 expression in vitro. We transduced KT5 and bEnd3 cells, mouse brain astrocytic and endothelial cell lines, respectively. mRNA expression levels of ADAM17 were measured using quantitative real-time PCR to assess the efficiency of the LV vector-mediated silencing. Our data revealed that ADAM17 mRNA expression was reduced by around 60% with LV-ADAM17seq1 compared to a control LV vector in KT5 cells transduced for 5 days (T+ 5). In bEnd3 cells, the same LV vector induced a down-regulation of ADAM17 expression of almost 50% at T + 5 and 60% at T + 11.
LV vector-mediated RNAi is efficient in reducing ADAM17 expression in murine brain endothelial cells and astrocytes in vitro. This silencing approach will now be applied to determine its effect in the Biozzi mouse model of EAE, using the LV-ADAM17 seq1 administered intravenously, this will further elucidate the role of ADAM17 in CNS inflammation. Recent studies of cerebral cortical pathology in secondary progressive multiple sclerosis (MS) have shown the presence of extensive sub-pial grey matter lesions in a significant proportion of cases. The association of sub-pial lesions with ectopic B-cell follicle-like structures in the cerebral meninges suggests that the diffusion of proinflammatory cytokines from the meninges into the brain parenchyma could be responsible for this pathology. To test this hypothesis we have established an animal model mimicking the cortical pathology observed in MS.
Female Dark Agouti rats were immunized with 5 mg recombinant myelin oligodendrocyte glycoprotein (MOG) in incomplete Freunds adjuvant (IFA). Twenty days post immunization, animals received an injection of tumour necrosis factor (TNF) and interferon-gamma (IFNG) into the sub-arachnoid space at the sagittal sulcus.
Immunohistochemistry revealed areas of subpial demyelination extending from the sulcus over the dorsal surface of the cortex, encompassing layers I-III, associated with extensive activation of microglia/macrophages. Lesions were first evident at 3 days post injection, were maximal after 7 days and had resolved by remyelina-tion at 14 days. The extent of demyelination correlated with the number of ED1+/Iba1+ microglia/macrophages in the cortical parenchyma. Cytotoxic CD8+ T cells were observed in the meninges, corpus callosum and scattered throughout the grey matter, whereas CD4+ T cells and CD79a + B cells were restricted to the meninges. This pattern of lymphocyte infiltration is similar to that seen in MS post-mortem tissue, indicating that the model has potential as a model of MS cortical pathology. Control animals (IFA immunised injected with cytokines) had increased numbers of meningeal macrophages and lymphocytes compared to naïve animals, but no demyelination despite microglial activation. Animals immunised with MOG and injected with phosphate buffered saline showed no increase in meningeal macrophages or lymphocytes and no demyelination. Thus the subpial demyelination was dependent on a pre-existing immune response against a myelin protein.
These findings support our hypothesis of a role for meningeal inflammation in the cortical pathology of MS are described for the first time an animal model that can be used to study the molecular mechanisms involved.
The fractalkine/CX3CR1 pathway is involved in the development of symptoms and allodynia in EAE through distinct mechanisms, and has both peripheral and central roles Fractalkine (FKN), a member of the fourth class of chemokines, plays important roles in both inflammatory responses and pain. Its sole receptor, CX3CR1 is constitutively expressed in the CNS (microglia) and lymphocytes including monocytes/macrophages, dendritic cells, NK cells and cytotoxic/effector T cells. We have reported that anti-FKN mAb showed an inhibitory effect on experimental autoimmune encephalomyelitis (EAE) models. Recently, we also demonstrated that anti-FKN mAb inhibited allodynia in EAE. In this study, we examined the mechanism of these inhibitory effects of anti-FKN mAb on both EAE symptoms and allodynia. Method: In the MOG (myelin oligodendrocyte glycoprotein)-induced EAE model, anti-FKN mAb (0.5 mg/mice) was administered every other day from 6 days after sensitization with MOG peptide. Allodynia was evaluated in a rat MBP-EAE model. Anti-CX3CR1 Ab was administered at 0.08 mg/site intrathecally at 23 days after sensitization of rats with myelin basic protein (MBP). Paw withdrawal threshold was evaluated by the von-Frey test. Result: To clarify the roles of CX3CR1 expressed in the CNS and peripheral lymphocytes, we established bone marrow transplanted (BMT) mice. WT, KO and BMT mice (WT to KO) showed EAE symptoms but BMT mice (KO to WT) did not. The administration of anti-FKN mAb ameliorated the EAE symptoms of WT and BMT mice (WT to KO) but did not affect those of KO mice. The effect of anti-FKN mAb on migration of lymphocytes in these WT, KO and BMT mice followed the same pattern as the EAE results. Regarding chronic pain in the EAE model, we established an allodynia model for the rat MBP-EAE model. In this model, the intrathecal administration of anti-CX3CR1 Ab clearly inhibited allodynia. To analyze the mechanism of this allodynia, immunohistochemical analysis of the spinal cord was performed. An elevation of mean intensity of OX-42 (CD11b) was observed. When anti-CX3CR1 Ab was administered, it inhibited this elevation at 6 h after injection of Ab. To study the effect of FKN on microglia, horizontal slices of L4 and L5 spinal cords from WT, KO and BMT mice were stimulated with FKN (50, 300 ng/ml). As the result, FKN induced the production of MCP-1 from the slices of WT and BMT mice (KO to WT). FKN/CX3CR1 pathway plays a key role in both EAE and EAE induced allodynia. Our results suggest that FKN/CX3CR1 is involved in EAE through migration of immune cells into the spinal cord, and in EAE-induced allodynia through microglia activation.
The profile of cytokine expression involved with virus persistence and virus-induced demyelination Ohara Yoshiro ⁎ , Himeda Toshiki, Okuwa Takako, Muraki Yasushi Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa, Japan
Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus and persists in the spinal cords of mice, followed by inflammatory demyelinating disease. This late chronic demyelinating disease serves as an experimental model for the human demyelinating disease, multiple sclerosis (MS). Virus persistence is a key determinant for the TMEV-induced demyelination. Macrophages are thought to serve as the site of TMEV persistence during the chronic demyelinating phase. However, the key factors of macrophage cells related to virus persistence and demyelination remain poorly understood. It is thought that the establishment of the macrophage cells persistently infected with DA strain of TMEV (PDAJ774) is useful to investigate the key factors of macrophages involved with virus persistence and virusinduced demyelination. In this study, we established PDAJ774 cells, and then analyzed the cytokine/chemokine expression pattern in PDAJ774 cells.
After DA-infection, J774 cells which showed a continuous growth were sub-cultured and handled as PDAJ774 cells. It was suggested that the type of infection of PDAJ774 cells is "chronic focal infection" which is similar to the type of DA-infection in vivo by analysis of viral antigen-positive rate, cell viability and production of infectious virus. Subsequently, we analyzed the cytokine/chemokine expression pattern in PDAJ774 cells by using RT-PCR and anti-cytokine antibody array. In this study, the upregulation of IL-10 and down-regulation of IFN-alpha4, IFN-beta and IFN-gamma was shown in PDAJ774 cells. Furthermore, the upregulation of B-lymphocyte chemoattractant (BLC/CXCL13) and granulocyte colony-stimulating factor (G-CSF) in PDAJ774 cells was first demonstrated.
The data suggest that the up-regulation of IL-10 and the downregulation of IFN-alpha4, INF-beta and IFN-gamma may contribute to the TMEV persistence, and the up-regulation of BLC and G-CSF may contribute to the acceleration of TMEV-induced demyelination. Therefore, the inhibition of BLC and/or G-CSF may be a promising novel therapeutic approach for not only TMEV-induced demyelinating disease, but also MS.
The role of alpha-1,2-mannosidase in regulation of central nervous system inflammation Hentschel Nicole 1 , Millward Jason ⁎ ,1 , Waiczies Sonia 2 , Schlickeiser Stephan 1 , Sawitzki Birgit 1 , Infante-Duarte Carmen 1 1 Charité-Universitätsmedizin Berlin, Berlin, Germany; 2 University of Malta, Msida, Malta Cell surface glycans play an important role in regulating immune responses, and are implicated in inflammatory diseases. Glycosidase alpha-1,2-mannosidase removes mannose residues in the process of protein n-glycosylation, initiating a shift from high-mannose glycans to complex glycans, a shift that seems to be crucial for the discrimination of self from non-self antigens.
Previously, we showed that alpha-1,2-mannosidase expression was reduced in patients with multiple sclerosis (MS) that responded well to immunomodulatory therapy. On the other hand, in transplantation, blockade of alpha-1,2-mannosidase inhibits allograft rejection, indicating that glycosidase inhibition may have anti-inflammatory effects also in neuroinflammation.
To prove this, we applied Kifunensine (KIF), a potent inhibitor of alpha-1,2-mannosidase, to mice suffering from experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We show that, in vivo, administration of KIF had a biological effect on dendritic cells (DC) and T cells as indicated by significant downregulation of complex glycans and an upregulation of high-mannose glycans. Further, we show that, unexpectedly, administration of KIF intraperitoneally during the induction phase of EAE led to a significant EAE exacerbation. However, when KIF was administered at the peak of disease no effect was observed. Thus, we hypothesise that alpha-1,2mannosidase inhibition may affect DC by enhancing their priming capacity or/and perhaps diminishing their potential to induce immune regulation. To explore this, we examined markers associated with antigen presentation and chemotaxis on DC treated in vitro with the alpha-1,2-mannosidase inhibitor. Preliminary data show that KIF treatment did not significantly affect expression of these molecules in DC generated in vitro. Work in progress is extending this analysis to different DC subsets analysed after in vivo KIF application.
These results underscore the need to consider alpha-1,2-mannosidase, and other regulators of glycosylation, as important mediators which can influence inflammatory disease in the CNS. In multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE) helper T cells (TH) primed in periphery orchestrate the pathogenic process in the central nervous system (CNS). Recently, innate immunity and foremost toll-like receptors (TLRs) were found crucial for shaping the TH response. However, data on the role of TLRs in MS are limited. Moreover, the results demonstrating the relevance of several TLRs (2, 3, 4 and 9) in EAE are controversial, whereas MyD88, adapter protein in TLR signaling, was indispensable for EAE development. The aim of this study was to analyze mRNA expression of TLR2, TLR3, TLR4, TLR9 and MyD88 in Dark Agouti (DA) and Albino Oxford (AO) rats, susceptible and resistant to EAE, respectively.
The rats were immunized with spinal cord (SC) homogenate in complete Freund's adjuvant. Draining lymph nodes (DLNs) and caudal portion of SC were taken in total for real-time-PCR analysis before onset, with first neurological signs, at the peak of disease and in recovery phase. EAE-susceptible DA strain had significantly higher TLR2, 4, 9 and MyD88 mRNA expression in SC at the peak of disease, while EAE-resistant AO strain exhibited increased TLR3 expression in SC during the induction phase of the disease. Nevertheless, DLN mRNA assessment did not reveal conclusive results.
Our data suggest that TLR2, 4, 9 and MyD88 expression in CNS correlates with the appearance of clinical signs of EAE, whereas interferon-β-inducing TLR3 signaling contributes to the resistance to disease. One-hundred-eighty-seven MS patients and 197 age-and sexmatched HLA-DRB1*15-positive healthy individuals from the same geographical region as well as 455 MS patients and 361 HLA-DRB1*15-negative healthy subjects were included in the study. Results showed that the rs731236 TT genotype and the (T) allele were significantly more present in HLA-DRB1*15-positive HC compared to patients (genotype: py = 0.004 OR:0.53 IC (95%) 0.33-0.83). Confocal microscopy showed a higher intracellular VDR expression in cells from rs731236 TT compared to CC genotype carriers. Statistical analysis of the possible phenotypic combinations of rs731236 TT and HLA-DRB1*15 markers in the entire cohort of MS patients and HC and a multivariate analysis, confirmed the HLA-DRB1*15/VDR rs731236 interaction.
Rs731236 is in linkage with the 3′UTR region, which regulates VDR expression via mRNA stability. The VDR rs731236 TT genotype in HLA-DRB1*15-positive individuals results in protection against MS, possibly because it is associated with an increased expression of VDR and/or HLA-DRB1*15. These findings offer a functional explanation of the interaction between major genetic (HLA-DRB*15) and environmental (vitamin D) factors involved in the pathogenesis of MS. Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system (CNS) characterized by recurrent severe attacks of demyelinating myelitis and optic neuritis. Recently, a biomarker for NMO, NMO-IgG, has been identified which diagnostically distinguishes NMO from typical Multiple Sclerosis (MS). It binds to aquaporin-4, a water channel expressed on astrocyte foot processes in the CNS and may participate in the pathogenesis of NMO.
MicroRNAs (miRNA) are short non-coding single-stranded RNA molecules that modulate gene expression at the post-transcriptional level by degradation or translational repression of target mRNAs. They play a role in physiologic development, cell differentiation and proliferation but also in pathologic conditions like autoimmune disorders including lupus and MS. We therefore want to know whether miRNAs and their putative mRNA targets are involved in the pathogenesis of NMO.
We have performed miRNA and mRNA analyses in post-mortem spinal cord (SC) tissue of 3 NMO patients and 3 age-matched controls. Using Luxol Fast Blue staining one demyelinating SC lesion from each patient and normal SC sections form controls were selected for RNA isolation. After quality control of miRNA and mRNA differences in miRNA and mRNA expressions were determined in duplicates using Agilent microarray systems. Unpaired t-tests with Benjamini-Hochberg adjustment (p b 0.05) were conducted to detect N=2-fold changes in miRNA and mRNA expression comparing demyelinating lesions with control material. Differentially expressed miRNAs were selected to assess their differentially expressed mRNA targets (FC N=2, p b 0.05).
The expression level of 796 miRNAs and 41,078 mRNAs were determined. As compared to control material, 28 miRNAs and 434 mRNAs were differentially expressed in NMO lesions. Their differentially and inversely expressed putative mRNA targets were assessed by joint analysis. These targets are known to be involved in myelopoesis, T-cell and dendritic cell differentiation, cancerogenesis, adipogenesis, smooth muscle homeostasis, neural differentiation, brain injury and neurodegeneration.
The alterations in miRNA and mRNA target expression in demyelinating lesions of NMO patients compared to controls suggest that miRNAs are involved in the pathogenesis of NMO. Further studies are required to investigate the targets and their functional involvement to further gain insight into the disease mechanism and reveal potential therapeutic targets.
Alterations in phosphorylation status of brain proteins in an animal model of multiple sclerosis Vanheel Annelies ⁎ , Daniels Ruth, Stinissen Piet, Noben Jean-Paul, Hellings Niels
Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system in which axons and myelin are damaged. The underlying molecular processes remain poorly understood, but are crucial in the search for new therapeutic options. Protein phosphorylation, important for protein function, may be involved in the pathology of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The identification of a panel of differentially expressed phosphorylated proteins along the disease course could provide valuable insights into global disease processes of EAE and MS.
Protein extracts from 'blood-free' brain stems of control and EAE Lewis rats were separated by 2D-gel electrophoresis. To detect phosphorylated proteins, gels were stained with a fluorescent dye, Pro-Q diamond phosphoprotein gel stain (Invitrogen). A fluorescent total protein staining was used as quality control and thus normalization of the phosphoprotein signals. Moreover total protein staining enables matching of different gels. Quantitative data were obtained by comparison to a previous two-dimensional difference ingel electrophoresis (2D-DIGE) study at different disease stages (control, onset, top and recovery, three biological replicates each). All identified differentially expressed (ANOVA = 0.05) proteins from this 2D-DIGE study (95) were checked for possible phosphorylation.
Both sensitivity and specificity of the Pro-Q diamond phosphoprotein gel stain were analyzed using different concentrations of the peppermintstick phosphoprotein standard (Invitrogen). Some background staining was detected, making it difficult to distinguish between differences in phosphorylation load.
The presence of phosphorylated, and differentially expressed proteins is currently being confirmed by immunohistochemistry (IHC) and 1D or 2D Western blotting (WB). These techniques allow us to determine the presence of the phosphorylated protein, but also the expression pattern if multiple time points are included. The global overview of phosphorylation during disease invites new studies to unravel the complicated molecular biological processes in the pathology of MS.
CSF proteomic pattern analysis of multiple sclerosis and its related disorders Multiple sclerosis (MS) related disorders are recognized as inflammatory autoimmune diseases of the central nervous system. We reported previously that cerebrospinal fluid (CSF) proteomic profiles using magnetic bead-based separation combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry significantly discriminate MS and neuromyelitis optica (NMO). Here we extend the study by increasing the number of samples of MS related disorders and other neurological diseases as well as trying to apply an updated statistic algorithm.
CSF samples from 107 patients with relapse-remitting MS (RRMS), primary progressive MS (PPMS), anti-aquaporin-4 (AQP4) antibody seropositive NMO (SP-NMO), and seronegative NMO (SN-NMO) were included. Control neurological diseases including amyotrophic lateral sclerosis (ALS) were also enrolled in this study. CSF samples were separated by ClinProt systemTM as reported previously with C8 magnetic beads and were analyzed by MALDI-TOF mass spectrometry (Autoflex IITM). Acquisition was in the mass range of 1000-15,000 m/z. The spectra were analyzed using ClinProTools 2.2TM. We first analyzed CSF ClinProt data sets using support vector machine (SVM). Then we cross validated with discrete wavelet transformation-support vector machine (DWT-SVM) algorithm. In addition, we applied a pattern matching method (BiotyperTM algorithm) from our data set to make a conceptual dendrogram of neurological diseases based on CSF proteomic patterns. SVM in general performed well on our CSF ClinProt data set and confirmed distinction of SP-NMO from RRMS in relapse phase as previously reported. From the dendrogram calculated by a pattern matching method, SP-NMO and SN-NMO had similar proteomic patterns both in relapse and remission. The spectral differences between RRMS and PPMS are much bigger than PPMS and ALS, raising a possibility that PPMS and RRMS have a bigger difference regarding inflammatory and neurodegenerative features than being considered.
Proteomic pattern analysis is a promising and a reliable biomarker strategy in neuroimmunological disease study representing multifactorial aspects of MS related disorders.
Gender-based systems biology in multiple sclerosis unravels distinct blood gene signatures but common epigenetic facets Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system (CNS) characterised by the presence of inflammatory cells and mediators within nervous tissue. It is a chronic disease with onset in young adulthood and gender bias in the relapsing-remitting (RR-MS) form of disease. Although MS affects the CNS, there are evidences of altered peripheral immunity in MS patients.
We investigated global gene expression in peripheral blood mononuclear cells of 23 RR-MS patients and 22 healthy individuals. In contrast to conventional studies, where the whole diseased population is compared to the healthy one, we performed genderbased analyses. This approach reduced the heterogeneity in human populations and, intriguingly, led to the definition of distinct MSrelated gene signatures in women and in men despite the same clinical course.
Gender-based functional annotation and systems biology studies showed that the differentially expressed genes shared molecular functions and participated to common biological processes. Furthermore, reconstruction of the gender-related MS interactomes demonstrated frequent sharing of interactors between the female and male gene sets. Issues regarding epigenetic control of gene expression appeared as the common basis for disease in women and men.
In conclusion, we propose gender-based systems biology as a novel tool to gain fundamental information on disease-associated functional processes. This approach highlighted distinct gene signatures but common processes in women and men with MS. Intense immunosuppression followed by autologous hematopoietic stem cell transplantation is a potential treatment option in a subset of patients suffering from multiple sclerosis (MS), a presumably autoaggressive immune disease of the central nervous system. Several gene expression analyses in peripheral blood mononuclear cells of MS patients have shown strong differences in the expression of immune genes compared to healthy controls. However, it is not known whether the immune deviation seen in MS patients develops in the peripheral immune system at later stages of immune development or whether these changes are already imprinted in hematopoietic precursor cells. This is highly relevant for the management of MS patients, because the rationale of autologous hematopoietic stem cell transplantation as therapy MS depends on it. Aims: To compare CD34+ hematopoietic stem cells from MS patients and healthy donors by gene expression analysis and epigenetic profiling.
CD34+ hematopoietic stem cells were collected by apheresis from healthy donors (n = 5, mean age 43 years, female 3), who were mobilized with G-CSF, from MS patients (n = 4, mean age 41 years, female 4), who were mobilized with G-CSF only and from MS patients (n = 4, mean age 38 years, female 2), who were mobilized with G-CSF and cyclophosphamide. CD34+ cells were isolated from the graft by immunomagnetic separation. Total RNA and genomic DNA were isolated using the Trizol reagent protocol. Global gene expression analysis was applied using the Human 4 × 44K Design Array from Agilent Technologies, which allows to analyse the expression of all known human genes and transcripts. Epigenetic analysis was done using the Affymetrix Platform (Human Promoter 1.0R Array).
We generated a list of genes differentially expressed between MS patients and healthy donors. Validation of candidates by RT-PCR is ongoing. Principal component analysis allowed clear discrimination between CD34 positive and CD34 negative cells. Significantly regulated genes were annotated to biological processes according to the Gene Ontology database. Analysing gene expression in CD34+ cells from MS patients did not reveal significant deviation in immune genes previously reported in MS.
In summary we did not find an inflammatory signature in CD34+ cells from MS patients. Thus, we provide evidence that the immune deviation seen in MS patients develops in the periphery during the evolution of the disease and is not imprinted in CD34+ hematopoietic precursor cells. The objective of this study is the inference and characterization of the protein-protein interaction (PPI) network in multiple sclerosis (MS) and in the antigen mediated immune cell response.
Using Ingenuity Pathway Analysis (http://www.ingenuity.com/), we selected candidates' genes for the different cell types involved in the antigen mediated immune response (B lymphocytes (lB), T lymphocytes (lT), dendritic cells (DC) and macrophages (Mph) and also those genes related to immune response in general (GIR). We then mined more than 11 external gene-to-disease databases to extract the list of candidate genes for MS. To identify network-and process-level similarities between MS and the different immune cell types and immune system in general, we constructed the protein-protein interaction (PPI) networks using STRING (http://string-db. org/; "Experiments" and "Databases" were selected as the interaction criteria, and 0.4 as the stringency score), and biological process enrichment scores from DAVID (http://david.abcc.ncifcrf.gov/). The topological analyses of the PPI networks were made with Cytoscape (http://www.cytoscape.org/).
The obtained results showed 220, 309, 208 and 403 genes related to the antigen mediated immune response in lB, lT, DC and Mph, respectively; 1671 genes were obtained as related to GIR, and 946 genes to MS. On the other hand, the number of genes included at each PPI network was 174, 248, 153, 301, 1369, and 704 for lB, lT, DC and Mph, GIR and MS, respectively. Functional analyses of the overlapped genes among MS and each immune cell type reveal that they are involved in biological processes such as immune system process, immune response, apoptosis, signal transduction, cell differentiation, inflammatory response, cell-cell signaling, locomotory behavior, response to stimulus, homeostasis and regulation of antibody isotype switching, among others (FDRb 0.05). Evaluating radiality as a node centrality index in the MS PPI network, and performing a functional analysis for the relevant genes obtained with a different radiality, we found that the MS genes involved in specific functional processes altered in MS are mainly located at the periphery of the network and are not hubs.
Further studies are being developed in order to obtain the modularity of the MS PPI network in the searching for critical MS genes candidates to be experimentally validated. Multiple sclerosis (MS), an immune-mediated disease of polygenic etiology, is characterized by individual clinical variability. The aim of the present investigation was to study an association of the immune response genes polymorphisms, which mainly code cytokine network components, with the early clinical features (first remission duration, FRD, and symptoms at onset), which may provide predictive value.
Unrelated 308 Russians (212 women and 96 men) with relapsingremitting MS were studied. We have performed complex association analyze of the polymorphisms in the functionally significant regions of the following candidate genes: DRB1 HLA class II, tumor necrosis factor (TNF, A-308G, rs1800629), interferon-gamma (IFNG, A874T, rs2430561), transforming growth factor-beta (TGFB1, C-509 T, rs1800469), interferon-beta (IFNB1, C153T, rs1051922), interferonalpha/beta receptor 1 (IFNAR1, C16725G, rs1012335) and CC chemokine receptor 5 (CCR5del32). The carriage of allele/genotype combinations associated with the FRD and symptoms at onset were identified using APSampler software, which is based on Bayesian MCMC method and provided validation by means of Fisher's exact test.
We revealed reliable differences in the means of Multiple Sclerosis Severity Score among patients stratified by the FRD: sFDR (short, b=12 months) vs lFDR (long, N12 months), p b 0.0001, and by symptoms at MS onset (sensitive and visual disturbances vs other initial symptoms), p b 0.0008. Positive associations of sFRD with TNF*G/G (p = 0.01) and its combinations with other alleles: (TNF*G/G; IFNB1*C), (TNF*G/G; IFNAR1*G) and (TNF*G/G; IFNG*T) (p =0.002-0.005) were found. Correspondingly, allelic combinations including alternate alleles TNF*A, IFNAR1*C, TGFB1*T were positively associated with lFRD. In the women subgroup sFRD was associated with TNF*G/G (p =0.005), IFNB1*C (p = 0.028), their combination (p = 0.00004) and in the men subgroupwith the combination (IFNG*A; IFNAR1*G), p = 0.005. Comparison of p-values for TNF and IFNB1 separately and in combination shows evidence for the cooperative effect of these genes.
We also compare allelic patterns in MS patients with different symptoms at MS onset. Initial sensitive and visual disturbances were considered as favorable prognostic factors, othersas unfavorable ones. We observed associations with unfavorable MS course of CCR5d32, TGFB1*C/C (p b 0.05) and their combinations with IFNG*T, IFNB1*C or TNF*G (p = 0.02-0.002).
Allelic variants of the immunoresponse genes are associated with MS early clinical features. This provides a possibility to use an individual genetic status of a patient for further MS prognosis.
Overlapping genes in autism and autoimmune diseases Previous studies have suggested an autoimmune role in the etiology and pathology of Autism Spectrum Disorders (ASD), although this role has not been fully characterized at the molecular level. The objective of this study is to look for a common gene expression signature between ASD and autoimmune diseases (AIDs).
To do this, we first selected a list of 150 AIDs from www.aarda.org. We then mined more than 11 external gene-to-disease databases to build ranked list of candidate genes for ASD and all AIDs. A comparative analysis between the ASD genes and the AIDs genes was then conducted, favoring those AIDs with more genes (over the mean (13.6) + 2SD (35)) in common with ASD as ASD sibling diseases for further analysis (Rheumatoid Arthritis (RA; #98), Multiple Sclerosis (MS; #91), systemic lupus erythematosus (SLE; #78), Ulcerative Colitis (UC; #62), cardiomyopathy (CAD; #61), Type I Diabetes (C1D; #59), Crohn Disease (CD; #59) and endometriosis (EM; #53). To identify network-and process-level similarities between ASD and the 8 sibling AIDs, we constructed the proteinprotein interaction (PPI) networks for each disease using STRING (http://string-db.org/), and biological process enrichment scores (FDR b 0.05) from DAVID (http://david.abcc.ncifcrf.gov/).
The PPI network analysis revealed a significant overlap between ASD and RA, MS, and SLE (64, 63 and 52 genes, respectively), indicating that these three AIDs are most closely related to autism. Seven genes involved in the regulation of antibody isotype switching were found in common across the 9 AID networks. The genes unique to ASD (#135) were significantly enriched for neurological processes as synaptic transmission, axon guidance, behavior and neurogenesis. Finally, we also obtained 797 genes as first neighbors of the overlapped (118) genes between autism and AIDs and calculated the biological enrichment of this gene set. These processes were compared to common significant biological functions between ASD and sibling neurological diseases (Wall et al., 2009) , and we obtained a list of 369 new gene candidates likely to be involved in ASD based on their functional implication in both neurological dysfunction and autoimmune response.
Our preliminary results suggest that there is a significant molecular signature of autoimmunity in autism, and that the signature is specific to a set of AIDs, rather than to autoimmunity in general. Our future work will focus on characterizing this molecular signature. Myasthenia gravis (MG), like other autoimmune diseases, has a multifactorial etiology determined by environmental and genetic factors. Advances in human genome knowledge, along with new systematic search tools, suggest the involvement in the autoimmunization process of some gene products central to T-cell tolerance induction or lymphocyte activation. Among these, LYP/PTPN22 is a non receptor-type protein tyrosine phosphatase which actively controls T-cell activation: it is a negative regulator of the Src family kinases, which mediate TCR signaling. Several different single nucleotide polymorphisms (SNP) of PTPN22 have been associated to different autoimmune diseases; most of the studies have been focused on the functional polymorphism rs2476601 (C1858T; R620W). Here we report our data on rs2476601 allele frequencies in an Italian MG population, as compared to an ethnically-matched control group.
SNP alleles were determined using the SNaPshot System (Applied Biosystems) on a flanking segment which was amplified by PCR from genomic DNA extracted from peripheral blood leukocytes. We analyzed 250 controls and 250 Italian MG subjects. The data were analyzed according to age at onset, thymus pathology and anti-AChR or anti-MuSK antibody presence. SNP distribution analysis showed an increased frequency of the major C allele in MG patients, as compared with controls; this increment was higher in early onset MG patients with anti-AChR antibodies.
Most of the previous studies, which have been focused on cellmediated or systemic autoimmune diseases, suggest a protective role of the C allele as compared to the T allele because of the gain-of-function determined by the amino acid substitution. In the present report we confirm the different distributions of C and T alleles in Italians as compared to northern Europeans. In contrast with studies on northern European MG patients, which have an increased frequency of the T allele, we found an increase of the C allele. Our findings can be explained either by a different role of PTPN22 in the autoimmunization process in ethnically different populations, or by the different pathogenic mechanisms underlying MG (a pure antibody-mediated disease) as compared with the other autoimmune diseases. These findings, all together, give new elements to debate the role of PTPN22 SNP in MG pathogenesis.
Validation of the CD6, TNFRSF1A and IRF5 genes in multiple sclerosis in Spain Multiple sclerosis (MS), a disorder of the central nervous system, is thought to be a chronic inflammatory disease with autoimmune basis. The pathogenesis of multiple sclerosis is as yet poorly understood. Amidst the plethora of data that are being generated by genome wide association studies, there is a need for replication studies to differentiate between false positive findings and genuine markers underlying the pathology of MS. Our study aimed at replicating candidate genes recently reported to be associated with MS, in our collection of samples from 4 different cohorts across Spain (Andalucía, Bilbao, Madrid and San Sebastián) totaling N2500 MS patients and N2900 healthy controls.
We found significant association of rs17824933 in CD6 (P CMH = 0.004; OR = 1.14; 95% CI 1.04-1.24) and of rs1860545 in TNFRSF1A (P CMH = 0.001; OR = 1.15; 95% CI 1.06-1.25) with MS, while the low-frequency coding non-synonymous SNP rs4149584 in TNFRSF1A displayed a trend for association (P CMH = 0.062; OR = 1.27; 95% CI 0.99-1.63). Furthermore, in our analysis of the data from 2 IRF5 SNPs (rs3807306 and rs4728142), we found association in a combined cohort of our study collections and earlier published data with P values of P CMH = 0.002 [OR = 1.14 (1.05-1.23)] and P CMH = 0.001 [OR = 1.14 (1.05-1.24)] respectively. We also detected trends for IRF5 to be associated with response to IFNbeta therapy and HHV6 infection.
In conclusion, CD6, TNFRSF1A and IRF5 have emerged from our study as risk factors for multiple sclerosis in Spain. Spinal cord tissue of 31 autopsied MS patients was compared to amyotrophic lateral sclerosis (ALS) and control subjects. Actively demyelinating MS lesions were identified according to the presence of phagocytes containing MBP positive myelin degradation products. Inactive demyelinated lesions were classified according to the density of KiM-1P + cells of macrophage and microglia phenotype. Meningeal and parenchymal CD3+ and CD8+ T cell distributions were determined according to the lesion stage. Axonal damage and neuroaxonal changes were studied by immunoreactivity (IR) for APP and variably phosphorylated neurofilaments (SMI312, SMI31, and SMI32).
Ventral horn neurons in the chronically demyelinated spinal cord showed altered neurofilament phosphorylation, namely a progressive loss of SMI32 immunoreactivity (IR) upon lesion and disease progression. Axonal loss in spinal white matter (WM) lesions was another progressive feature in the inactive lesion stages, correlating with both disease duration and severity. A selective reduction of axonal phosphorylated neurofilaments (NF-H and SMI31) took place in both acute and chronic inactive WM lesions. In ALS, the loss of neuronal SMI32 IR was even more severe, whereas the relative axonal reduction resembled that observed in MS.
Progressive neuronal and axonal neurofilament alterations in the context of chronic inflammatory demyelination reflect changes in neuroaxonal metabolism, contributing to chronic neuroaxonal dysfunction and axonal loss as likely substrates of clinical progression. The pathological hallmark of multiple sclerosis is the formation of chronically demyelinated lesions in the CNS. Although remyelination may be extensive in some patients resulting in shadow plaques, remyelination fails in others. Why some lesions fail to remyelinate is unclear, as the CNS contains an endogenous repair mechanism that normally acts to ensure rapid and essentially complete remyelination. Our goal is to identify potential mechanisms inhibiting remyelination of those MS lesions that failed to remyelinate.
We used frozen autoptic tissue, identified shadow plaques and dissected them along with active and chronic inactive MS lesions. Then we performed an expression profiling using quantitative PCR with low density arrays. Thereby we detected a complex alteration of growth and differentiation factors for oligodendrocyte lineage cells such as FGFs, PDGFs, semaphorins, IGFs, chemokines and IL6 family. Most striking was an up-regulation of FGF9 in a subset of chronically demyelinated lesions, but in none of the remyelinated shadow plaques. The potential functional implication of this observation was investigated by treating myelinating rat CNS cultures with exogenous FGF9. In this experimental setting, FGF9 inhibited the ability of mature oligodendrocytes to myelinate and ensheath axons; an effect associated with a corresponding decrease in transcripts encoding myelin proteins and myelin related transcription factors.
The expression pattern of factors regulating oligodendrocytes is altered in a complex way in different types of MS lesions. The induction of FGF9 in some chronic demyelinated MS lesions might be one of the inhibitory mechanisms accounting for the failure of remyelination.
Immunopathogenesis of brain lesions of Nasu-Hakola disease Satoh Jun-ichi ⁎ ,1 , Tabunoki Hiroko 1 , Kim Seung U. 2 Meiji Pharmaceutical University, Tokyo, Japan; 2 University of British Columbia, Vancouver, Canada; 3 International Medical Center, Chiba, Japan; 4 Hyogo-Cyuo National Hospital, Hyogo, Japan; 5 National Center of Neurology and Psychiatry, Tokyo, Japan Nasu-Hakola disease (NHD), also designated polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), is a rare autosomal recessive disorder distributed worldwide, characterized by a combination of progressive presenile dementia and formation of multiple bone cysts. NHD is caused by genetic mutations of DAP12 or TREM2 expressed on myeloid and lymphoid cells, including microglia and osteoclasts. A defect in the TREM2/DAP12 signaling causes impaired osteoclast differentiation. Although the number of microglia is reduced in the brain of DAP12-deficient mice (Nat Immunol 10: 734, 2009), it remains unknown whether microglial differentiation and activation are impaired in NHD brain lesions. We attempted to characterize the immunopathogenesis of NHD brain lesions.
We studied brain lesions of a DAP12 141delG patient by immunohistochemistry. We also reanalyzed a GEO microarray dataset of IL-4/ GM-CSF-induced dendritic cells (DC) of NHD patients (GSE3624). Finally, we determined DAP12 and TREM2 expression in PMA-exposed THP-1 monocytic leukemia and HMO6 microglial cells by real-time RT-PCR. We identified massive infiltration of CD68 and Iba1-positive microglia/ macrophages in the cerebral cortex and white matter lesions of NHD. The substantial number of microglia/macrophages also expressed HLA-DR, while a very small population of macrophages but not microglia expressed TREM2. We identified upregulation of AIF1 (Iba1) and SIGLEC1 in DC, shared by DAP12 and TREM2 defective patients. Following exposure to PMA, THP-1 but not HMO6 upregulated substantially expression of TREM2 and DAP12.
Activated microglia/macrophages might induce chronic inflammation-mediated neuronal injury in NHD brain lesions, supporting the view that the TREM2/DAP12 signaling plays an inhibitory role in microglial activation. Cortisol is the end product of the hypothalamus-pituitary-adrenal (HPA) axis and is routinely used in its synthetic form, prednisone, to treat multiple sclerosis (MS) relapses. Post-mortem and in vivo studies show that the HPA axis is chronically activated in MS. Interestingly, in a post-mortem study of 16 MS brain donors, we recently found a highly significant inverse relationship between responsivity of the HPA axis and severity of MS, revealing a group of donors with very low HPA-axis activity and severe MS (Huitinga et al., Ann. Neurol. 2004) . A follow-up study of 30 female MS brain donors was performed to further determine what pathological mechanisms may be responsible for this observation.
The study clearly confirmed that severe MS is associated with low HPA-axis activityas indicated by low cortisol levels in donors with severe MS. Analysis of neuropathological reports of all MS brain donors revealed that more demyelination and less remyelination was present in low cortisol donors with severe MS. The opposite was found for high cortisol donors with relatively mild MS. To identify molecular mechanisms underlying these findings, low cortisol donors with severe MS were compared to those with high cortisol and relatively mild MS for gene expression in normal appearing white matter (NAWM). Highly significant cortisol-associated expression was found between severity of MS and genes involved in inflammation, immunosuppression, myelination and blood-brain barrier functioning.
These data indicate that high levels of endogenous cortisol modulate a variety of molecular mechanisms in NAWM of MS patients, thereby rendering it less vulnerable to new MS lesions.
Tissue was obtained from the Netherlands Brain Bank (www. brainbank.nl) and the study was financed by the Dutch foundation MS research.
Species differences in origin of reactive microglia Microglia serve homeostatic functions in normal CNS and they sense threats to and modulate neuronal function in the injured and diseased CNS. At present, our understanding of microglial biology and involvement in disease is influenced by the observation that exogenous immigrating bone marrow (BM)-derived cells may contribute to reactive microgliosis in the mouse. We set out to investigate possible species-differences in microglial recruitment and transformation of BM-derived cells into microglia in the injured CNS.
We analyzed microglial origin in the brain of irradiation BM chimeric mice and rats subjected to transient global cerebral ischemia, known to elicit a predictable, strong microglial reaction. Both species displayed the characteristic microglial hyperplasia and rod cell transformation in the regio superior of the hippocampus 6-7 days after surgery. In the mouse a subpopulation of lesion-reactive microglia originated from transformed BM-derived cells, in contrast to BM chimeric rats, where no recruitment and microglial transformation of BM-derived cells was observed.
The results suggest that reactive microglia in the rat originate from resident microglia, while they have a mixed BM-derived and resident origin in the mouse. Stress has been related to cognitive deficit. The hippocampus, a limbic area involved in learning and memory, is particularly sensitive to the effects of chronic stress. Cytokines have been shown to affect some behaviour, including memory. Moreover, IL-2, IFNgamma and IL-6 have been implicated in psychiatric disorders. Glatiramer acetate (Copaxone®) is a synthetic amino acid polymer that can weakly cross-react with CNS-resident autoantigens and can safely simulate the protective and reparative effects of autoreactive T cells. The aim of the present work was to study Copaxone effects in the behaviour and in the TH1/TH2 balance induced by chronic stress in BALB/c mice.
We found that BALB/c mice exposed to chronic stress had a poor learning performance with respect to control mice in both, alternation behaviour in Y-maze task and habituation in open field. The lymphoid production of cytokines analysed by ELISA showed a decrease of IFN-gamma and not changes in IL-2 (TH1-cytokines) and an increase of IL-6, IL-4 and IL-10 (TH2-cytokines) in stressed BALB/c mice. These effects induced by chronic stress were reverted by administration of copaxone (100 μg per injection s.c. to four times during three weeks).
These results indicate that copaxone is able to reverse both the memory impairment and the TH1/TH2 cytokine balance. These results suggest that TH1 response could constitute a protective mechanism preventing behaviour impairment.
Supported by UBACyT-MO24 and PIP 00281 from CONICET. Copaxone was a generous gift from laboratory Teva-Tuteur from Argentina.
Effect of prenatal stress on immune response after acute and chronic stress exposure Pascuan Cecilia Gabriela, Wald Miriam Ruth, Palumbo María Laura ⁎ , Genaro Ana María
Prenatal stress (PS) has been associated with changes in immune response but the mechanism involved has not been fully elucidated. The aim of this work was investigate alterations in neuroimmune interaction in adults animals subjected to PS.
For this purpose, pregnant mice were individually restrained 2 h a day, since gestational day 14, until delivery. Stressed offspring mice were tested at 2-months of age together with control matched mice. A group of animals were submitted to acute or chronic stress. Results shown that PS did not induce significant changes either in proliferative response in vitro or in antibody levels after immunization in vivo, but lead to a Th2/Th1 imbalance. On the other hand were not observed changes in corticosterone and catecholamine levels. However, these animals had a lower immune response with respect to control mice when they were submitted to acute and chronic stress. Moreover, PS animals respond to acute stress with lower levels of corticosterone. But, not significant changes were observed under chronic stress. On the other hand, PS animals respond to both acute and chronic stress with similar changes of catecholamine levels than control animals. In addition an increased sensitivity of inhibitory effect of corticosterone was found in lymphocytes from PS animals.
These results indicate that PS induces a disruption in hyphothalamic-pituitary-adrenal axis (HPA)-immune interaction.
Supported by UBACyT-MO24 and PIP 00281 from CONICET. The University of Queensland Centre for Clinical Research, Brisbane, Australia; 2 Queensland Brain Institute, The University of Queensland, Brisbane, Australia; 3 School of Medicine, The University of Queensland, Brisbane, Australia Schizophrenia affects~1% of the global population. It is a severe debilitating psychiatric disorder resulting from disrupted neurotransmission. Evidence from several research groups suggests that there may be immune abnormalities present in some people with schizophrenia (PwS), including alterations in T cell distribution and circulating cytokine levels, and the presence of autoantibodies directed against the brain. The aim of this study was to investigate whether autoantibodies directed against the brain, particularly against molecules involved in neurotransmission, are elevated in PwS, and to identify the antigenic targets.
Serum samples from PwS and age-and sex-matched healthy controls were screened by immunohistochemistry for reactivity against tissue from 2 regions of the brain that are commonly affected in schizophrenia. Approximately 20% of samples from PwS showed significantly elevated levels of reactivity against brain tissue when compared to healthy controls. To identify potential autoantigenic targets, human brain tissue was separated by 2D gel electrophoresis, blotted, and probed with the sera. Spots that were recognized by the positive sera from PwS, but not by control sera, were excised and the proteins identified by matrix assisted laser desorption/ionizationtime of flight (MALDI-TOF) mass spectrometry. One of the proteins frequently identified was neurofilament medium (NFM), a dopamine receptor interacting protein (DRIP). DRIPs are pivotally involved in regulating dopamine receptor signal transduction. Subsequently, sera from 110 PwS and 47 healthy controls were tested in ELISA against NFM. In addition, sera from 128 PwS and 106 healthy controls were tested for reactivity against an extracellular portion of the muscarinic acetylcholine receptor, which has previously been suggested to be a target of autoantibodies in schizophrenia. Over one quarter of PwS had significantly elevated levels of autoantibodies to one or both of these proteins, compared to b1% of unaffected individuals. A subgroup (15%) of PwS showed extremely high reactivity to NFM. We are currently investigating whether there are correlations between this strong NFM reactivity and specific clinical features.
Our results provide support for the hypothesis that autoantibodies against neurotransmitter receptors or molecules involved in regulation of neurotransmission may be of pathogenic relevance in a subpopulation of people with schizophrenia.
Gender differences in the correlation of behavior, redox and immunological parameters in very old C57b/129 mice Aging is a complex process that affects many regulatory systems including the immune system. Age-related neuroimmunological network alterations can lead to the loss of homeostasis and may contribute to the progress of aging. Behavioral phenotypes and gender-dependent differences are also known to contribute to the status of the neuroimmunological network. The aim of the present work was to analyze the predictive validity of different behavioral tests correlating with parameters of oxidative stress and immune function in old (22 months) C57b/129 male and female mice.
Behavioral screening assessed several aspects: 1) sensorimotor abilities and locomotion, 2) neophobia, emotional responses and other anxiety-like behaviors, 3) exploratory behavior and noveltyseeking, 4) behavioral despair. Thereafter, several parameters of redox state (extracellular anion superoxide levels and activity of the antioxidant enzyme catalase) and immunological function (natural killer activity, phagocytosis, basal and concavaline A and LPS-induced lymphoproliferative response) were studied in peritoneal leukocytes. The results show gender-dependent effects. The redox state of both males and females correlates with most of the behavioral aspects (1, 2, 3, and 4) studied in the different tests although the correlations are more evident in males than in females. By contrast, the immunological function parameters show a better correspondence with some or other behavioral variables according to the function and gender studied. In general, females show higher correlations than males. Regarding the immunological functions, NK activity and phagocytosis seem to be more related to anxiety-like behaviors, while lymphoproliferation correlate better with the levels of exploratory and noveltyseeking activities exhibited in the different tests.
In conclusion, the results reinforce the relevance of taking gender into consideration when assessing redox state and neuroimmunological functions as well as the need to consider other behavioral traits in addition to those typically related to anxiety.
Financial support: BFU2005-06777, BFU2008-04336; SAF2006-13642, Fundació LaMarató Tv3-062930, UCM Research Group (910379ENEROINN); RETICEF (RD06/0013/0003).
Neonatal maternal deprivation induces long-term increases in cytokine plasma levels that are modulated by a high fat diet Viveros Maria-Paz ⁎
On the basis of diverse behavioural, neurochemical, immunological and endocrine effects, we have proposed that a procedure of maternal deprivation (MD) in neonatal rats (24 h at PND 9) may be a useful animal model for the study of developmental neuroimmunoendocrine interactions. In the present study we evaluated the effects of MD and/or a high fat diet (HFD) [D12450B (10% fat) as control and D12451 (45% fat) as HFD (Research Diets, Brogaarden)], from weaning (PND 22) until adulthood (PND 101) on the levels of diverse cytokines in the plasma of adult male and female Wistar rats.
Cytokines were measured by multiplexed ELISA using a Luminex 200 machine (Millipore). Maternal deprivation induced a long-term increase in the plasma levels of the pro-inflammatory cytokine IL1b in males and females that were reversed by the high fat diet. Maternally deprived males (but not females) also showed a significant increase in TNF levels and, also in this case, the effect was diminished by the special high fat diet. Similar trends were observed with respect to IL 6. A sexual dimorphism was found in the levels of IL 13 and IL 1a, with control females showing a significantly higher content of both cytokines when compared with control males. In females, the levels of IL 13 were significantly decreased by the high fat diet. The results indicate that cytokine plasma levels are modulated by both, a neonatal stress of maternal deprivation that appears to induce a long-term increase in at least some pro-inflammatory cytokines, and also by a diet with a high fat content. This latter factor tended to counteract the MD effect.
The results show diverse sexual dimorphisms in the baseline plasma levels of certain cytokines, as well as in the effects of the two treatments. Maternal deprivation, "per se" induced a decrease in body weights throughout the experimental period, whereas the special high fat diet counteracted this effect. Moreover, at adulthood, MD males on the high fat diet weighed more than control non-deprived males receiving the same special diet. Individual differences in premorbid behaviours and in those exhibited in the course of an infection disease may be useful to explain the individual susceptibility to infections, the underlying neuroimmunological mechanisms and be helpful to get a better prediction of pharmacological reactivity. Age (old age) and gender (being a male) are also considered vulnerability factors.
In the present work, locomotor activity, emotionality and anxiety were assessed in adult (6 month-old) and old (18 month-old) C57Bl6 male mice prior to the study of LPS (150 mg/kg, i.p.)-induced sickness behavior and lethality. The results showed equal neophobic response to novelty in the corner test (corners: 9.38 +0.86 and 8.4 + 1.3; rearings: 4.9 + 0.9 and 3.2 + 0.9, respectively) but a 2-fold increased freezing behavior (latency of movement, s: 7.9 + 1.6 vs 3.9 + 0.4) in old animals when confronting a more anxiogenic environment (openfield test). They also differed in the development of the sequence of behavioral events and the exploratory activity (faster and reduced with aging). Measures related to anxiety and emotionality such as number and duration of grooming or the presence of urination showed also a 2-fold increase in old animals as compared to adult mice. In the LPS-induced sickness behavior (measured as its locomotor effects during 30 min), both groups developed equal activity during the first minute of the test. Then, activity dropped to 66-77% and reached the maximum effect during the next 6 min. In the old group, the motor depressant effect lasted until the end on the test, while in the adult animals a recovery of both horizontal and vertical locomotor activities was experienced in the last 10 min interval. Mortality rate, recorded every 30 min over a total period of 72 h, was 100% in both groups of age, but in the old animals the mean survival time was reduced exactly to one half (survival, h: 26.88 + 3.55 vs 57.63 + 7.86, respectively) .
Age (− .680**) and LPS-induced sickness behaviour (mainly horizontal activity, but all N.778*) showed a direct correlation with survival while premorbid behavioral individual differences were not predictive enough to reach statistical significance but it cannot be excluded that they are related to survival. Tel Aviv University, Tel Aviv, Israel; 2 Oklahoma University, Oklahoma, United States
Group A streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders, including Sydenham's chorea (SC), obsessive-compulsive disorder and Tourette's syndrome. SC is the classic post-streptococcal neurological disorder, characterized by involuntary movements and neuropsychiatric disturbances. The leading hypothesis suggests that an antecedent GAS infection induces a cross-reactive immune response directed against neuronal brain determinants. The aim of the present project is to identify the neural mechanisms that are altered by the antibody response in GAS-related neuropsychiatric disorders (GRND), and lead to neuropsychiatric symptoms.
We recently developed a new animal model of GRND disorders using immunization of rats with a crude GAS extract. The immunized rats showed increased compulsivity and motor disturbances. Autoantibodies in immunized rats targeted the striatum and thalamus, two brain regions implicated in the pathophysiology of GRND. Sera taken from these rats demonstrated strong immuno-reactivity to GAS antigens and to neural tissue including dopamine receptors. Interestingly, the levels of antibodies against dopamine receptors were correlated with the severity of behavioral symptoms. Based on these results, we are currently testing whether immunization can lead to alterations in different neurotransmission systems. Male Lewis rats are inoculated with GAS extract, followed by a comprehensive behavioral examination: (a) Rats' compulsive behavior is evaluated in the induced grooming assay, and (b) rats' motor disturbances are evaluated by the Food Manipulation and the Beam Walking assays. Subsequently, we will evaluate the pathogenic changes in the brains of immunized rats by assessing changes in mRNA and actual protein levels (using RT-PCR and Western blot) of proteins of the dopaminergic, glutamatergic, serotonergic, cholinergic and GABAergic systems. Based on the results found in brains of immunized rats, we will assess correlations between the different biological measures and measures of behavioral dysfunction.
This project can provide the basis for developing novel therapies for GRND and improve the diagnosis and prevention of these disorders. An understanding of the neural mechanisms underlying autoimmunerelated disorders will provide clues as to the pathogenesis of similar movement and behavioral disorders that are not autoimmune.
Single electrical stimulation of the bed nucleus of the stria terminalis and the medial septal nucleus increases peripheral blood natural killer cell cytotoxicity in rats Myslinska Dorota ⁎ , Plucinska Karolina, Redzimska Dorota, Ciepielewski Ziemowit, Glac Wojciech, Badtke Piotr, Wrona Danuta Department of Animal Physiology, University of Gdansk, Gdansk, Poland
In our previous study we found that electrolytic lesion of the bed nucleus of the stria terminalis (BST) as well as the medial septal nucleus (MS) caused depression of the peripheral blood natural killer cell cytotoxicity (NKCC) and the number of leukocyte in rats. In the respective sham operated groups, the mere insertion of electrodes into the BST and the MS evoked transient enhancement of NKCC, probably resulting from mechanical stimulation of the brain tissue. We also found that chronic electrical stimulation of both the BST and the MS caused significant augmentation of blood NKCC and large granular lymphocytes (LGL) number.
In the present study we evaluated both spleen and blood NKCC after single electrical stimulation of the BST and the MS in conscious, freely behaving rats.
Male Wistar rats implanted with stimulating electrodes at the BST (n = 16) and at the MS (n = 18) area were divided into groups subjected to single electrical stimulation (constant current 0.1 ms duration cathodal pulses delivered at a frequency of 50 Hz during 30min) of the BST (n = 8) and the MS (n = 9) and the sham stimulated control (n = 8 and n = 9, respectively). Blood samples were collected by heart puncture (halothane anesthesia) seven days before and one hour after the stimulation. NKCC was quantified using 51Cr release assay. Target cells (5 × 10^6) were labelled with 100 μCi of Cr^51, adjusted to 1 × 10^5 with concentration of effector cells E:T = 50:1. After determination of experimental (Exp), spontaneous (Sp) and maximal (Max) Cr^51 release percentage cytotoxicities was obtained from the following equation: [(Exp − Sp) / (Max − Sp)]. We found that single electrical stimulation of the BST caused significant NKCC augmentation of blood NKCC (36.84 +/− 7.07%, P b 0.01) in comparison to the sham operated group (17.99 +/− 1.71%) and to the baseline (23.35 +/− 7.69%). Similarly, single electrical stimulation of the MS resulted in augmentation of blood NKCC in comparison to the sham operated group (P b 0.01) and to the baseline (P b 0.05) (34.43 +/− 8.72% vs 18.02 +/− 4.82% and 22.40 +/− 5.27% respectively). No such effects were found in the spleen.
The results obtained indicate that the BST and the MS belong to the limbic structures involved in the regulation of immune responses: their lesion causes depression while stimulation causes enhancement of cellular immunity. This work was supported by University of Gdansk Grant BW/L125-5-0412-0. It is known that depressive disorders are associated with an impairment of the immune system. In the present study, we investigated spleen lymphocyte subpopulations after chronic desipramine (DEZ) treatment followed by an acute, white and illuminated open field (OF) stress in order to induce behavioral depression in rats. Animals used in the experiments differed in spontaneous locomotor activity, which may reflect different psychobehavioral types in human.
A fourteen day injection of DEZ followed by an exposure to the acute OF resulted in a decrease in the spleen percentage number of NK cells (7.14 ± 1.75%; p b 0.01/mean ± SD) and subpopulation of TCD4+ lymphocytes (43.08 ± 4.51%; p b 0.01) in the non-divided into HRs and LRs as compared to their saline controls (10.41 ± 2.65%; 52.02 ± 8.48% for NK and TCD4+, respectively). There were no significant influences of OF stress and DEZ injections on spleen T, B and TCD8+ lymphocytes. In behaviorally different groups, the lower level of percentage of T (56.36 ± 9.72% vs. 67.25 ± 9.92%) and TCD4+ (41.57 ± 4.59% vs. 57.06 ± 5.56%) was observed in HRs with DEZ injection than in controls. As compared to the controls, NK cell percentage decreased in both HRs (7.00 ± 1.74% vs. 9.93 ± 2.95%) and LRs (7.25 ± 1.92% vs. 11.18 ± 2.14%). Moreover, splenocyte apoptosis following OF stress was significantly lower in all DEZ-injected groups (non-divided = 4.04 ± 1.44%, HRs = 4.35 ± 1.18%, LRs = 3.66 ± 1.82%) as compared to the controls (non-divided = 8.07 ± 2.77%, HRs = 10.18 ± 3.80%, LRs = 8.15 ± 2.23%).
The results obtained suggest that individual differences in locomotor activity in novelty play the important role in immune effects of desipramine influences during OF stress-induced behavioral depression.
This work was supported by a research grant N 303 3335336 from the Ministry of Scientific Research and Information Technology (former State Committee for Scientific Research, KBN).
The effect on salivary biomarkers in awakening response after a two week sleep tutorial Suguri Kazumichi ⁎ ,1 , Tanaka Hideki 2 , Okamoto Yuko 2 , Nomura Tomoyo 2 , Nomura Shusaku 1 1 Nagaoka University of Technology, Nagaoka, Japan; 2 Hiroshima International University, Higashihiroshima, Japan
Recent developments in molecular analysis techniques have enabled scientists to study a tiny amount of biochemical substances contained in a variety of secretory fluids, and it has been revealed that there is a close relationship between the secretion of such substances and human mental state. Among that, there is growing evidence suggesting that the magnitude of cortisol awakening response (CAR), which is characterized by a profound increase of salivary cortisol after awakening, plausibly reflects the level of chronic stress, social stress, anxiety, etc. Such an elevation after awakening has also been reported in a variety of biomarkers. However, there is still a small number of studies in this field, little is known about the alternation of the magnitude of these biomarkers and its' relevance to CAR. We then investigated such awakening responses of cortisol and immunoglobulin A (IgA) at the start and at the end of two weeks of sleep tutorial; by which we anticipated that the tutorial would bring forth a positive affection with better sleep management, and also a result in an altered CAR, with illustrating the difference in such awakening response between endocrine and immune system.
Eleven healthy students aged from 20 to 21 voluntarily participated in this study. They were instructed to individually go through our originally-developed tutorial for taking good sleep just before going to bed for two weeks. The tutorial consists of a literature material introducing proper sleep time, diet, exercise and a check list of 18 items relating to the material. At the start and at the end of the experiment, salivary cortisol and IgA were assessed at the time of awakening, and 30 and 45 min after awakening, for estimating the awaking response of these biomarkers. It should be noted that subjects were not forced to change their behavior nevertheless their sleep cycle and the other behaviors were checked by the check list. In result, subjects did not change their daily sleep, diet, and other behaviors as anticipated. However, the awakening response of cortisol (CAR) significantly increased (p b .01) whilst that of IgA did not change.
The difference in the awakening response of the endocrine and immune systems was successfully demonstrated in this study. However, the higher CAR observed in this study implies that the procedure of experiment, i.e. having a tutorial every night before going to bed, might be merely taken as a stressful task for subjects.
Neuroinflammation in neurological diseases I Chairs: A. Bar-Or and P. Villoslada Accumulating evidence demonstrates that inflammatory and oxidative processes together play a fundamental role in the onset and evolution of Parkinson's disease (PD). Indeed, inflammatory processes associated with microglial activation and cytokine release have been suggested by human postmortem analyses and animal toxin models of PD. Overactive microglia release the damaging oxidative radical, superoxide, which has been shown to be toxic to midbrain dopamine (DA) neurons. We presently propose that the pro-inflammatory cytokine, interferon-gamma (IFN-gamma), can mediate microglial reactivity and release of oxidative species in response to environmental toxin exposure.
Immunohistochemical methods were used to co-localize IFN signaling factors and oxidative regulatory elements upon microglia and neurons following exposure to the pesticide paraquat. Similarly, rigorous stereological techniques enabled quantification of loss of midbrain DA neurons following paraquat exposure RT-PCR and Western blot were used to evaluate mRNA and protein changes influenced by paraquat exposure in IFN-gamma deficient mice and their wild type littermates.
IFN-gamma null mice were found to be resistant to the loss of DA neurons and enhanced microglial response provoked by the pesticide, paraquat (which has been epidemiologically linked to PD) in wild type littermates. Paralleling the neuroprotective actions of IFNgamma deficiency, these mice failed to display the increased expression (and localization on microglia) of NADPH oxidase subunits required for oxidative radical production that was evident in wild types. Additional cytokine signalling elements, including STAT1 and JNK, were also elevated within the substantia nigra of paraquat exposed wild type mice but were inhibited by IFN-gamma deletion.
We suggest that the environmental toxin, paraquat, can provoke a loss of nigral DA neurons through activation of IFN-gamma pathways which regulate NADPH oxidase activity upon local microglia. These data should have important implications for the development of novel targets for therapeutic actions in PD and enhance our understanding of the neuronal and inflammatory mechanisms through which environmental toxins can act to promote pathology. Heinrich-Heine-University, Duesseldorf, Germany; 2 German Diabetes Center, Duesseldorf, Germany
In chronic inflammatory demyelinating polyneuropathy (CIDP) an autoimmune mediated damage to peripheral nerves causes chronic progressive pareses and sensory impairments. The immunological targets remain elusive and further studies are impeded by the lack of adequate animal models. We searched for an adequate animal model for inflammatory neuropathies. The 'non-obese diabetic' (NOD) mouse strain spontaneously develops autoimmune diabetes. NOD mice deficient in 'intercellular adhesion molecule 1' (ICAM-1)mediating costimulation and endothelial cell adhesionare protected from diabetes.
We surprisingly observed that these ICAM-1 deficient NOD miceinstead of diabetesdeveloped hind limb pareses and gait disturbances progressing with age and predominantly affecting females. We studied ICAM-1 deficient NOD mice by phenotypic analysis, peripheral nerve electrophysiology, immunohistochemistry and semi-thin sections. We analyzed for lymphocyte cytokine expression and performed adoptive cell transfer studies. Electrophysiology demonstrated signs of chronic demyelinating neuropathy which correlated with the clinical impairments. Histopathology and cell extraction from peripheral nerves revealed T cell and macrophage infiltration. Semi-thin sections demonstrated consecutive loss of myelin and to a lesser extent of axons in the peripheral nervous system. All other organs including brain and spinal cord did not exhibit any abnormalities. The adoptive transfer of CD4+ T cells induced an inflammatory neuropathy in immunodeficient recipients. T cells in neuropathic ICAM-1 deficient NOD mice exhibited a predominant Th17 bias.
We conclude, that in mice generally prone to autoimmunity, deficiency in ICAM-1 shifts autoimmunity specifically from endocrine pancreas to peripheral nerves. This introduces a novel animal model of human CIDP and may allow us to study mechanisms and test preclinical treatments in chronic inflammatory neuropathies in the future.
The role of interleukin-7 receptor alpha in experimental autoimmune encephalomyelitis
Jopek Ashbaugh Jessica ⁎ , Brambilla Roberta, Malek Thomas, Bethea John
Experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), is a neurodegenerative disease characterized by extensive inflammation, demyelination, and axonal damage. Recent studies in MS patients have shown a genetic linkage between the disease and mutations in the interleukin 7 receptor (IL7R) alpha chain locus. IL7R alpha is common to both the IL7 and thymic stromal lymphopoietin (TSLP) receptors and is important for lymphocyte development and homeostasis, as well as dendritic cell function. Therefore, the ultimate goal of our studies is to identify how IL7R alpha is functioning through specific cell populations in the EAE disease setting, which can then be applied to therapeutic strategies and translated to the clinical setting.
Studies using genetically altered mice that express IL7R alpha only in the thymus (IL7RTg^IL7R −/− ) show significant neuroprotection in the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide model of chronic progressive EAE. Based upon these results and the genetic analysis in humans we wanted to determine if targeting IL7R alpha signaling would be therapeutic in EAE. Our studies show that systemic blockade of IL7R alpha via neutralizing antibody after disease onset promoted a significant decrease in EAE severity and lymphocyte infiltration into the central nervous system, which was accompanied by an increase in the resident microglia population. Conversely, in vivo neutralization of IL7 did not show any protection from disease, despite the ability to inhibit IL7 function in vitro. Next, we performed chimera studies whereupon bone marrow (BM) from either IL7RT-g^IL7R −/− mice or wild type (WT) controls were transplanted into Workshops irradiated Rag −/− recipients. Interestingly, the disease severity was equivalent between both groups.
Taken together, both the genetic and therapeutic approaches to systemically block IL7R alpha reduce EAE severity, however, blocking IL7 does not yield protection, implying that alternative pathways such as TSLP may be involved. Additionally, inhibition of IL7R alpha specifically on small lymphocytes is not sufficient to promote the protection observed in our IL7RTg^IL7R −/− mice, suggesting that other leukocytes and/or cells of a non-hematopoietic lineage are involved in IL7R alpha mediated neuroinflammation. Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by inflammation, myelin damage, and axonal degeneration. MS disease relapses are markedly reduced during pregnancy, with the greatest suppression in disease activity observed during the third trimester. A serum factor is implicated as responsible for pregnancy-associated disease suppression. Exosomes are small lipid-bound vesicles that function as facilitators of intercellular communication and are augmented in the serum during pregnancy. Exosomes are able to modulate cells of the immune and central nervous systems by relaying molecular signals from their cell of origin to target cells bearing specific adhesion molecules. Therefore, the goal of this study is to elucidate the role of serum exosomes in pregnancy-associated suppression of MS.
We have studied pregnancy in murine experimental autoimmune encephalomyelitis (EAE). Disease severity is significantly reduced when pregnancy is induced during established EAE. Additionally, we observe a post-partum flare in disease course with disease suppression observed only during gestation. We have administered pregnancy-derived serum exosomes to mice with established EAE and observed reduced clinical severity following exosome treatment. Further, we have shown that exosomes are able to suppress the activation of myelin-specific T cells measured by a reduction in proliferation and interferon-gamma expression. We have also demonstrated a role for serum exosomes in the proliferation and maturation of oligodendrocyte precursor cells (OPC) in vitro and in vivo. To determine which proteins are expressed in pregnancy-versus control-derived exosomes, we performed differential gel electrophoresis followed by mass spectrometry. Proteins enriched in pregnancy exosomes facilitate the local action of corticosterone, scavenge oxygen-derived free radicals, inhibit matrix metalloproteases, and provide survival signals to oligodendrocytes.
These data suggest that serum exosomes are critical modulators of both the immune and central nervous systems during pregnancy and govern pregnancy-associated suppression of EAE and MS. Harnessing the mechanism by which exosomes suppress immunity, enhance the function of OPCs, and consequently suppress clinical EAE, can have extraordinary implications for therapy development in MS.
Macrophage migration inhibitory factor promotes central nervous system pathology in a model of neuroinflammation Cox Gina Mavrikis ⁎ , Alexander J., Kithcart A., Williams J., Smith K., Shawler T., Satoskar A., Whitacre C.C.
The Ohio State University, Columbus, OH, United States Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Cerebrospinal fluid collected from MS patients during relapse was found to contain greater levels of macrophage migration inhibitory factor (MIF) than samples collected during remission. We have shown that induction of experimental autoimmune encephalomyelitis (EAE) in MIF-deficient mice results in reduced clinical signs and CNS inflammatory infiltrates relative to wild type controls. However, there was no difference in T cell function in the periphery between the two groups. These findings suggest that MIF acts within the CNS to potentiate disease progression. The potent pro-inflammatory properties of MIF are well established; however, the precise contribution of MIF to neuroinflammation is not well defined. MIF is expressed by many cell types in the CNS including infiltrating macrophages and resident microglia, suggesting that MIF may enhance the inflammatory environment of MS lesions. Thus, we sought to determine the relative contribution of MIF to neuroinflammation and identify MIF as a therapeutic target for the treatment of MS.
To assess the contribution of MIF to CNS inflammation, primary microglial cultures were treated with increasing doses of rMIF, which upregulated inflammatory mediators associated with MS and EAE: IL-1, IL-6, TNF-a, iNOS, and CCL2. In addition, rMIF induced morphological changes in microglial cultures in a dose-dependent manner, consistent with an activated phenotype. To explore the role of MIF in vivo we used an intraspinal model of neuroinflammation. Stereotactic spinal cord microinjection of rMIF was performed, resulting in transient microglial reactivity and cellular accumulation within the CNS. We are currently utilizing this model to explore the role of MIF in the progression of EAE.
These studies implicate MIF as an inflammatory mediator within the CNS and suggest that MIF may contribute to the development and maintenance of MS lesions. Taken together, these data suggest that inhibition of MIF may serve as a therapeutic strategy for resolving CNS inflammation. Interaction between adhesion molecules expressed by blood-brain barrier endothelial cells (BBB-ECs) and their cognate ligand expressed by lymphocytes promotes transmigration of inflammatory cells to the central nervous system (CNS), an early phenomenon in multiple sclerosis (MS) lesion formation. Melanoma cell adhesion molecule (MCAM/CD146) is an adhesion molecule that can reportedly interact with itself and was identified by proteomic using lipid raft membrane microdomains of human BBB-ECs grown in primary culture. Our goal is to define the role of MCAM in both Th17 and BBB-ECs, and thus in CNS immune infiltration such as seen in MS.
Our data demonstrate that MCAM is expressed by a subset of inflammatory effector memory CD4+ T lymphocytes, co-expressing CD95, CD147, CD11a, CD49d and CCR6. MCAM+ lymphocytes also express more RORg, IL-17, IL-22 and Granzyme B than MCAMneg cells, both ex vivo and after in vitro activation. IL-23-driven in vitro polarization induces MCAM expression on as much as 60% of CD4+ CD45RO + lymphocytes and MCAM + lymphocytes display a significantly higher proliferation and IL-17 production as compared to MCAMneg cells. Furthermore, the proportion of MCAM+ lymphocytes is higher in the blood of MS patients than in healthy controls, and is enriched in the CSF of MS patients. When compared to MCAM+ cells from healthy donors, MCAM + lymphocytes from MS patients consistently produce more IL-17, in percentage and intensity. In addition, we report that MCAM is expressed on the surface of human BBB-ECs in vitro and in situ in MS lesions and colocalizes with lymphocyte surface markers during diapedesis. Preliminary data on MS and EAE tissue reveal that MCAM+lymphocytes are present in immune infiltrates and colocalize with inflammatory cytokines, especially IL-17. Moreover, cross-linking of MCAM induces a brief calcium influx similar to that obtained with anti-CD28. Our data further show that MCAM antibodies can influence the proportion of activated CD4 +CD45RO+expressing IL-17 and IFN-g as compared to control, and that MCAM neutralization restricted the migration of inflammatory lymphocytes across human BBB-ECs.
Our data indicate that MCAM is expressed by both BBB-ECs and by a subset of IL-17-expressing effector memory CD4 lymphocytes, and is a potential adhesion molecule for Th17 entry in the CNS of MS patients and that MCAM engagement influences IL-17 and IFNg release or production. Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS, associated with complex anti-myelin autoimmunity. A number of CNS myelin proteins (myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), myelin oligodendrocyte basic protein (MOBP) and oligodendrocyte specific protein (OSP)) have been implicated as potential primary target antigens in MS. The multiplicity of potential primary target antigens in MS, together with the likely possibility of 'epitope spread' whereby the pathogenic autoimmunity can expand to other myelin target antigens in the same patient, with disease progression, suggest that MS can be associated with complex pathogenic anti-myelin autoimmunity, imposing major difficulties for devising immune-specific therapeutic approaches for MS. In view of such potential complexity, our aim was to establish a new animal model that better reflects the complex autoimmunity associated with MS.
Here, we present a novel model for chronic "complex" EAE associated with well defined multiple pathogenic anti-myelin autoreactivities induced in (SJL/JxC57Bl/6J)F1 mice by immunization with the "mouse multi-epitope encephalitogenic protein" (mMEP), a protein product of synthetic gene encoding in tandem only the disease-related epitopes of MBP, PLP, MOG, MOBP and OSP. "Complex" EAE induced by mMEP is associated with pathogenic autoreactivities against multiple myelin antigens that result in severe chronic clinical manifestation, accompanied by massive inflammation, extensive demyelination and neuronal damage in the CNS and the optic nerve.
Overall, our results suggest that the "complex" EAE induced by mMEP better simulates the complexity of pathogenic autoimmunity in MS, and is a useful model for devising immunospecific-therapies for MS. A total of 24 C57BL6 8-week old female mice were injected with mouse recombinant myelin oligodendrocyte glycoprotein (r-mMOG) to induce EAE. Twenty one of them (87.5%) developed clinical disease symptoms starting at 11 days post immunization (dpi). Disease peaked at 21 dpi, showed partial remission at 28 dpi, and in some animals relapsed by 50 dpi. Mice were sacrificed at 3 different time points (14, 28 and 50 dpi) and the spinal cord was examined by immunohistochemistry, immunoblotting and real-time PCR. At 14 dpi, EAE mice showed 5% demyelination of the total white matter area (TWM) and 14% axonal loss compared to controls. Inflammatory (microglia) cells infiltrated 16.5% of the TWM. Cx32 and Cx43 protein levels were reduced, whereas at RNA level the expression of Cx32 was upregulated in contrast to marked downregulation of Cx43. Cx47 expression remained unchanged at this stage. At 28 dpi, demyelination and axonal loss reached 3.5% and 33%, respectively, and inflammation diminished (7.7%). Cx32 and Cx47 were overexpressed while Cx43 expression remained downregulated. Levels of all 3 GJ proteins were reduced. At 50 dpi, demyelination and axonal loss were 12% and 36%; inflammation increased again to 8.7%. Expression of Cx43 was markedly upregulated with increased protein levels. Cx32 expression remained slightly upregulated with increasing protein levels compared to controls, while Cx47 showed no significant changes.
Our study shows distinct alterations in the expression of major oligodendrocytic and astrocytic GJ proteins at different stages of inflammatory demyelination, and suggests that GJ proteins may be differentially involved in this process. Impaired GJ connectivity in oligodendrocytes and between oligodendrocytes and astrocytes resulting from these changes may have a significant impact on the outcome of EAE and MS pathology. This project is funded by the Cyprus Research Promotion Foundation (Grant HEALTH/BIOS/0308/01) and by Cyprus Telethon. University of Oxford, Oxford, United Kingdom N-methyl D-aspartate receptor (NMDAR) is a class of ionotrophic receptor for glutamate, the predominant excitatory neurotransmitter in the central nervous system, and is implicated in learning and memory as well as several diseases. NMDAR antibodies (NMDAR-Abs), principally against the constitutive NR1 subunit, have recently been detected in the sera of patients with encephalitis who present with psychosis, memory loss and seizures, usually progressing to a reduction in level of consciousness and marked dyskinetic movement disorders. Early immunotherapy with plasma exchange, corticosteroids or intravenous immunoglobulin's can result in clinical improvement correlating with a reduction or disappearance of NMDAR-Abs (Dalmau et al. 2008; Irani et al. 2010) . NMDARs are transmembrane heteromers with extracellular domains that are accessible to the circulating antibodies. The NMDAR IgG antibodies bind to the hippocampus of rodent brain tissue and to the surface of live hippocampal neurons. They have been shown to decrease the number of NMDARs on these hippocampal neurons (Dalmau et al. 2008 ). However, it has not yet been shown that the patient's NMDAR IgG antibodies can passively transfer cognitive or behavioural changes to live animals.
We purified IgG from a patient with NMDAR-Abs and from a healthy individual. C57Bl6 mice were coded and tested over a forty day period following a single intracerebroventricular injection of either patient or control IgG. They were observed, with the observer blinded, for the presence of species-typical behaviours, motor activity, seizure activity and cognitive performance. Mice displayed normal light-dark activity cycles and no differences in motor performance on the rotarod task and static rods. However, some NMDAR-Ab-injected mice displayed visual signs of seizures, and 5/7 exhibited a phenotype which involved dyskinetic hind limb clasping when suspended by their tail, which was not observed in the control mice (p b 0.0001; video available) Cognitive ability was examined throughout the 40 days using the T-maze, a task of working memory which assesses the natural tendency of mice to alternate their choice of goal arm (spontaneous alternation). NMDAR-Ab treated mice showed a significant impairment (p b 0.0043) compared to mice receiving control IgG. This is the first evidence that passive transfer of NMDAR-Ab positive IgG can elicit a neurological phenotype in the mouse that reflects some aspects of NMDAR-Ab antibody encephalitis. Multiple sclerosis (MS) is an inflammatory disease for which 17 susceptibility loci have been identified. The goal of the project is to explore the functional consequences of these susceptibility loci in genome-wide RNA expression data generated from peripheral blood mononuclear cells (PBMCs) of subjects with MS.
We assessed our data for cis and trans effects on RNA expression by each of the 17 validated MS loci variants. The dataset consists of genome wide RNA expression data on PBMCs from 255 MS subjects. They were either untreated (n = 83) or treated with immunomodulatory drugs (n = 105 interferon-beta; n = 67 glatiramer acetate). Analyses were performed using linear regression and additive model for each SNP as an independent variable and adjusting for SNP-treatment interactions.
We have identified: (1) a robust cis association of MPHOSPH9 RNA expression with the MPHOSPH9 variant rs1790100 (p = 1.04 × 10 − 6 ), (2) several significant trans associations within the MS loci modulated by the susceptible SNPs in IRF8, TNFRSF1A, IL12A, IL2RA, CD226, IL7R, CXCR4 and RGS1. Further, using pathway analyses (IPA, Ingenuity Systems), we find that 7 different loci (CD6, TNFR1, IL12A, CD226, CLEC16A, IRF8, IL2RA, and RGS1) affect RNA expression of multiple genes in the same 4 signaling pathways: CD28 signaling, TCR signaling, iCOS_iCOS-Ligand and IL2 signature. For example, rs2760524 on RGS1 affects the expression of multiple genes in the CD28 pathway (P = 1.479 × 10 − 9 ).
We show a remarkable level of interconnectedness among a subset of MS susceptibility loci: some of these loci affect RNA expression within other susceptibility loci and, more striking, multiple different loci have a broad effect on signaling pathways that are critical for T cell function and proliferation. These observations provide a strong bridge between the recent genetic discoveries in MS and the wealth of immunological data that has been accumulated in this disease. Multiple sclerosis is a chronic inflammatory autoimmune disease of the central nervous system. The exact cause and working mechanism of the disease is not known, which makes specific treatment difficult. The aim of this study is to identify disease-related proteins that help us unravel fundamental disease processes. To identify differentially expressed proteins in the brain stem of EAE-animals and control rats in different stages of the disease, a quantitative study by two-dimensional difference in-gel electrophoresis (2D-DIGE) was performed.
Three rats were CFA injected controls, the other 9 rats were immunized with MBP to induce acute EAE. The EAE animals were divided into three groups, representing onset, top and recovery of the disease (3 animals in each group). 'Blood-free' brain stems of all 12 Lewis-rats were collected and detergent-soluble brain protein extracts were used for the 2D-DIGE analysis. Differential proteins were selected and thereafter identified using mass spectrometry. We found 70 differentially expressed protein spots over the four conditions (control, onset, top and recovery, 1-ANOVA 0.01). With these protein spots, we were able to discriminate between early and late groups (control and onset samples versus top and recovery samples). Moreover, a set of the 8 most discriminating proteins was selected from the discriminant analysis. This classifier was able to discriminate all four groups. Ingenuity pathway analysis with all identified proteins was performed to find molecular relationships between proteins. This analysis revealed a role for proteins involved in neuronal signalling, inflammation, BBB damage and mitochondrial dysfunction.
Apart from commonly identified differentially expressed proteins, also known brain markers and new candidates or interesting pathways were identified to be differentially expressed.
Quantitative analysis of differentially expressed brain proteins in an animal model of multiple sclerosis was the first successful step to identify interesting disease-related proteins that may be targets for further fundamental research.
Brain microRNAs targeting neurosteroidogenesis in multiple sclerosis mediate demyelination and neurodegeneration Noorbakhsh Farshid 1 , Ellestad Kristofor K. 1 Multiple sclerosis (MS) is the prototypic autoimmune neurodegenerative disease of the central nervous system for which the underlying disease mechanisms remain uncertain. Neurosteroids are synthesized within the nervous system and exert diverse effects on brain growth, repair and viability although their involvement in neuroinflammatory disorders has not been previously studied.
Using a multiplatform approach including autopsied brain tissues, in vitro cultures and the MS animal model, experimental autoimmune encephalitis (EAE), we investigated microRNA profiles in brain-derived white matter from MS and non-MS patients. Based on principal component and gene ontology analyses, predicted targets of dysregulated miRNAs were limited to specific biological functions including neurosteroid biosynthesis, which showed a targeting bias toward induced miRNAs (p b 0.05). The neurosteroid, allopregnanolone (ALLO), was suppressed in MS brains, which was complemented by diminished neurosteroid enzymatic machinery transcript and protein quantities in the same brain tissues (pb 0.05). Transduction of human astrocytes with select miRNAs suppressed enzyme expression responsible for ALLO synthesis (pb 0.05). Exposure of oligodendrocytes to nonimmunosuppressive ALLO protected them from the cytotoxic effects of activated macrophages (pb 0.05). Reduced ALLO and the enzymes responsible for its synthesis were observed in C57/Bl6 mice with MOG/ PTX-induced EAE (pb 0.05) together with induction of miRNA neurosteroid-specific enzymes. Treatment of EAE animals with ALLO diminished neuroinflammation, demyelination, axonal loss together and suppression of neurobehavioral abnormalities (pb 0.05). microRNA dysregulation potentially contributes to MS pathogenesis by altering neurosteroid expression leading to selective suppression of ALLO expression and ensuing adverse effects on oligodendrocyte viability. ALLO represents a plausible neuroreparative therapy for MS. The transcription factors CCAAT/enhancer binding protein beta (C/ EBPbeta) and C/EBPdelta regulate the expression of some of the genes most often associated with deleterious effects of glial activation, i.e. NOS2, COX-2, TNFalpha, IL-1beta, IL-6. If C/EBPbeta and C/EBPdelta regulate the expression of these genes in activated glial cells, the inhibition of C/EBPbeta and/or C/EBPdelta could result in an attenuated, less neurotoxic glial activation. To test this hypothesis we have undertaken a study with two aims: 1) to study the expression of C/EBPbeta and C/EBPdelta in glial activation 2) to analyze the effects of the inhibition/absence of C/EBPbeta or C/ EBPdelta in the proinflammatory and neurotoxic profiles of activated glial cells.
1) Using primary mixed glial, astroglial and microglial murine cortical cultures we have observed that toll-like receptor (TLR) agonists upregulate C/EBPbeta and C/EBPdelta, both at the mRNA and protein levels. The effect was observed in astrocytes but was particularly strong in microglia. By immunoprecipitation we observed the association of C/ EBPbeta and C/EBPdelta in the nuclear fraction of activated microglial cells, indicating the presence of C/EBPbeta-delta heterodimers.
2) To analyze the role of C/EBPbeta and C/EBPdelta in glial activation we used three experimental approaches: a) Overexpression of LIP, a dominant negative form of C/EBPbeta, in microglial cells.
b) Primary glial cultures from C/EBPbeta deficient mice. c) Treatment of glial cultures with chrysin, an anti-inflammatory flavonoid which reduced C/EBPdelta expression and did not affect NFkappaB or C/EBPbeta nuclear levels or DNA-binding.
In all three paradigms, the inhibition or absence of the C/EBPs markedly reduced the proinflammatory gene expression induced by LPS or LPS +IFNg. Interestingly, the absence of C/EBPbeta or the pharmacological reduction of C/EBPdelta levels markedly attenuated the neurotoxic effects of microglial activation in microglial/neuronal co-cultures.
These results indicate that the upregulation of C/EBPbeta and C/ EBPdelta is a common feature in TLR-induced microglial activation. They also suggest that these transcription factors play a key role in the regulation of proinflammatory gene expression in glial activation. In consequence, the inhibition of C/EBPbeta and C/EBPdelta could be useful in the treatment of neurological disorders in which glial activation plays a pathogenic role.
Supported by ISCIII PI07/0455 and PI08/1396.
Type I interferons as signals for axonal injury-induced glial response
Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark
Insult to the central nervous system (CNS) leads to initiation of a glial response that is innate to the CNS. This innate glial response is critical for the induction of cytokines and chemokines that direct leukocytes entry to the injury site. These responses may contribute to repair processes in the damaged CNS, but may also exacerbate neurodegeneration. To better understand these processes, it is important to study the mechanism of signaling in glial responses to injury. The glial response may involve cytokines, such as type I interferons (IFNa/β), which are known to regulate innate immune responses against viral infections. IFNa/β signal through a receptor (IFNAR), which involves activation of STAT1/2 and interferon regulatory factor-9 (IRF9), leading to activation of interferon stimulated genes including IRF7. The induction of IFNa/β is enhanced by IRF7. We have examined the involvement of IFNa/β signaling in the hippocampus after transection of the entorhinal afferents.
Axonal injury induced up-regulation of IFNa/β receptor associated signaling in hippocampus. Double immunofluorescent co-localization studies showed that IRF7 was induced in Mac-1/CD11b positive macrophages/microglia in the denervated molecular layer of dentate gyrus of the hippocampus. In addition, IRF7 mRNA was detected in FACS-sorted microglia from lesion-reactive hippocampi. The induction of IRF7 mRNAs was IFNAR-dependent. Furthermore, lack of IFNa/ β signalling resulted in increased leukocyte infiltration into the lesionreactive hippocampus. Unlike WT mice, axonal lesion did not induce an increase in CXCL10, but it induced an increase in matrix metalloproteinase 9 gene expression in IFNAR-KO mice.
Our findings point to a role for type I IFN signalling in the regulation of innate immune response to sterile injury.
Microglial cells in acute EAE may play a key role in the regulation of lymphocyte dynamics Almolda Beatriz, Costa Manuela, Montoya Maria, Gonzalez Berta, Castellano Bernardo
Experimental autoimmune encephalomyelitis (EAE), a wellestablished model of multiple sclerosis, is characterised by microglial activation and lymphocytic infiltration. Lymphocyte activation through the antigen presentation process involved two main signals, the first provided by TCR-MHC engagement and the second by the binding of co-stimulatory molecules, such as B7.1/B7.2 with CD28/ CTLA-4. A wide number of reports have described microglial reactivity and T-cell infiltration after EAE, however the major part of studies are focused on the peak, and less is known about aspects taking place during the inductive and recovery phases. In this work we perform a detailed analysis of microglial activation and T-cell dynamics along the acute Lewis rat EAE model.
MBP-injected rats were sacrificed at different phases, attending exclusively to their clinical score, and spinal cords analysed by flow cytometry and immunohistochemistry. Our findings revealed that during the inductive phase and peak, microglial cells became activated and a subpopulation displayed an immature dendriticcell-like phenotype (CD1+, MHC-class I+, MHC-class II+, B7.1−, and B7.2−). In parallel, a high infiltration of CD3+ lymphocytes with a Th1 phenotype takes place from the first signs of symptomatology until peak. Coinciding with the beginning of clinical improvement, a high decrease in the number of Th1 cells occurred. This decline can be mediated by a mechanism of apoptosis induced by microglial cells with an immature dendritic cell phenotype. We cannot discard, however, that Th1 cells differentiate into other types of lymphocyte, because during the recovery there is not a reduction of the total numbers of lymphocytic cells. In fact, during the recovery phase we found an important increase in Th17 and T-reg cell populations. Also during this recovery phase, microglial cells remained highly activated and those found in the neighbouring areas of blood vessels displayed a CD1+, MHC-class I+, MHC-class II+, B7.1−, and B7.2+ phenotypes. The presence of activated microglial cells and infiltrated lymphocyte populations extended during the post-recovery phase.
In conclusion, our results indicate that microglial cells may regulate the dynamics of lymphocyte subpopulations and may be involved not only in leading the resolution of the pathological process, but also in the induction of the subsequent tolerance characteristic of this model of EAE.
IL-17 enhances CCL20 production in primary astrocytes Meares Gordon ⁎ ,1 , Ma Xiangyu 2 , Qin Hongwei 1 , Benveniste Etty N. 1 1 University of Alabama at Birmingham, Birmingham, United States; 2 Qilu Hospital of Shandong University, Jinan, China Multiple sclerosis (MS) is a debilitating autoimmune disease characterized by the progressive and selective destruction of the myelin sheath and subsequent degeneration of axons. During the onset of MS, pathogenic T cells, including IL-17 producing Th17 cells, invade the CNS and contribute to demyelination. Chemokines including chemokine (C-C motif) ligand 20 (CCL20), through its binding to CCR6, promote migration of T cells to sites of inflammation. Astrocytes have an important role in the regulation of inflammation within the CNS and astrocytes respond to a number of cytokines. Recently, we demonstrated that astrocytes express the IL-17 receptor and respond to IL-17 with enhancement of IL-6-induced signaling in an NF-kappaB and MAPKdependent fashion. During experimental autoimmune encephalomyelitis (EAE), astrocytes produce CCL20; however the influence of IL-17 on astrocyte production of CCL20 is currently unknown. In this study, we examined the role of IL-17 on CCL20 production in primary mouse astrocytes.
IL-17 alone or IL-6 alone had only a modest effect on CCL20 expression. However, the combination of IL-17 and IL-6 led to a robust increase in CCL20 mRNA expression and secretion in astrocytes as measured by RT-PCR and ELISA, respectively. This increase in CCL20 expression was not due to IL-17-induced mRNA stabilization. Instead, IL-17 increased the activation-associated phosphorylation of NF-kappaB; and inhibition of the NF-kappaB pathway ablated IL-17/IL-6induced CCL20 expression. Additionally, chromatin IP (ChIP) revealed that stimulation of primary astrocytes with IL-17/IL-6 increased the recruitment of active NF-kappaB to the CCL20 promoter, increased p300 binding and histone acetylation, consistent with a transcriptionally active gene. Interestingly, the IL-17-induced enhancement of CCL20 production is not unique to IL-6. IL-17 also synergized with IL-1beta, a well known inducer of CCL20, to increase CCL20 expression in a human glioma cell line.
Collectively, these results suggest that astrocytes, in response to IL-17, may shift chemokine production to that favoring T cell recruitment to the CNS.
The microglia phenotypes involved in central nervous system deand remyelination
Olah Marta 1 , Biber Knut 2 , Boddeke Erik ⁎ ,1 1 University Medical Center Groningen, Groningen, The Netherlands; 2
It has been proposed that microglia, the resident immune cells of the central nervous system (CNS), have a multifaceted role in CNS disorders affecting the white matter. They are believed to be critically involved in both the demyelination and remyelination phase of leukoencephalopathies, such as multiple sclerosis. Microglia are therefore a potential therapeutic target in demyelinating diseases, rendering the understanding of the exact microglia phenotypes that are involved in de-and remyelination highly desirable. Our aim was to investigate the role of adult murine microglia in the process of de-and remyelination in the cuprizone model.
With a protocol recently developed in our laboratory we isolated microglia from the brains of adult mice at different time points of the cuprizone diet (demyelination phase) and after withdrawal of cuprizone (remyelination phase). Microglia total RNA was subjected to gene expression experiment, using Illumina MouseRef8 v2.0 bead arrays. The results of our gene expression analysis reveal the microglia phenotypes that are involved in the process of primary demyelination and remyelination in the central nervous system. The microglia phenotype that is associated with demyelination is characterised by the expression of mRNA for proteins that are involved in the phagocytosis of myelin debris and apoptotic cells.
Nevertheless, during the remyelination phase microglia highly expressed transcripts of genes involved in tissue remodeling, oligodendrocyte precursor cell recruitment and differentiation, and trophic support.
Since we have confirmed selected gene expression results at the protein and functional level, we suggest that in addition to a microglia phenotype involved in clearance a specific microglia phenotype that supports the remyelination process exists.
IRF-1/caspase 1 signaling in central nervous system glial cells regulates inflammatory demyelination Balabanov Roumen ⁎
Interferon regulatory factor 1 (IRF-1) is an interferon-dependent transcription factor that has been implicated in the pathogenesis of multiple sclerosis (MS) and its animal model, the experimental autoimmune encephalomyelitis (EAE). In the present study, we investigated the involvement of IRF-1 signaling in the mechanisms of inflammatory demyelination in MS and EAE.
For the purpose of this study we employed a number of molecular methods focusing on the expression and interactions of IRF-1 and caspase 1 in MS and EAE tissues and cell cultures. In addition, we generated bone marrow chimera mice that differentially expressed IRF-1 in the central nervous system (CNS) and tested their responses in EAE.
We found that IRF-1 was expressed in MS and EAE tissues by microglia, oligodendrocytes and astrocytes. Our mouse bone marrow chimera experiments demonstrated that mice lacking IRF-1 in the CNS were protected against EAE. Investigation of IRF-1 signaling identified caspase 1, a pro-inflammatory and pro-apoptotic gene, as an important target of its transcriptional activities.
Based on these results we concluded that IRF-1/caspase 1 signaling in glial cells regulate inflammatory demyelination in MS and EAE. Our findings are clinically relevant and may provide a basis for development of novel therapeutic strategies in MS. Nitric oxide (NO) and the NO synthases (NOS) are present in a wide range of brain regions involved in the modulation of defensive and adaptative behaviours, including in the dorsal hippocampus. Recent evidence have suggested that systemic or hippocampal administration of non-selective NOS inhibitors causes anxiolyticand antidepressant-like effects in animal models. However, the NOS isoform involved in these effects is not clearly defined. Considering that mediators of the inflammatory response as well as upregulation of NO production can be induced by exposure to stress, the aim of the present study was to investigate the differential involvement of hippocampal inducible NOS isoform (iNOS) and neuronal NOS isoform (nNOS) in the modulation of defensive behaviors related to depression and anxiety, using the Forced Swimming Test (FST) and Elevated Plus Maze (EPM), respectively.
Male Wistar rats with guide-cannulas aimed at the dorsal hippocampus were submitted to FST or EPM. In the FST, rats were submitted to pretest (PT: 15 min swimming) and received a local administration of n-propyl-L-arginine (NPLA, selective nNOS inhibitor: 0.001, 0.01, 0.1 or 1.0 nmol/0.5 μL), 1400 W (selective iNOS inhibitor: 0.001 nmol/0.5 μL) or vehicle (0.5 μL). One day later, the immobility time (IT) was registered at a 5 min swimming test. In the EPM, rats received hippocampal administration of NPLA (0.01 nmol/0.5 μL), 1400 W (0.001 nmol/0.5 μL) or vehicle (0.5 μL) 5 min before being exposed to EPM, where the time spent in the open and enclosed arms were scored for 5 min. Diazepam (2.5 mg/kg, IP) was used as positive control. All protocols were approved by a local ethical committee.
NPLA, but not 1400 W, reduced the IT in the FST (F55,5=11.22; pb 0.001), an antidepressant-like effect in this model. In EPM, the treatments (NPLA, 1400 W and Diazepam) increased the time spent in the open arms (F30,3=5.35; pb 0.05), indicating an anxiolytic-like effect. NPLA, 1400 W or diazepam did not induce significant locomotor effects since no differences between treatments were detected in the total number of entries in the arms of the EPM (F30,3=1.526; p=0.228).
These results indicate that hippocampal nNOS and iNOS may be differentially involved in the regulation of defensive behaviors related to anxiety and depression.
The effect of chronic stress on male university students in their final examinations Nomura Shusaku ⁎ ,1 , Morishima Mika 2 , Migita Masao 3 , Mizuno Tota 4 , Nozawa Akio 5 , Suzuki Ikuo 6 , Izawa Shuhei 7 , Imai Jun-ichi 8 1 Nagaoka University of Technology, Nagaoka, Japan; 2 Gifu City Women's College, Gifu, Japan; 3 Shiga University, Hikone, Japan; 4 Tokyo Polytechnic University, Atsugi, Japan; 5 Meisei University, Tokyo, Japan; 6 Hokkaido University, Sapporo, Japan; 7 National Institute of Occupational Safety and Health, Tokyo, Japan; 8 Chiba Institute of Technology, Chiba, Japan
The influence of chronic stress on the immune-endocrine system of male university students who were engaged in their graduation examination was investigated. Recent developments in molecular analysis techniques have enabled scientists to study tiny amounts of biochemical substances contained in a variety of secretory fluids, and it has been revealed that some hormones and immune substances elevate in the level against the short-term and acute experimental stressors. Therefore such substances could also be considered as possible biomarkers for human mental stress levels. However studies investigating the effect of chronic stress on these biomarkers frequently showed inconsistent results. Such a discrepancy might attribute to the difficulty in controlling a variety of background factors, duration, and strength of the chronic stress. We then focused on male university students who were engaged in their graduation examination. The graduation examination consists of a vital oral defense at the end, following six months of preparation. Thus the duration or at least the end point of the chronic stress level caused by the exam would be highly expected to be parallelly aligned with each of the other participants.
Forty six male university final-year students who were engaged in their graduation examination (target) and 23 male university students in their second grade (control) voluntarily participated in this study. With regard to biomarkers, salivary secretory substances, immunoglobulin A (IgA), cortisol, chromogranin A (CgA), and dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S), testosterone (TE), alpha-amylase (AMY) were assessed at seven distinct points in time series; about three months, two months, a month, two weeks, and a week before the oral defense, and a week and two weeks after the oral defense. As a developing result, during the whole period of observation IgA, CgA, DHEA, and DEHA-S were significantly higher in the target group than the control (p b .01), whilst TE and AMY were significantly lower in the target group (p b .01), and there was no significant difference in cortisol.
In conclusion, the difference in the effect of chronic stress on the immune-endocrine system was illustrated in this study. The variations observed in biomarkers in this study imply that there might be a difference in physiological reaction mechanism against chronic stress.
Biomarkers Chairs: C. Whitacre and X. Montalban In multiple sclerosis (MS), the clinical course in an individual patient is largely unpredictable. The future development of a prognostic test for MS via biomarker identification would have much clinical value in that it would allow the physician to tailor his treatment regimen to the pathology of each individual patient. Gene expression signature has become one of the most promising methodologies for identifying biomarkers in complex diseases. The aim of this project was to develop a prognostic test for MS using DNA arrays and bioinformatic tools.
First, we performed an expression screening using the HG-U133 Plus arrays to analyze the gene expression of PBMC from 3 patients with aggressive MS, 3 patients with benign MS and 3 controls without the pathology. We identified 45 genes that were differentially expressed between the three groups. We then performed a validation of these genes by real time PCR in a prospective cohort of 60 MS patients classified in good (no relapses and not change in the EDSS) and bad outcome (N2 relapses or increase of EDSS) and 20 controls, followed for two years.
We were able to validate a gene signature pattern of 25 genes, which we used to create a Bayesian classifier with a diagnostic accuracy of 95% for distinguishing patients with a good or bad prognosis.
Mining the T cell miRNome for multiple sclerosis biomarkers Guerau-de-Arellano Mireia ⁎ , Smith Kristen, Godlewski Jakub, Whitacre Caroline, Lawler Sean, Lovett-Racke Amy, Racke Michael K.
While myelin-specific T cells are present in peripheral blood mononuclear cells (PBMC) of both healthy controls and multiple sclerosis (MS) patients, they only become pathogenic in MS patients, suggesting that MS T cells are predisposed to autoreactive activation. microRNAs (miRs) regulate a number of cellular processes through mRNA degradation and/or translation inhibition. We hypothesized that miRNAs underlie the susceptibility of MS T cells to autoimmune activation.
To investigate the role of miRs in predisposing to MS, we determined the miR profile of naïve CD4 T cells of healthy, relapsing/remitting (RRMS), secondary progressive (SPMS) and primary progressive (PPMS) individuals with a real-time PCR Taqman Array assay that covers 95% of all described human miRs. More than 25 miRs were found to be significantly different in MS compared to healthy controls. RRMS and SPMS patients had an almost identical miRNA profile, in which down-regulated miRs predominated. In contrast, PPMS patients had a distinct and highly unique profile dominated by up-regulated miRs. Ingenuity pathway analysis of genes predicted to be regulated by up-regulate miRs revealed the cooperative targeting of Th2 cell differentiation, including silencing of STAT-6 and GATA-3 transcription factors, favoring Th1 differentiation. MSassociated miR-transfection resulted in reduced T cell GATA-3 expression.
These results identify miRs as MS biomarkers of biological significance and potential clinical value, and reveal new possible therapeutic targets in MS. For children presenting with an initial episode of an acquired demyelinating syndrome (ADS), biomarkers are lacking to predict whose illness will remain monophasic, and who will develop recurrent disease consistent with the diagnosis of multiple sclerosis (MS).
Using the Luminex LX100 multiplex system, we investigated a selected panel of 22 immune molecules (previously implicated as putative biomarkers of disease activity in adult-MS) in serum collected at time of ADS from 176 children (age = 10.5 ± 4.3 years; F/M: 87/89) who were then followed with comprehensive, standardized clinical and imaging studies as part of the prospective Canadian Pediatric Demyelinating Disease Study. Over an average follow-up of 3.6 ± 1.1 years from ADS, 38 children exhibited clinical or imaging evidence of recurrent disease (MS), while 138 had no evidence of recurrent disease (monophasic-ADS). Comparing samples from ADS onset to 3 months following ADS, serum levels of MIG, TRAIL and VCAM increased (p b 0.0001 each), while levels of IL-10, HGF and RANTES decreased (p b 0.0001 each), though no differences were observed between groups. However, serum levels of osteopontin (p = 0.03), and E-selectin (p = 0.03) were lower both at time of ADS, and at 3 months following ADS in children subsequently diagnosed with MS (p = 0.006; p = 0.01, respectively) compared to those who remained monophasic; serum MMP-3 levels were also higher 3 months following ADS in children with MS (p b 0.001).
While several immune molecules previously implicated in established adult MS may represent non-specific markers of acute inflammation rather than early predictors of disease outcome, further study is warranted to confirm whether levels of osteopontin, Eselectin, and MMP-3 will serve as predictive biomarkers distinguishing MS from monophasic-ADS.
The switch between relapse and remission in multiple sclerosis: Continuous inflammatory response balanced by Th1 suppression Gurevich Michael ⁎ ,1 , Dolev Mark 1 , Magalashvili David 1 , Achiron Anat 2 1 Sheba Medical Center, Ramat-Gan, Israel; 2 Tel-Aviv University, Tel-Aviv, Israel
Multiple sclerosis (MS) is characterized in most patients by a relapsing-remitting disease course. However, the trigger of relapse and the transformation switch into remission are not clearly understood. We aimed evaluated key molecular pathways operating in MS relapse and in MS remission to better understand the associated repairing mechanisms.
Microarray gene-expression analysis (Affymetrix U133A-2 array) was applied to study peripheral blood mononuclear cells (PBMC) signatures of 123 relapsing-remitting MS patients: 89 patients in remission (age 36.18 ± 11.6 years, disease duration 7.1 ± 7.9 years, F: M 53:36, EDSS 1.8 ± 1.3), 34 patients in acute relapse (age 34.1 ± 8.4 years, disease duration 6.0 ± 4.1 years, F:M 25:9, EDSS 3.2 ± 1.3) compared with data from 41 healthy subjects (age 35.5 ± 8.8 years, F: M 21:20). PARTEK (www.partek.com) and Ingenuity software (www. ingenuity.com) were applied for statistical analysis of most significant genes (MIGs,) with p b 0.01 and fold change N2.0.
Differential gene-expression of 315 MIGs distinguished MS patients in relapse from healthy subjects, while only 97 MIGs differed between MS patients in remission and healthy subjects. MS relapse signature was characterized by suppression of genes related to the caspase-apoptotic pathway (FADD, FASL, caspase 3), activation of MMP9 that enhances macrophage penetration through the bloodbrain barrier and in accordance stimulation CXC-modulated inflammation (CXCL8) known to recruit macrophages and neutrophils. In concert with this intensified inflammatory response, we identified suppression of Th1 response (TBX21 and IFNg) indicating the initiation of remission. MS remission signature was characterized by a further suppression of Th1 response (TBX21 and CD58) associated with activation of negative regulators of NFkB dependent inflammation (TNFAIP3) and cytokine signaling (SOCS). There was still an ongoing over-expressed pro-inflammatory activity (MMP9, ICAM5, and CCL19) promoting lymphocyte chemotaxis and adhesion. Comparison between MS relapse and remission, demonstrated elevated levels of neurotrophic factors like BDNF and its downstream signaling molecules (TNR, NTRK3, and ARHGEF10).
MS relapse and MS remission constitute a continuous process where transformation switch occurs by imbalance between activation of CXC-modulated inflammation related to trafficking and adhesion that is more evident in relapse, and by Th1 suppression and neurotrophic activation that are more prominent in remission. Type I interferons (IFN) are crucial antiviral cytokines released in response to viral infection including herpes simplex virus type 1 (HSV-1). Studies have shown children unable to produce type I IFNs following HSV-1 brain infection are highly susceptible to HSV-1induced encephalitis. We sought to elicit the role of IFNs in the CNS following HSV-1 infection using mice lacking the alpha chain of the type I IFN receptor (CD118 −/− ).
Following ocular infection, we found a significant increase in viral burden in the cerebral cortices and brain stem (BS) of CD118 −/− mice 5 days post infection (pi) when compared to wild type (WT) controls. A lack of HSV-1 containment in CD118 −/− mice correlated with a decreased T (both CD4+ and CD8+) and NK cell infiltration into the BS as seen in WT mice. The loss of NK and T cell recruitment in CD118 −/− mice corresponded with a decrease in CXCL10 production. However, there was a significant increase in the expression of the chemoattractants CXCL1 and CCL2 in the BS of CD118 −/− mice that corresponded with an increase in macrophages (F4/80+, Gr1−, and CD45Hi). In order to evaluate blood-brain barrier integrity, matrix metalloproteinase 9 (MMP-9) levels were determined by ELISA and were significantly elevated in CD118 −/− as compared to WT mice in the brain and BS by day 5 pi. Using T1-and T2-weighted magnetic resonance images, we found a significant enlargement of the lateral ventricles in CD118 −/− mice by day 5 pi in comparison to WT counterparts.
In conclusion, the absence of type I IFN signaling results in rapid dissemination of the virus throughout the CNS resulting in gross morphological changes associated with an aberrant immune response. Consequently, we feel that this is an excellent model to dissect the contribution of cells and the soluble factors they elicit in response to viral pathogens and the ensuing neuropathology to better define viral-mediated encephalitis at the cellular and molecular levels.
Consistent detection of EBV RNA in immune infiltrates in the multiple sclerosis brain using combined laser capture microdissection and real-time RT-PCR Rosicarelli Barbara, Severa Martina, Coccia Eliana, Serafini Barbara, Aloisi Francesca ⁎ Istituto Superiore di Sanità, Rome, Italy An increasing number of epidemiological and immunological studies support an association between Epstein-Barr virus (EBV) infection and multiple sclerosis (MS). However, the mechanisms linking EBV infection to brain pathology are still unknown. The possibility that EBV establishes a persistent infection in the CNS and reactivates periodically bolstering an immunopathological response has been explored by our and several other groups with contrasting results. In particular, inability to detect EBV nucleic acids in whole MS brain sections could be due to absence/low frequency of CNSinfiltrating B cells (the main viral reservoirs) and/or limited sensitivity of conventional real-time PCR techniques. Using brain sections from MS cases with established B-cell containing immune infiltrates (tissue samples obtained from the UK MS Tissue Bank) we confirm inability to detect EBV latency and lytic transcripts using conventional real-time RT-PCR whereas selective cDNA pre-amplification allowed to detect EBV latency transcripts in samples with the highest content of B cells, as assessed by CD19 mRNA level. To increase the sensitivity of our assay, we next applied laser capture microdissection in conjunction with real-time RT-PCR. Selective cDNA pre-amplification allowed to perform a quantitative analysis of cellular (CD19) and viral (LMP2A, EBNA1, and BZLF1) genes even if the amount of starting RNA was very small. With these optimized techniques, LMP2A and EBNA1 mRNAs were detected in all immune infiltrates analyzed (perivascular cuffs from active and chronic active white matter lesions, meningeal infiltrates and B-cell follicles) whereas BZLF1 mRNA was detected in acute lesions and B-cell follicles, thus confirming our previous immunohistochemical findings ( Serafini et al., J Exp Med 2007; 204:2899) . No EBV transcripts were detected in laser-cut normal-appearing and lesioned white and grey matter parenchyma or B-cell follicles from a normal lymph node.
Without the sensitivity of laser capture microdissection and/or pre-amplification real-time PCR techniques we expect that detection of EBV nucleic acids would be unattainable in MS brain samples. Lipocalin 2 (Lcn2) is an iron-siderophore binding bacteriostatic factor that is produced during the host innate immune response to bacterial infection. However, it is unclear whether Lcn2 is expressed and has a function in the host response to viral infections. In addition to antimicrobial actions, Lcn2 may also regulate cellular iron metabolism and apoptosis. Little is known concerning the function of Lcn2 in the CNS host response, therefore we investigated the regulation and function of Lcn2 in the CNS following systemic lipopolysaccharide (LPS) injection or during West Nile Virus (WNV) encephalitis.
Although undetectable under physiological conditions, both Lcn2 mRNA and protein levels were highly induced by either LPS injection or WNV infection in choroid plexus epithelium, endothelial cells and microglia in the brain of C57BL/6 mice. Interestingly, Lcn2 protein but not mRNA was found to be present at high levels in subsets of neurons consistent with the uptake and accumulation of Lcn2 in these cells. Although the CNS expression of various cytokine, chemokine and other inflammation-associated genes was increased in the brain following systemic LPS injection, there was no significant difference in these or markers of iron-metabolism in WT control versus Lcn2 KO mice. However, compared with WT, Lcn2 KO mice showed significantly increased mortality following intranasal infection with a low dose of WNV.
1. Lcn2 is induced strategically at key gateways to the CNS in the host response to not only bacterial but also viral infection.
2. Lcn2 protein distribution in the brain after bacterial and viral infection suggests that neurons, which are the dominantly infected cell type with WNV, may be a key target for the actions of Lcn2.
3. While no clear functional role for Lcn2 was established in the LPS-induced inflammatory response in the brain, our data suggest that Lcn2 has an important protective function in WNV encephalitis. Malaria is an infectious systemic disease caused by the protozoa Plasmodium. Nitric oxide (NO), produced in large amounts by enzyme iNOS, has been considered to play a major role in malaria. However, NO effects are controversial in malaria pathogenesis. The present study aims to evaluate iNOS role in the cerebral and hepatic inflammatory response in mouse infected by Plasmodium berghei NK65.
All animals used were female C57Bl/6 mice, wild-type (WT) or deficient in iNOS (iNOS-KO), 6-8 weeks old. Mice were infected by intraperitoneal (i.p.) injection of 106 parasitised erythrocytes. Parasitemia levels were monitored on Giemsa-stained blood smears. Survival rate was analyzed during the infection. The leukocyteendothelial interactions in the brain and hepatic microvasculature were evaluated, by intravital microscopy, in wild-type, iNOS-KO and also animals treated (i.p.) with aminoguanidine (50 mg/kg), an iNOS inhibitor.
The parasitemia was significantly higher in iNOS-KO (n = 6) compared with wild-type group (n = 6) on 6°(iNOS-KO 29.86 ± 3.14%; WT 17.70 ± 3.35%) (p b 0.05) and 7°day post-infection (p.i.) (iNOS-KO 43.67 ± 7.21%; 24.07 WT ± 5,87) (p b 0.001). However the survival rate showed no significant difference between infected wildtype and infected iNOS-KO. Infected mice showed a significant increase in leukocyte rolling and adhesion in the brain microvasculature compared to control mice. However, infected iNOS-KO and infected wild-type treated with AG mice showed no significant difference compared to infected wild-type mice in the cerebral leukocyte recruitment. On the other hand, infected iNOS-KO exhibited a significant increase in leukocyte rolling (2.80 ± 0.73 rolling cells/ min) (p b 0.01) and adhesion (3.04 ± 0.2 adherent cells/100 μm) (p b 0.001) in hepatic microvasculature compared to infected wildtype (1.08 ± 0.22 adherent cells/100 μm) (0.50 ± 0.1 rolling cells/ min). Mice treated with AG had also presented a significant increase in the rolling (5.43 ± 1.7 rolling cells/min) (p b 0.05) and adhesion (2.00 ± 0.21 adherent cells/100 μm) (p b 0.05) compared with wildtype treated with saline (1,44 ± 0,43 rolling cells/min) (1.05 ± 0.23 adherent cells/100 μm).
The higher levels of parasitemia in the infected iNOS-KO mice indicate that nitric oxide can inhibit the proliferation of Plasmodium berghei NK-65. In addition, P. berghei NK-65 infection was able to induce leukocyte recruitment in the brain microcirculation. However, the absence or inhibition of iNOS enzyme only increased leukocyte rolling and adhesion in the hepatic microvasculature, suggesting an important role of iNOS in the peripheral inflammation after P. berghei NK65 infection.
Financial support: CAPES and FAPEMIG.
Bone marrow-derived cells are the primary source of CXCL10 regulation of antigen-specific CD8+ T cell recruitment to the CNS
The University of Oklahoma Health Sciences Center, Oklahoma City, United States
The chemokine CXCL10 is crucial for the control of viral replication through the regulation of mobilization of antigen-specific T cells to sites of infection. CXCL10 is highly expressed both at sites of inflammation as well as constitutively within lymphoid organs by both bone marrow (BM)-derived and non-BM derived cells. However, the relative immunologic importance of CXCL10 expressed by these divergent sources and at differing anatomic sites is unknown.
Using mouse chimeras reconstituted with either wild-type or CXCL10 deficient bone marrow (BM), we show that BM-derived cells were the dominant source of CXCL10 within the draining lymph nodes and spleen following herpes simplex virus type-1 (HSV-1) infection but not in non-lymphoid organs. CXCL10 expressed by BMderived, radiation sensitive cells regulated the mobilization of HSV-1 specific CD8+ T cells into the CNS and provided the dominant contribution of CXCL10 towards control of virus.
Our results establish the site-specific contribution of leukocyte expressed CXCL10. The study also challenges the view that this chemokine functions through simple recruitment of leukocytes along a concentration gradient established by CXCL10 production by non-BM derived, resident cells at sites of inflammation.
Whilst the contribution of microglia to pathogenetic pathways associated with neuroinflammation has been the focus of intense investigation, that of astrocytes has been largely ignored. It is generally assumed that astrocytic reactivity is secondary to the microglial one, despite rapidly accumulating evidence of the high capability of these cells for instantaneous and robust responses to changing conditions, as well as for communication with other central nervous system cell types. Therefore, in order to improve the mapping of effector pathways triggered in neuroinflammatory conditions such as multiple sclerosis (MS), we have investigated the timing and nature of astrocytic reactivity in relation to microglial reactivity in the experimental autoimmune encephalomyelitis (EAE) MS model. This was performed in the NOD/Lt mouse with myelin oligodendrocyte glycoprotein 35-55 peptide as neuroantigen and evaluated by confocal microscopy, from pre-clinical stages. Astrocytic reactivity, as evidenced by glial fibrillary acidic protein (GFAP) is observed from the time of earliest meningeal accumulation of inflammatory cells, extending across both grey and white matter and manifested by different morphological changes in the two compartments. It is most prominent around blood vessels and neuronal cell bodies. Quantification of GFAP shows a significant increase over controls. The above findings overlap, but are not identical with those of microglia. Coincident with the earliest parenchymal invasion, direct contact with T cells, identified by the marker CD3, can be seen. This occurs at least in two forms, firstly as interactions between T cells and astrocytic processes, as protrusions which appear to interlock and secondly, as intimate associations between cell bodies. In the latter case, 3-D reconstructions demonstrate that contact involves a substantial area of both cell bodies, where T cells become enfolded by astrocytes which undergo considerable morphological change. Current investigations address the question as to whether interactions between astrocytes and T cells are restricted to sampling of the environment as part of the homeostatic role of astrocytes, or are evidence of the formation of immunological synapses between these cell types, or both.
These data demonstrate pre-clinical astrocytic reactivity in parallel with that of microglia and have implications for the development of therapies for neuroinflammation.
With the accumulation of data from diverse high throughput platforms, the analysis of each dataset independently is only part of the solution. Following the axiom that the whole is more than the sum of its parts, the hypothesis that much knowledge about the pathogenesis of complex diseases is to be gained thorough the systematic and creative combination of omics results can now be empirically tested. In this presentation I will discuss new ways of integrating genomics, gene expression, and imaging data in a large cohort of MS patients and controls. I will also present analyses on the genetic similarities and differences between MS and other complex diseases, using an original network-based approach.
Meaningful data integration will become the next frontier in the analysis of complex datasets.
What have we learned from the other omics: Transcriptomics, proteomics, lipidomics, as compared to genomics
Stanford University, CA, USA
I shall compare what we have learned from the 'other omics' to what we have learned from GWAS studies to date. Obviously we need 'all the omics' to understand MS. Resource allocation however is key in these times when research budgets are shrinking. I shall argue the case for increased funding at this point for the 'other omics' besides genomics.
Brain-blood barrier regulation of brain inflammation Chairs: A. Prat and Elga de Vries 651 Novel adhesion molecules involved in the preferential migration of leukocyte sub-sets to the CNS
The blood-brain barrier (BBB) protects the central nervous system by regulating molecular and cellular exchanges between the brain and the blood. The BBB is made of a network of tightly adherent endothelial cells (ECs) surrounded by astrocytic processes which provide factors that contribute to BBB maintenance. Several proteomic based-profiling of human and animal BBB endothelial cells revealed the presence of unique regulatory proteins involved in BBB physiology and transendothelial leukocyte migration. We and others found that human BBB-EC specialized membrane microdomains (lipid rafts) are enriched for proteins involved in cellular adhesion, cell structure, BBB development, immunity and defense, transport and trafficking and signal transduction. Our recent work, using animal models of MS and spinal cord contusion, as well as human in vitro, in situ and ex vivo analyses revealed that these new BBB candidate proteins, including ALCAM, the Hedgehog pathway, ninjurin-1 and MCAM are involved in the regulation of immune cell trafficking across vascular structures of the CNS. More importantly, while ALCAM-CD6 interactions regulate the trafficking of CD4 lymphocytes and monocytes-macrophages, it does not impact on the recruitment of CD8 lymphocytes. Conversely, expression of ninjurin-1 is restricted to the cells of the myeloid lineage and regulates the recruitment of monocytes and DCs to the CNS. Finally, MCAM (CD146) is expressed primarily on TH17 lymphocytes and is highly expressed within active MS and EAE lesions. Our recent data, based on proteomic analysis of human BBB-associated lipid raft membrane microdomains, identified novel adhesion molecules involved in the recruitment of specific immune cell subsets to the CNS.
Role of ABC transporters in neuro-inflammatory events at the blood-brain barrier Background and goals: At the blood-brain barrier (BBB), the ATP binding cassette (ABC) transporters drive cellular exclusion of a variety of compounds, thereby protecting the brain from neurotoxic compounds. Recently it is described that ABC transporters may also be involved in removal of inflammatory agents from immune cells. In a variety of neuroinflammatory disorders including multiple sclerosis (MS), a defective function of the BBB has been described, based on structural differences. However, lowered expression of ABC transporters at the BBB may lead to enhanced exposure of the brain to inflammatory mediators, thereby aggravating the inflammatory process at the vasculature. Methods and results: We therefore set out to study the regulation of ABC transporters at the BBB in MS using our well-defined post-mortem patient material. Dominant loss of P-glycoprotein (P-gp: ABCB1) was detected in active MS lesions. Moreover, we have identified that the interaction of activated T-cells with brain endothelial cell cultures leads to a reduction of P-gp expression and function through regulation of the NF-kB signalling route (Kooij et al., J Autoimmun 2009). Our data therefore indicate that in MS not only at the structural level of the bloodbrain barrier alterations occur but that also the endothelial efflux barrier properties are affected, exposing the brain to higher levels of inflammatory agents. Thursday October 28 th , 2010
Concurrent Symposia Strikingly, in MS lesions, reactive astrocytes start to express a number of these ABC transporters including multi-drug resistance protein-1 (MRP-1) and P-gp. Expression was not solely localized to the astrocytic endfeet contacting the vasculature but was found distributed along the whole cell body. Importantly, in vitro cultures of primary human astrocytes derived from MS lesions were found to have enhanced function of MRP-1 and P-gp compared to astrocytes isolated from non-neurological controls. In vitro data using human astrocytes further revealed that both efflux pumps are involved in the secretion of monocyte chemoattractive protein 1 (MCP-1/CCL-2), which was found to act as the leading chemoattractant for monocyte recruitment across the BBB. Blocking the activity of astrocytic ABC transporters reduced cellular migration across the BBB in vitro. Conclusions: Together our data emphasize that ABC transporters have a modulatory role during inflammation at the BBB.
A role for leukocyte trafficking mechanisms in the pathogenesis of epilepsy
A seizure is a paroxysmal hypersynchronous discharge from central nervous system (CNS) neurons. Repeated seizures can lead by unknown mechanisms to epilepsy, a chronic neurological disorder that affects 1 percent of the world population. Leukocyte recruitment is a hallmark of and a point of potential therapeutic intervention in tissue inflammation. Our results show that seizures induce elevated expression of vascular adhesion molecules and enhanced leukocyte rolling and arrest in brain venules mediated by the leukocyte mucin Pselectin glycoprotein ligand-1 (PSGL-1) and integrins alpha4beta1 and alphaLbeta2. Interestingly, intravital microscopy studies show that Th1 and Th17, but not Th2, cells are preferentially adhere in inflamed brain venules after seizures, suggesting that adaptive immunity may have a role in epilepsy. Inhibition of leukocytevascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduces seizures and leads to a significant decrease in neuronal cell loss. Vascular leakage, which is known to enhance neuronal excitability, is induced by acute seizure activity and is prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, vascular damage and seizure generation.
Leukocyte motility behavior inside CNS parenchyma during neurological diseases and how leukocytes interact with neural cells is largely unknown. Adhesion molecules able to bind leukocyte counterreceptors have been previously shown to be upregulated on neural cells under inflammatory conditions, suggesting potential adhesive interactions between immune cells and neural cells. Leukocyte trafficking followed inside CNS parenchyma by using two-photon microscopy show that Th1 and Th17 cells migrate inside brain parenchyma after seizures. Particularly, Th17 cells display enhanced ability to penetrate inside brain parenchyma and exhibit remarkable motility aptitude and capacity to interact with neural structures after seizures.
In conclusion, the identification of the molecular mechanisms controlling vascular inflammation phenomena and leukocyte motility behavior and cell-cell contacts with neural cells inside CNS parenchyma in experimental models of epilepsy will open new avenues to the understanding of neural damage and immune responses during epilepsy and will help to identify new therapeutical approaches able to complement existing anti-epileptic drugs.
Carman Christopher V.
Inflammation challenges the integrity of the endothelium through the formation a micron-scale discontinuities that could be defined as 'micro-wounds', which require healing in order to maintain homeostasis. Micro-wounds are produced either as para-cellular gaps resulting from endothelial contraction in response to inflammatory mediators (e.g., histamine and thrombin) or as either para-cellular gaps or trans-cellular pore generated during the diapedesis of inflammatory leukocytes. Mechanisms for pore/gap resolution (i.e., micro-wound healing) remain poorly characterized. Here we show for the first time that lamellipodium-like structures, which are formed explicitly on the ventral aspect of the endothelium, rapidly reseal pores/gaps and precede either pore constriction or re-annealing of adherence junctions. The leading edge of these novel ventral lamellipodia-like structures (VLS) exhibited strong enrichment of actin, cortactin, NADPH oxidase subunit p47phox and hydrogen peroxide. Small molecule and dominant negative constructs demonstrate that VLS require signaling by Rac, cortactin and to a lesser extent Cdc42, but not RhoA. Furthermore, Nox-specific inhibitors and ROS scavengers also robustly and reversible inhibited VLS formation. These findings define Rac1-and reactive oxygen species-dependent VLS activity as critical determinants for maintenance and recovery of endothelial integrity. Immunotherapy for high grade glioma (HGG) is an innovative treatment approach that entered into clinical practice with promising perspectives. Understanding of the mechanisms of the immune responsiveness will open strategies to improve the antitumor immunotherapeutic effects. Clinical observations in well-designed clinical trials add evidence to make immunotherapy part of the standard treatment approach for patients with HGG.
The orthotopic GL261 mouse model was used to unravel the immune responses upon DC vaccination. DC was loaded with tumor antigens from GL261 cells. We demonstrated the role of effector, memory and regulatory T (Treg) cells, and found in the tumor an enrichment of myeloid-derived suppressor cells. However, these MDSC were not suppressive in case of Treg depletion prior to tumor inoculation.
Treg were observed also in patient samples. The Foxp3 expression in the infiltrating lymphocytes correlated with CD127dim expression, and was demonstrated to be functionally active Treg.
In clinical practice, we used autologous mature Dendritic Cells loaded with autologous tumor lysate: DCm-HGG-L. Boost vaccines were performed with HGG-L. Patients with relapsed HGG were treated in the cohort comparison trial HGG- IMMUNO-2003. 117 adults with relapsed HGG have been grouped according to an adapted RPA classification reflecting integrated risk factors. The PFS and OS for the different classes were significantly different. Two-year surviving adults were observed in classes I-IV patients. The treatment was feasible without major side effects.
Next, immunotherapy was integrated into the multimodal primary treatment in 8 pilot adults. Median PFS and OS were 17.8 m and 24.3 m with a 2-year OS of 50%. Subsequently, 78 patients with first diagnosis of HGG grade IV were enrolled in HGG-2006. Their median PFS and OS were 9.5 m and 20.7 m, with 6-m PFS of 70.5% and 2-year OS of 42.8%. These patients were per definition RPA classes III-V. The additive value of immunotherapy compared to the historical control was significant for classes III and IV patients. The therapy was feasible without major side effects.
Preclinical research continuously supports strategies to improve immunotherapy. Its efficacy should be confirmed in a prospective randomized double blind clinical trial HGG-2010. Approaches for further immune modulation should be looked for, based on the insights obtained in the preclinical models. Glioblastoma (GBM) remains uniformly fatal, and conventional therapies are limited by non-specific damage to normal brain or systemic tissue. The inherent biologic specificity of immunologic reactivity could meet the clear need for more specific and precise therapy. However, immunotherapy has been thwarted by a lack of tumor-specific targets and an inability to generate potent anti-tumor immune responses in the context of a profoundly immunosuppressive host environment. Over the years, our work has addressed both of these limitations.
Towards that end, we and others have identified several potential targets -EGFRvIII, mutant IDH1 and the antigens of Cytomegalovirus. Our peptide vaccines targeting EGFRvIII have led to nearly universal generation of EGFRvIII-specific immune responses, eradication of EGFRvIII-expressing tumor cells and an apparent prolongation in progression-free and overall survival. IDH1 mutations have not been targeted immunologically but represent a parallel paradigm with the exception that this mutant protein is not expressed on the surface of tumor cells. This and the predilection for lower grade gliomas for this mutation may have significant implications for approaching this mutation immunotherapeutically. We and others have now reproducibly confirmed the presence of multiple CMV antigens -pp65, IE1, and GBin almost all patients with GBM. We have now conducted two clinical trials targeting these antigens in patients with GBM. In these trials we have been able to increase immune responses to pp65 based on tetramer analysis, but the induction of polyfunctional T cell responses has proven difficult as patients with GBM appear to have immunologic deficits specific to pp65.
We believe that the general specific immunologic deficits that can be identified in patients with GBM are a result of multiple immunosuppressive pathways. We have shown that regulatory T cell levels are significantly elevated in patients with glioblastoma, and the level of CD4-positive T cells is dramatically diminished. More recently, we have identified a dramatic ability to enhance this immune response with temozolomide-induced lymphodepletion, but have also seen that temozolomide actually increases regulatory T cell levels. We have shown in both animal and human studies that CD25 antibodies can deplete regulatory T cells and these results are associated with dramatic prolongations in PFS patients with GBM.
The virtues and vices of youth: Age, immunity, gene expression and malignancy in glioma High-grade glioma (HGG) outcomes have not improved substantially over the last 25 years, despite advancements in non-CNS tumors. Novel research and clinical paradigms are thus needed to develop better HGG therapies. Patient age outweighs other metrics in predicting survival, treatment sensitivity, and prognostically favorable gene expression patterns in gliomas. We previously showed that age-dependent outcome and chemosensitivity in human HGG are linked to levels of age-sensitive CD103 + CD8+ T cells, which dominate anti-tumor responses following dendritic cell (DC) vaccine therapy. Since age-dependent outcome and chemosensitivity are linked to patterns of gene expression, we examined the role of anti-tumor T cells generally, and CD103 + CD8+ T cells in particular, in glioma gene expression and malignancy. We found that nascent CD103 + CD8+ T cell levels correlated inversely to the worst prognostic group (mesenchymal) of the 3 major subclasses of HGG, and that immune responsiveness in DC vaccinated patients abrogated age-dependent survival, a property conferred by a more favorable prognostic group (proneural). This raised the possibility that T cell activity might directionally alter subclass-specific gene expression in gliomas. Consistent with this, HGGs subjected to standard therapy up-regulated mesenchymal genes, whereas those subjected to DC vaccination up-regulated non-mesenchymal genes. In direct support of directional alteration of glioma gene expression by CD103 + CD8+ T cells, GL26, a murine HGG model, exhibited selective down-regulation of mesenchymal genes, immune resistance, and invasiveness upon exposure to wild-type but not CD103deficient T cells. This was linked to down-regulation of a subset of master transcriptional activators of mesenchymal genes in GL26. Moreover, proliferative subclass genes were up-regulated in GL26, as was chemosensitization, upon exposure to endogenous CD8+ T cells, whereas distinct genes and stem-like properties were enhanced upon exposure to vaccine-induced anti-tumor CD8+ T cells. Thus, favorable as well as potentially unfavorable changes in age-related gene expression and associated phenotypes were elicited by different strengths of anti-tumor T cells against gliomas. Further elucidation of the master regulators of such genetic programming should help optimize therapeutic synergy, enhance vaccine responsiveness, and increase benefits to HGG patients in the context of immunotherapy. Three distinct inflammatory myopathies are currently distinguished: dermatomyositis (DM), inclusion body myositis (IBM), and polymyositis (PM). The infiltrates in DM muscle contain B cells, plasma cells and plasmacytoid dendritic cells. The pathogenetic mechanism of perimysial atrophy, the pathological hallmark of DM, remains controversial. One hypothesis assumes that endothelial antibody and/or immune complex deposition cause capillary injury, leading to perimysial atrophy. Another hypothesis is based on the observation that several type-1 interferon-induced genes, e.g., interferon-stimulated gene 15 (ISG15), are strongly upregulated in DM muscle, triggering a cascade of pathogenic changes. In IBM and PM, the mechanisms of muscle fiber injury seem to be different from DM. IBM is the most common inflammatory myopathy in adults, whereas "pure" PM is rare. In PM and especially IBM, clonally expanded, CD8-positive T cells invade nonnecrotic muscle fibers which strongly express MHC class I antigens.
Using laser-assisted microdissection, putatively pathogenic autoimmune CD8+ T cells were "resurrected" from inflammatory lesions of muscle biopsy tissue. The revived T cells can be used as probes for searching their unknown target antigen(s). In the special case of gamma-delta T cell-mediated myositis, this strategy recently allowed the molecular identification of a novel autoimmune target "motif" which is expressed on several proteins of the translational apparatus, including several aminoacyl-tRNA synthetases.
Plenary Symposium -Stem cells at the crossroad of neuroimmunology Chair: G. Martino
The role of neural stem cells in brain regeneration
Inflammation and degeneration are the usual pathological processes occurring in the central nervous system (CNS). They are only apparently distinct process because as soon as the pathological process becomes chronic they have the tendency to become strictly interrelated. As such, primary neurodegeneration triggers a secondary inflammatory reaction while primary inflammatory reactions lead to neurodegenerative phenomena. Several molecular and cellular events sustaining intrinsic brain repair mechanismsoccurring within the CNS as a consequence of chronic inflammatory and/or degenerative processeshave been described so far. They can be divided into three distinct categories: inflammation-driven processes, CNS plasticity and neuro(glio)genesis. By one hand, humoral and cellular inflammatory components shift sense (function) over time from a tissue-damaging mode to a mode promoting tissue repair (e.g. neurotrophic support from inflammatory cells). By the other hand, the recruitment of alternative "non-damaged" functioning neuronal pathwaysoccurring mainly via axonal branching and synaptogenesistakes place as a consequence of brain damage. Whether or not (and to what extent) the recapitulation of precise developmental pathways underlies the whole phenomenon of brain plasticity is still a matter of investigation. Finally, endogenous neural stem/precursor cells (NPCs)the self-renewing and multipotent cells of the CNS capable of driving neurogenesis and gliogenesis in adult lifemay adapt targeted migration into damaged areas and promote repair via several mechanisms of action (e.g. neuro and gliogenesis, immunomodulation, and neuroprotection). Recent evidence suggests that these intrinsic repair mechanisms are only apparently distinct processes. Immunological, developmental and cellular events aiming at restoring tissue integrity within the CNS are strictly interrelated and NPCs are at the cross road of these interrelated phenomena. Understanding the relationship between repairing mechanisms and the reason why they fail over time in chronic CNS disorders, can be considered as a way of developing alternative therapeutic approaches promoting brain repair.
Mesenchymal stem cells for brain repair: Does neural differentiation really matter?
Uccelli Antonio ⁎
Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated and expanded ex vivo from many adult tissues including the bone marrow and can differentiate into mesodermal tissues. Despite some potentiality for transdifferentiation recent data suggest that MSCs therapeutic potential is due mostly to significant paracrine effects and cell-to-cell interactions. MSCs have been shown to interact with cells of both the innate and the adaptive immunity and modulate their function. Upon intravenous administration in experimental autoimmune encephalomyelitis, MSC rapidly leave the blood flow and migrate into the lymph nodes and the injured central nervous system where they engraft for a short time and rapidly vanish. Despite this fast clearance from tissues, MSC administration induce an immediate state of immune tolerance and exert a significant neuroprotective effect promoting survival of damaged cells through paracrine mechanisms. This therapeutic plasticity is sustained by striking immunomodulatory, anti-oxidant, trophic and anti-apoptotic activities with no evidence of differentiation into neural cells. Due to these striking therapeutic features, the relative simplicity of growing them in vitro and their relative safety arising from hemato-oncological studies, MSCs have been chosen for an incoming international for the treatment of multiple sclerosis patients refractory to conventional therapies.
Immunology of the brain: Intracranial immune cells are part of the functioning brain and link physiology to pathology
The Weizmann Institute of Science, Rehovot, Israel
Background: For decades, the central nervous system (CNS) was viewed as an autonomous unit preserved behind a wall, and shielded from immune cells and from pathogens present in the circulation. Accordingly, the local inflammation seen in brain pathologies has been considered as a major contributor to disease progression, which should be mitigated. Therefore, the conundrum is that systemic antiinflammatory drugs fall short in most neurodegenerative diseases. Results: We found that the CNS is critically dependent on circulating immune cell support (provided by monocytes and self-reactive T cells) for its normal functioning and repair. Breakdown of the dialogue between the brain and circulating immune cells may occur at any level, starting from the immune cell composition in the circulation, the nature and the number of immune cells recruited cells to the borders of the CNS, the phenotypes acquired by the recruited cells, and their location and time of arrival within the CNS territory; defects in these processes can impact cognitive performance, resilience to stress, emergence of developmental neuropsychological disorders, onset and progression of neurodegenerative diseases, and repair following acute injury. This concept unifies the role of immune cells in the healthy brain (as manifested by their function in supporting neurogenesis, expression of brain-derived neurotrophic factors, expression of growth factors, and of synapse-associated proteins), and under pathological conditions, as well as explaining their role in protective autoimmunity. We further propose, based on experimental evidence that the dialogue between the CNS tissue and the circulating immune cells occurs in the healthy brain in discrete compartments within the brain's territory, but not within the parenchyma, and is mediated by a selected subpopulation of circulating immune cells that operate under the control of the brain's milieu. Conclusions: These findings introduce new principles of brain immunology that were not hitherto acknowledged, call for re-definition of the brain as an immune privileged site, and suggest potential immune-based means of intervention in supporting recovery from injury, and coping with stress, depression, aging or neurodegenerative conditions. Schwartz and Shechter, Mol Psychiatry. 2010 15:342, Schwartz and Shechter, Nat Rev Neurol. 2010 Jun 8.
Brain-blood barrier 444 A4-Integrin-independent mechanisms are involved in initiating T cell interaction with the blood-brain barrier during experimental autoimmune encephalomyelitis in vivo Sathiyanadan Karthik ⁎ , Coisne Caroline, Engelhardt Britta
In multiple sclerosis (MS), and in its animal model, experimental autoimmune encephalomyelitis (EAE), circulating immune cells gain access to the central nervous system (CNS) and cause inflammation, blood-brain barrier (BBB) breakdown and demyelination. Immune cell recruitment across the BBB has been recognized as a major pathophysiological hallmark of MS. In vitro and in vivo studies highlighted the predominant role played by the -integrins in T cell trafficking into the CNS.
Direct visualization of the multistep interaction of T cells with the non-inflamed spinal cord white matter microcirculation by means of intravital fluorescence videomicroscopy (IVM) allowed us to demonstrate that T cell interaction with the healthy BBB is unique due to a lack of rolling and the predominant involvement of integrins in mediating the G-protein independent capture and subsequently the G-protein dependent adhesion of encephalitogenic T cells to spinal cord microvascular wall. In contrast, during EAE we saw that although -integrins are still critically involved in mediating T cell adhesion to the inflamed spinal cord microvessels, -integrins are no longer required for initiating T cell interaction with the inflamed spinal cord microvasculature, which can now be initiated by rolling or capturing. Thus, during EAE additional adhesion mechanisms become available on the inflamed BBB mediating this initial step.
To define the -integrin independent mechanisms involved in T cell capturing and rolling on the BBB during EAE, we observe T cell interaction with the inflamed spinal cord microvasculature during EAE by IVM in the functional absence of a number of different adhesion molecules such as P-selectin and PSGL-1. Our data will allow to delineate the integrin independent molecular mechanisms involved in T cell capturing and rolling on the inflamed BBB during EAE in vivo.
Evaluation of vascular adhesion protein-1 (VAP-1) expression in multiple sclerosis brain and its involvement in disease pathogenesis in EAE Airas Laura ⁎ ,1 , Langen Barbara 2 , Dost Rita 2 , Österman Thua 2 , Röyttä Matias 1 , Smith David 2 1 Turku University Hospital, Turku, Finland; 2 Biotie Therapies Corp., Turku, Finland
Two putative routes exist for the entry of lymphoid cells into the CNS. The initial contact of the immune system with the CNS has been suggested to take place via the vessels of the choroid plexus, whereas the second wave of activated T cells likely enters via stimulated cerebral blood vessels.
Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule capable of mediating lymphocyte binding and migration through, the vascular vessel wall at sites of inflammation.
The goal of this study was to evaluate the expression of VAP-1 in inflamed human brain, and its role in experimental autoimmune encephalomyelitis in the mouse.
Expression of VAP-1 in multiple sclerosis (MS) brain and in control brain was studied by immunohistochemistry. Frozen tissue sections of post-mortem brain from seven MS patients and from four control patients were obtained from the UK Multiple Sclerosis tissue bank. These sections were stained for the expression of VAP-1, VCAM-1, CD31 and CD3. The level of VAP-1-expression was evaluated by counting the number of VAP-1-expressing vessels in different microscopic fields (size of field = 1 mm 2 ). The mouse model of MS, experimental autoimmune encephalitis (EAE), was used to study the functional role of VAP-1 in the development of CNS inflammation.
All of the studied MS patients had SPMS, and the lesions under examination were chronic active lesions. Strong VAP-1 expression on meningeal and choroid plexus vessels was seen both in MS brain and in control brain. In MS brain parenchyma, the range of VAP-1+ vessels was 4.8-14/mm 2 , and in control brain it was 3.6-5 vessels/mm 2 . On average there were 9.3 VAP-1+ vessels/mm 2 in MS brains, whereas in the control brains the density of VAP-1+ vessels was 3.6/mm 2 . This difference was statistically highly significant (p b 0.0001). In EAE, targeting of VAP-1 with a VAP-1 antibody reduced disease activity.
VAP-1 was strongly expressed on choroid plexus and meningeal vessels both in MS brain and control brain, but in MS brain parenchyma the prevalence of VAP-1-expressing blood vessels was increased compared to control brain. The data from EAE experiments suggest that VAP-1-function might contribute to the immunopathogenesis of MS.
Inflammation mediated changes in blood spinal-cord barrier (BSCB) permeability following sciatic nerve injury Echeverry Stefania ⁎ , Wu B.Y., Zhang Ji
Peripheral nerve lesion triggers alternations in the spinal cord microenvironment that contribute to the pathogenesis of neuropathic pain. While neurons and glia have been implicated in these functional changes, it remains largely unexplored whether the blood-spinal cord barrier (BSCB) is also involved in the pathological events and its potential roles in neuropathic pain development. The goal of this study is to examine the functionality of BSCB following peripheral nerve injury, and possible underlying mechanisms, consequences of such changes in BSCB.
Alterations in BSCB permeability were examined 3, 7, 14 and 60 days after partial ligation of the left sciatic nerve using Evans Blue (EB) as index. EB content within the spinal cord parenchyma was significantly increased 3 and 7 days after nerve injury. This increased permeability was confirmed by the leakage plasma proteins, IgG and fibronectin, into the spinal parenchyma. MCP-1 is an important neuron-to-glia signaling molecule in the spinal cord after nerve injury. Intrathecal administration of MCP-1 (2.5 μg) evoked an increase of EB in the spinal parenchyma in naive animals, similar to that seen in animals at 3 days post-injury. Intrathecal treatment with antiinflammatory cytokine TGF-b1 (2 μg) for 3 days blocked the EB extravasation induced by peripheral nerve lesion. To further explore the role of inflammatory mediators on BSCB function we injected systemically IL-1b in naïve rats. We observed a dose dependent increase of EB extravasation that was significant after an intravenous injection of 2 ng of the cytokine. In addition, we observed that low Poster Sessions dose (b2 ng) of IL-1b in the circulation was able to cross the compromised BSCB associated with nerve injury, which provided a new pathway for neuroimmune interaction. To assess the structural integrity of the BSCB, the levels of tight-junction protein expression were also evaluated using western blot. Finally, mechanical and thermal hypersensitivities developed after nerve injury was assessed for all time points (day 0-day 60) and there is a close temporal correlation between BSCB disruption and the initiation of neuropathic pain.
Our results suggest that sciatic nerve injury impaired the permeability of the BSCB. This phenomenon was most likely mediated by nerve injury associated inflammatory response. Our findings highlight the importance of inflammation in BSCB function and the potential consequences of BSCB disruption in the development of neuropathic pain. Cerebral microvascular endothelial cells (CMECs) constitute the physiological barrier of the central nervous system (CNS). A number of transmembrane proteins such as occludin and claudin-5 are integral constituents of tight junctions (TJs) in CMECs and restrict the free passage of molecules from blood to brain. Blood-brain barrier (BBB) dysfunction as a result of alterations in the CMEC phenotype is a major hallmark of neuroinflammatory diseases such as multiple sclerosis. Cellular factors released by activated leukocytes and/or brain-resident cells (e.g. TNFa, IFNg) alter TJ organization leading to increased BBB permeability of CMECs and facilitate leukocyte infiltration into the CNS parenchyma. Here, we investigated the changes in the pattern of gene and microRNA (miR) expression induced by inflammatory stimuli in an immortalized human cerebral microvascular endothelial cell line, hCMEC/D3. Transcriptome analysis was performed using an Illumina human v8 microarray. Under basal conditions,~11,000 transcripts were indentified in hCMEC/D3 cells. Treatment with TNFa and IFNg increased mRNA levels of 464 genes N2 fold, with the highest increases (N30 fold) observed for chemokines (CCL2, CCL5, CCL8, CXCL8, CXCL9 and CXCL10) and adhesion molecules (VCAM1), and decreased mRNA levels of 242 genes N2 fold, many of them associated with regulation of TJs. Cytokine-induced changes in mRNA levels correlated with: 1) an increased endothelial permeability associated with decreased levels of occludin and claudin-5, and 2) an increased adhesion of the monocytic THP1 and the T cell Jurkat cell lines to hCMEC/D3 monolayers. We then hypothesised that miRs may be involved in the fine tuning of the brain endothelial inflammatory response. The miR expression profile of cytokine-activated hCMEC/D3 was analysed using an Agilent v13 microarray identifying 10 miRs up-regulated and 118 down-regulated at different time points following the inflammatory stimuli.
Alterations in miR levels might play important role in regulating the CMEC phenotype, ultimately leading to BBB breakdown. We are currently investigating whether modulation of miR levels by transfection with specific pre-miRs or anti-miRs affect BBB permeability and/or leukocyte adhesion and migration. Understanding the molecular mechanisms leading to cytokine-induced BBB breakdown might constitute an important strategy in the development of new therapeutic targets for neuroinflammatory disorders.
Nascent multiple sclerosis lesions are associated with early disturbances in the blood-brain barrier Alvarez Jorge ⁎ , Godschalk Alisha, Terouz Simone, Prat Alexandre
Early changes in normal appearing white matter of multiple sclerosis (MS) patients precede the appearance of gadolinium-enhancing lesions on brain magnetic resonance imaging (MRI) scans. Although these MRI studies suggest the breakdown of the blood-brain barrier (BBB) as an important feature in MS pathogenesis, limited information is available on the pathological changes occurring at the BBB during lesion genesis. Hence, this work was designed to study the differential changes of BBB components, particularly during the early stages of lesion formation.
MS lesions from n = 10 donors were categorized as nascent, active, chronic active and chronic inactive. Sections were immunostained for distinct cellular markers (CD3, CD68 and GFAP) and BBB disruption was determined by evaluating the expression pattern of fibrinogen, laminin, GFAP and the junctional proteins occludin, claudin-5, ZO1, VE-cadherin, a-catenin and p120-catenin. In addition, to correlate BBB disruption and endothelial activation, the expression of the cell adhesion molecules (CAMs) ICAM-1, VCAM-1 and ALCAM was studied. Nascent lesions displayed moderate leukocyte infiltration, very limited demyelination, moderate gliosis, discrete basement membrane proteins abnormalities, considerable changes in the expression of junctional proteins and increased expression of CAMs. BBB junctional proteins and basement membrane components were more severely disrupted in active lesions, as compared to nascent lesions and the expression pattern of most junctional components was partially reestablished in chronic active and inactive lesions.
Our pathological findings indicate the presence of early disturbances in the expression of junctional, CAMs and basement membrane proteins at the level of the BBB in non-demyelinating MS specimens. These early vascular changes coincide with perivascular immune cell infiltration and bring pathological support for important BBB disruption in the first stages of lesion formation in MS and prior to demyelination.
Netrins regulate blood-brain barrier function Podjaski Cornelia ⁎ ,1 , Lebeurrier Nathalie 1 , Antel Jack P. 1 The microvasculature of the central nervous system is very complex and highly structured in order to maintain proper brain function. This specialised endothelium generates a highly selective and dynamic physical blood-brain-barrier (BBB). Only a small number of factors that regulate barrier function have been identified. Several studies show that netrins are expressed by endothelial cells and contribute to the embryonic development of the vasculature. We hypothesised that netrins, best known as axonal guidance cues, are expressed by the endothelial cells (ECs) of the human adult BBB and govern regulatory functions on the BBB.
Screening of primary human brain-derived endothelial cells (BBB-ECs) for netrin and netrin receptor expression by quantitative real time PCR demonstrated that BBB-ECs express multiple netrin and netrin-receptors. Netrin expression was regulated differentially by barrier-promoting (astrocytes) or -destabilizing conditions (tumor necrosis factor alpha). Netrin expression was confirmed by western blot and immunochemistry and was co-localized to the microvasculature of adult human brain sections.
The functional significance of netrin at the BBB was determined by characterizing the expression of tight junction molecules and evaluating the diffusion of tracer-molecules using an in vitro transwell model of the BBB. Netrin-treatment promoted a functional decrease in BBB-permeability and an upregulation of tight junction molecules.
In summary, netrins and their receptors are expressed by the specialized brain microvasculature, regulating junctional complexes and controlling blood-brain barrier permeability. We propose that netrin is a novel BBB regulatory molecule with importance to the maintenance of a properly functioning BBB.
Role of angiotensin II receptors in controlling BBB integrity and macrophage entry to the brain Füchtbauer Laila, Groth Rasmussen Maria, Toft-Hansen Henrik, Khorooshi Reza, Owens Trevor ⁎
The blood-brain barrier (BBB), a complex of endothelial and glial barriers, controls passage of cells and solutes between the blood and the central nervous system (CNS). We were interested whether the renin-angiotensin-system is involved during BBB breakdown.
Expression of type 1 angiotensin receptor AT1 by astrocytes increased in the hippocampus dentate gyrus of C57BL/6 mice, after stereotactic transection of entorhinal afferents. Systemically-applied candesartan, which blocks the AT1 receptor, led to increased macrophage infiltration to the dentate gyrus 1-2 days after axonal transection. T-cell infiltration, which is known to increase after lesion, was not affected by AT1 blockade. There was no discernible effect on microglial or astroglial response and no induction of BBB breakdown (horseradish peroxidase (HRP) leakage). Changes in expression of the chemokine CCL2, which is known to control leukocyte entry in this system, did not correlate with increased macrophage entry.
We also studied the type 2 angiotensin receptor AT2, using two models of brain inflammation, to distinguish solute versus cellular barrier functions. Both leukocytes and HRP accumulated in the perivascular space of transgenic mice expressing the chemokine CCL2 in the CNS, indicating selective endothelial effects. Cellular infiltration and HRP leakage across the glia limitans to the parenchyma were induced by pertussis toxin treatment. By contrast, there was no detectable HRP leakage in the hippocampus dentate gyrus after transection of axonal afferents, despite that leukocytes infiltrate to this site. Immunoreactivity for AT2 was increased on glia limitans astrocytes in pertussis toxin-treated CCL2-transgenics whereas AT2 immunostaining was not induced in the lesion-reactive dentate gyrus.
Our results suggest that AT1 and AT2 control BBB integrity in and leukocyte entry to the brain. AT1 influences macrophage infiltration whereas AT2 induction correlates with solute leakage rather than cellular infiltration.
TET-induced expression of claudin-1 in brain endothelium reduces edema formation and clinical disease in an animal model of multiple sclerosis Engelhardt Britta ⁎ , Pfeiffer Friederike, Schäfer Julia, Tauber Silke, Deutsch Urban
Theodor Kocher Institute, University of Bern, Bern, Switzerland
In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, leukocytes migrate across the bloodbrain barrier (BBB) causing central nervous system (CNS) inflammation and edema formation, which both contribute to the disabling clinical picture of the disease. Paracellular permeability of the BBB is regulated at the level of its complex tight junctions (TJ), where the concerted actions of claudins-3, -5 and -12 seem to maintain TJ integrity. During EAE, loss of claudin-3 from BBB TJs correlates with cellular infiltrates and focal BBB leakiness. As leukocyte diapedesis leaves TJ morphologically intact we hypothesized that modulating TJ integrity will not affect immune cell recruitment across the BBB during EAE but may control edema formation.
We established a transgenic mouse model that allows for TETinducible, endothelial cell-specific expression of claudin-1, which is absent from TJ of CNS parenchymal microvessels. Severity of chronic EAE was significantly reduced in double transgenic claudin-1expressing C57Bl/6 mice compared to single transgenic control mice. Immunofluorescence analysis of brain and spinal cord sections from mice with chronic EAE revealed that expression of TET-induced claudin-1 at the BBB does not influence the number and size of cellular infiltrates within the CNS. Rather, vascular permeability was found to be significantly reduced around CNS microvessels with TET-induced claudin-1. Our findings show that TET-induced claudin-1 reseals BBB TJs during CNS autoimmune inflammation.
Taken together our study demonstrates the possibility for claudin-1 mediated resealing of BBB junctions during inflammation and suggest that claudins are good therapeutic targets for controlling BBB permeability during CNS autoimmune inflammation.
The study of adhesion molecules via a novel modified migration assay modelling the blood-brain barrier The loss of blood-brain barrier (BBB) integrity, which is associated with an increase trafficking of leukocytes into the central nervous system (CNS), is a hallmark of multiple sclerosis (MS). However, the mechanisms by which these cells gain access to the CNS are still not fully understood. Previous reports have shown that BBB dysfunction is associated with an upregulation of cell adhesion molecules (CAMs) (selectins, integrins, and members of the immunoglobulin family) allowing the reciprocal attachment of blood-borne leukocytes to BBB endothelial cells (BBB-ECs), which is a crucial step for infiltrating cells. In order to better understand the molecular mechanisms underlying recruitment and transmigration of immune cells to the CNS, we developed a new in vitro model of the human BBB that allows research of novel CAMs.
This model consists of a modified migration assay in which human immune cells migration occurs across human primary culture of BBB-ECs (hBBB-ECs) under pressure-controlled flow, which more adequately recapitulates the in vivo conditions. Migration experiments are being performed using different CAM ligands/antibodies in combination with immune cells. The ability of these cells to adhere to hBBB-ECs and their velocity is assessed using the VolocityTM software. We have been able to corroborate the importance of ICAM-1/LFA-1 and VCAM-1/VLA-4 for the recruitment of CD4+ T lymphocytes across human BBB-ECs. We have further found a 50% increase in velocity and a 40% diminution in adhesion of T cells following blockade of the latter combination. In addition, we have explored the influence of new CAMs such as melanoma cell adhesion molecule (MCAM/CD146), activated leukocyte cell adhesion molecule (ALCAM/CD166) as well as Ninjurin-1 for the adhesion and migration of CD4+ T lymphocytes and CD14+ monocyte across brain endothelium. Our data show a 38% increase in velocity and 34% reduction in adhesion of monocytes following ninjurin-1 steric inhibition. As for MCAM, preliminary data using antibody neutralization revealed a 16% increase in the velocity of human CD4+ T cells after 20 min under flow conditions. This new experimental human and dynamic setup has allowed us to study the role of human CAMs in the migration of specific immune cell subset. This system allows us to study new potential therapeutic targets to block entry of specific human immune cells across hBBB-ECs in order to reduce neuroinflammatory processes. Multiple sclerosis (MS) is a demyelinating disease in which acute and chronic inflammation associate with glial scarring and neuroaxonal damage. In early phases of the disease, inflammation caused by mononuclear cells infiltrating the central nervous system, is a prominent pathological feature.
The cannabinoid system consists of endogenous (endocannabinoids) and exogenous (natural and synthetic) cannabinoids, receptors and enzymes. Cannabinoids mediate their physiological effects basically by activating specific receptors; cannabinoid receptor 1 (CB1) mainly expressed in the CNS, and cannabinoid receptor 2 (CB2), expressed predominantly in immune cells, according to a rank order of B cells N NK cells N monocytes N polymorphonuclear leukocytes N T cells. Many data suggest that exogenous cannabinoids, might be beneficial in the treatment of MS symptoms like spasticity, tremor, ataxia, bladder control and pain. Beyond symptomatic relief, there is an increasing body of evidence supporting the involvement of the cannabinoid system in immunosuppression and neuroprotection.
Considering the importance of the immune response in MS, and the extensive CB2 expression in immune cells, the possibility exists that the cannabinoid system might regulate in part the extent of the immune response in the CNS. In the periphery, this would be mediated through a modulation of autoreactive cells via CB2. In this investigation, we decided to examine, using real-time quantitative PCR, the expression pattern of CB2 in immune cells from patients with active MS, as well as the effect of interferon beta therapy on CB2 expression.
Here we show a differential CB2 expression in B, NK and T cells as reported. Although the rank order of CB2 expression in these cell populations was similar in patients and healthy controls, MS patients had an increased expression of CB2 in B, NK and T cells compared to controls. Furthermore, we observed that interferon beta therapy induced a significant and progressive decrease of CB2 expression in those cells.
The differential expression of CB2 mRNA in MS patients points to a relevant role of the cannabinoid system in the immune response in this disease. A better understanding of the cannabinoid system, in particular CB2, might result in the development of new drugs with therapeutic potential in MS.
Consequences of immune complex formation in the brain Pathogenic antibodies exist in several neuropathological conditions, but little information exists on how they infiltrate the brain and initiate CNS tissue damage. Antibodies can mediate cell death by either direct signalling, or by recruiting cytotoxic effector cells via Fc receptors (FcR) and complement activation. We developed a novel mouse model to characterize antibody-mediated inflammation in the brain to further investigate the underlying mechanisms of neurological complication mediated by antibodies.
Balb/C mice were immunized against ovalbumin (OVA), followed by an intracerebral OVA challenge. Brain-tissue was analyzed by immunohistochemistry for the presence of immune complexes and inflammation. Fcg chain or C1q deficient mice were used to study the role of FcR and complement in antibody-mediated brain inflammation.
Inflammatory responses characteristic for immune complexes were detected in the injection site and around blood vessels within the cortex and the meninges. Increased expression of several macrophage and microglial activation markers, but no neutrophil activity was detected in the brain. The inflammatory responses were absent in Fc deficient mice, while C1q deficient mice were not different from wild type mice. Apart from histopathological changes, which were detected up to 4 weeks, we show that immune complexes induce transient neurobehavioural changes, which can be reactivated following systemic inflammation. We also found increased microglia activation, expression of FcR, in particular FcRIV, and increased levels of IgG in the brains of mice with chronic neurodegeneration, lupus and experimental acute encephalitis.
These results show that immune complexes formed in the brain can induce inflammation and behavioural changes that are initiated by FcRs. Therefore, in addition to a direct attack on neuronal cells, brain-specific (auto) antibodies, can recruit and activate microglia through their activating FcR, resulting in a release of pro-inflammatory cytokines which induce neuronal damage. These observations have important implication for the use of therapeutic antibodies for example anti-Abeta, possibly explaining the side effects observed in animal models and clinical trials. It has been proposed that immune senescence, the age-associated restructuring changes of immune functions, participates in the rate of aging through modulating oxi-inflamm-aging. Moreover, age-related changes in the immune system can be biological age markers and predictors of longevity. Gender differences in oxidation status and immune functions have been observed in rats, with males showing higher oxidation and immune senescence than females of the same age. Estrogens are sex hormones that actively participate in modulating the mammalian immune function and, therefore, the age-related impairment of the immune response is drastically accelerated in females during the menopausal transition. Ovariectomy in rodents constitutes a good model for mimicking human estrogen loss and thus the menopausal situation. Recently, we have shown the deleterious effects of estrogen loss on several functions of leukocytes from immune organs in rats and mice. In addition, ovariectomized rats show similar levels in these immune functions to those in males. The aim of the present work was to study in mouse peritoneal macrophages and lymphocytes from old sham and ovariectomized females, as well as in males of the same age, several functions as well as inflammatory and oxidative stress parameters.
The following parameters have been studied: chemotaxis, phagocytosis, intracellular ROS levels, lymphocyte proliferation in response to mitogens, IL-2, Il-10 and IL-6 secretion, natural killer cytotoxicity, antioxidant defence such as reduced glutathione (GSH) levels and glutathione peroxidase activity, oxidants such as oxidized glutathione (GSSG) and GSSG/GSH ratio and lipid peroxidation damage. The results show that females, which have a higher immune response and lower oxidation and inflammation than males, appear similar to males in the parameters studied when they have lost estrogens by ovariectomy.
Thus, these data support the positive role of estrogens in the immune function through the aging process.
Financial support: MCINN (BFU2008-04336); UCM Research Group (910379ENEROINN); RETICEF (RD06/0013/0002; RD06/0013/0003).
Old mice treated with pulsed high-energy potential-vortices improve behavior and immune cell function parameters, which reach similar levels to those in adult animals The pulsed high-energy potential-vortices (nanopulses), since they are produced by natural thunderstorm lightning, supply the cells with energy and thus, affect many cellular and physiological functions. Although the anti-inflammatory and analgesic actions of nanopulses are known, their effects on immune function and behavior, especially regarding the aging process, are yet unclear. Aging is a consequence of oxidation and inflammation stress affecting all cells but specially those of the nervous and the immune systems.
Recently we have proposed that immune senescence is involved in the rate of aging and that several immune functions are markers of biological age. The aim of the present work was to study the possible positive effects of a special nanopulse treatment (only possible with the technology of the medical instrument Pulsarion®) in several behavior and immune function parameters in old mice.
Three groups of female mice were used. A group of old mice (21 ± 1 months of age) (old treated group: OT), received 8 sessions of nanopulses, with a frequency of 60 pulses/min, for 10 min, twice a week for 4 weeks using Pulsarion®. Another group of old mice were given the same handling but did not receive treatment (old control group: OC). A group of adult mice (7 ± 1 months) was used as an age control. After 4 weeks all animals performed an exploration test of a T-maze. In this behavior test the vertical activity (percentage of animals performing rearing and number of rearings) and the horizontal activity (exploratory efficiency) were analyzed. Then, the peritoneal suspensions were obtained and the phagocytosis capacity of macrophages, the levels of superoxide anion, the proliferation capacity of lymphocytes (both basal and in response to mitogens), and the anti-tumor NK activity, were studied. The results show an improvement in OT mice of the exploratory parameters with respect to OC animals. Moreover, all the immune functions studied improved, reaching values similar to those obtained in adult mice.
In conclusion, treatment with these special nanopulses seems to be a good strategy to improve the nervous and the immune systems in old mice. Since the immune functions studied are markers of health, biological age and predictors of longevity, this treatment could be used to maintain a younger biological age and consequently a better and longer longevity. We thank Mr F. Pricci (Pulsartec SLU) for the financial support; RETICEF (RD06/0013/0003). Visceral leishmaniasis (VL) is an anthropozoonosis caused by parasitic protozoans of the Leishmania donovani complex. There are few studies focusing on the neurological manifestations of Leishmania infection. In dogs there are reports of signs of generalized central nervous system (CNS) involvement. Microglia is considered the first line of defence within the CNS and, once stimulated by inflammatory mediators, become activate. Activated microglia is identified by their morphological pattern, which is related to changes on their immunophenotype and function.
Twenty mixed-breed adult dogs were selected from the Control Zoonosis Center in Araçatuba, São Paulo State, Brazil, which is an endemic area for VL. Samples of nervous tissue and cerebrospinal fluid (CSF) of dogs with positive diagnosis for VL, as assessed by ELISA and/ or parasitological test (infected group, n = 10) and dogs with negative diagnosis (control group, n = 10) were collected. CSF titers of anti-Leishmania antibodies were detected by ELISA. Brain samples were stored in 10% buffered formalin and submitted to identification of microglial cells using RCA-1 lectin-histochemistry. The microglial reactivity, observed in light microscopy, was quantified using the software Image-Pro Plus 6.1. Significant differences between groups were determined by Student's t test with Welch's correction. A value of P b 0.05 was considered statistically significant. The infected group presented higher levels of antibody titers in CSF than the control group (P = 0.0005).Concerning the microglial reactivity, a significant difference between infected and control group was detected (P b 0.0001). In the infected group, microglial cells in the subependymal area were more intensely detected than those at cortical areas (P = 0.0044), and a positive correlation between microglia and antibody titers was detected (P = 0.0411).
Perivascular cells, including microglia, can act as an interface between the blood and the brain, bringing inflammatory stimuli from periphery. The presence of circulating antibodies within the nervous milieu may activate microglia contributing for the establishment of an inflammatory process, as described in immune and degenerative diseases of CNS. The results presented herein indicate a proinflammatory status in the brain and strongly suggested the microglial involvement in the pathogenesis of the brain lesions associated with visceral leishmaniasis in dogs. Human apolipoprotein E (apoE) is a glycoprotein with three isoforms (apoE2, apoE3 and apoE4). Experimental autoimmune neuritis (EAN), an animal model for Guillain-Barré syndrome is an immune-mediated experimental disorder in the PNS. Increased susceptibility to EAN in apoE KO mice has been previously found.
To elucidate the isoform-dependent effects of apoE on EAN, we used human apoE 2, 3, and 4 transgenic (Tg) mice immunized with P0 peptide 180-199 to induce EAN, along with macrophage culture, T cell proliferation test, flow cytometry and ELISA, etc. to investigate the effects of apoE isoforms on the functions of macrophages and T cells both in naive conditions and during EAN. The clinical signs of EAN during the course are the most severe in C57BL/6 mice and apoE4 Tg mice, followed by ApoE2 Tg mice, and the least severe in ApoE3 Tg mice (WT = E4 N E2 N E3, p b 0.01). At the nadir, either spleen weight or mixed T cell proliferation test stimulated with P0 peptide or interleukin (IL)-23 is in line with clinical severity of EAN. Proliferation test of purified naive T cells stimulated with ConA or IL12 shows isoform-specific difference (WT = E4 N E3= E2, p b 0.01). Macrophages from both naive and EAN mice produce nitric oxide (NO) upon inflammatory stimulations with LPS, or IFN-, or poly I:C, or combinations thereof, in an isoformdependent manner (WT = E4 N E2 N E3, p b 0.01). Furthermore, generalized intervention with 1400 W, a specific inducible nitric oxide synthase (iNOS) inhibitor, could significantly suppress the clinical course of EAN in WT mice as well as human ApoE2, 3 and 4 Tg mice.
The preliminary data supported a subtype-dependent effect of apoE on EAN. This might be due to the isoform-specific effects of apoE on macrophage functions, and/or T cell functions, which contribute to the distinct clinical severity of EAN. Cannabis compounds, cannabinoids, have been shown to exert anti-inflammatory activities in certain experimental models of inflammatory CNS degenerative diseases. The main obstacle for clinical application of those materials is their psychoactive properties. We evaluated the effects of a non-psychoactive cannabinoid, cannabidiol (CBD), in myelin oligodendrocyte glycoprotein (MOG)-induced EAE murine model of multiple sclerosis (MS) and determined the mechanisms underlying these properties, specifically in microglial cells.
We observed that peripherally given CBD (administered at the time of disease onset) ameliorates the clinical EAE symptoms as evaluated using behavioral and pathological scores. Histochemical analysis of spinal cords of MOG-injected EAE mice treated with CBD vs MOG-only treated mice revealed that CBD down regulates infiltration/ proliferation (Iba-1 staining) and activation (Mac-2 staining) of macrophages and microglia into spinal cord white matter.
Using the BV-2 mouse microglial cell line and lipopolysaccharide to induce inflammatory response, we were screening for intracellular mechanisms that might be involved in the CBD anti-inflammatory activity. We observed that CBD decreased the release of interleukin (IL)-1β and IL-6 proinflammatory cytokines from activated microglial cells. CBD inhibited the activation of STAT1 proinflammatory transcription factor and up-regulated the STAT3 factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events.
In conclusion, we observed that CBD exerts anti-inflammatory activities in vivo (using the in EAE model of MS) as well as in vitro (using microglial cells). These activities may be mediated via STAT dependent pathways. The relation between prolonged stress and the development of psychiatric disorders such as depression and anxiety has been well established (Ader and Cohen, 1993) . Given the role of the HPA axis in depression, a recent work (Miller et al., 2009 ) suggested a critical role for the endocannabinoid system on its regulation. The aim of the present work was to study the effect of restraint stress on some neuroimmunoendocrine, behavioral and endocannabinoid parameters.
Restraint stress was applied to adult Sprague-Dawley rats two hours daily during 7 or 21 consecutive days. The behavioral tests were open field and elevated plus maze. Blood samples were collected and the hippocampi were dissected. Total mRNA and protein levels were analyzed by real time PCR and Western blot respectively.
The results showed that stressed animals have increased IL-6 and cannabinoid receptor type 1 (CB1) total mRNA and protein levels in the hippocampus. Oxytocin and corticosterone plasma levels were increased as well as adrenal gland weight. Also, the animals showed a reduced habituation in the open field test but no changes in anxiety parameters.
These findings suggest a possible interaction between neuroimmunoendocrine and endocannabinoid systems in the present stress paradigm (BID-OCR-AR, PICT-06-0258).
Cognitive-enhancing and antidepressant effects of chronic celecoxib and memantine treatment in an OBX rat model of Alzheimer's disease Borre Yuliya ⁎ , Lemstra Susan, Oosting Ronald, Olivier Berend Utrecht University, Utrecht, The Netherlands
Immune dysfunction plays a role in the pathogenesis of various neurodegenerative disorders. Increase in the proinflammatory cytokines in Alzheimer's disease (AD) is associated with cognitive deficits and depressive symptoms. Removal of the olfactory bulbs (OBX) from a laboratory rat leads to long-lasting behavioral, neurochemical changes and deficits in learning and memory; and as such OBX may model certain aspects of AD. OBX have been reported to have elevated levels of pro-inflammatory cytokines and have been used to study the relationship between immune dysfunction and behavioral/cognitive abnormalities. In the present study, OBX were used to assess whether chronic celecoxib and memantine administration starting presurgery, would prevent/delay abnormal behavioral and cognitive developments.
Pharmacological effects of an anti-dementia drug, memantine, an uncompetitive NMDA receptor antagonist, and celecoxib, a COX2 inhibitor, were evaluated in the OBX induced cognitive impairments. Chronic administration of memantine (20 mg/kg) and celecoxib (10 mg/kg) starting 2 days before OBX surgery and continuing for 4 weeks, attenuated passive avoidance (PA) memory deficits and normalized the hyperactivity in the open field (OF) associated with OBX; yet had no effect on the spatial memory as measured in the holeboard. When tested 1 week after treatment cessation, the celecoxib treated OBX animals still showed improved PA memory in comparison to the OBX treated vehicle and the sham operated controls. Peripheral interleukin levels were measured at various time points: before, during, and post chronic drug administration.
Possible mechanisms by which celecoxib and memantine improved PA memory and hyperactivity in OBX rats may result from the suppression of inflammation and normalization of the glutamatergic system. Celecoxib has been found to reduce both blood and brain PGE2 concentrations and decrease blood IL-1 levels resulting in attenuating PA memory deficit and normalizing hyperactivity mediated by OBX. Memantine's antagonistic properties to diminish the sustained increase of extracellular glutamate levels may have resulted in cognitive protection and prevention of the pathological microglial activation. These findings suggest that pharmacological intervention during the early phases of the neuroinflammation development not only rescues the behavioral and cognitive abnormalities, but also slows down the progression of the cognitive impairment. Idiopathic intracranial hypertension (IIH) is a neurological condition whose aetiology is unknown. The factors thought to contribute to the raised intracranial pressure include oversecretion or impaired absorption of cerebrospinal fluid (CSF).
A few studies have investigated the composition of CSF in IIH, but the condition has most commonly been used as a "non-inflammatory control". Here we show findings that may challenge this assumption of non-inflammation.
We compared 14 cytokines in the CSF of 17 patients with IIH and over 50 patients with other neurological diseases, including 33 with multiple sclerosis (MS), 8 with inflammatory neurological diseases (5 with chronic inflammatory demyelinating polyneuropathy (CIDP) and 3 with neurosarcoidosis) and 5 with functional illnesses, using a combination of multiplex arrays and ELISA. The cytokines chosen were those suggested to be of importance in T cell differentiation and function, focussing on Th1, Th2, Th17 and Treg cells.
CSF IL17 was significantly elevated in the IIH population compared to both the MS and functional groups; interferon-gamma, IL2 and IL4 were also significantly higher in the IIH group than the MS group. However, IL10 was significantly lower in the IIH group compared to the "neuroinflammation" group (CIDP and neurosarcoidosis). No significant differences were seen between the groups when levels of IL1b, IL6, IL8, IL12p70, tumour necrosis factor-alpha, transforming growth factor-beta or osteopontin were compared.
Positive correlations were seen between the levels of certain cytokines in CSF, including IL2 and IL4, IL2 and IL17, IL2 and interferon-gamma, IL4 and IL17, and IL17 and interferon-gamma.
It is unclear whether the dysregulated cytokines in IIH CSF are causal or consequent to the raised intracranial pressure. However, this raises an interesting question about possible pathogenic mechanisms and whether targeting inflammation may in fact be a future treatment for this potentially disabling condition. Increasing studies have shown that patients with diabetes mellitus have an increased risk of cognitive impairment, dementia, and neurodegeneration. Inflammation is now recognized as a prominent feature in the pathology of diabetes mellitus and Alzheimer's disease. The present study was designed to evaluate the effect of Danshensu on diabetes-associated learning and memory decline in mice.
Diabetes was induced by a single intraperitoneal injection of streptozotocin (150 mg/kg body weight) in C57BL/6 mice. Animals with blood glucose levels exceeding 20 mmol/L were selected as the diabetic mice. Then the mice were administered with Danshensu at doses of 15, 30, or 60 mg/kg by intragastric administration once a day for 10 weeks continually. Afterwards, the effect of Danshensu on diabetes-associated learning and memory decline in mice was investigated by evaluating the mean escape latency and the time spent in target quadrant. The TNF-a, IL-1β contents, acetylcholinesterase activity, and NO levels in hippocampus were assayed. Western blot was used to analyze the expression of iNOS. The results showed that diabetes mellitus induced a significant learning and memory decline. Administration with Danshensu at doses of 15, 30, or 60 mg/ kg not only reduced the mean escape latency but also increased the spent time in target quadrant. Furthermore, Danshensu decreased the acetylcholinesterase activity, the TNF-a, IL-1β and NO levels in hippocampus. Compared with diabetes group, the expression of iNOS significantly decreased in Danshensu groups.
The study demonstrated that Danshensu could attenuate diabetesassociated learning and memory decline in mice and the mechanism may be related to the anti-inflammatory action of Danshensu. The most common phenotype of multiple sclerosis (MS) is the relapsing-remitting (RR) form, which is characterised recurrent acute-onset episodes of neurological dysfunction, followed by complete or partial recovery. The long-term prognosis of RRMS is, usually, unfavourable, since patients enter the so-called secondary progressive (SP) phase of the disease and accumulate irreversible neurological disability. A different disease pattern, primary progressive (PP) MS, is seen in patients showing a progressive course from the onset; an even rarer form of disease is called benign MS (BMS). In the latter case, there is absent or minimal neurological impairment many years after the disease onset. The course of RR-MS appears to be largely driven by the inflammatory process; conversely neurodegeneration, which at least in part develops independent from inflammation, drives chronic brain injury in patients with PP and SP-MS. Antiinflammatory therapies and immune modulation have shown a beneficial effect only in RR-MS patients. This discrepancy could be explained by different pathogenetic mechanisms acting in different disease phenotypes. We investigated the immunologic correlates of these diverse disease manifestations in the attempt to clarify the pathogenesis of MS.
Thus, we performed an extensive immunophenotypic and functional analyses of MBP-stimulated T lymphocytes in 10 BMS, 15 RR-MS, 10 SP-MS and 10 PP-MS patients, comparing the results to those obtained in a group of healthy controls (HC). Results showed that trascriptional factors and cytokines associated with the activity of TH17 and TH9 cells are significantly upregulated in RR and SP-MS patients whereas PP-MS is associated with an increase of TH1-associated markers alone. Thus, MBP-stimulated: 1) IL-9, IL-21, and IL-22, as well as GATA-3 and RORg, were up-regulated in RR and in SP-MS; 2) T-bet and IL-12 were upregulated in PP-MS. Interestingly, no significant differences were observed in BMS when were compared to HC.
Data herein reinforce the pivotal role for inflammation in the pathogenesis of MS, for the first time show that different patterns of disease are associated with the activation of different T helper cell subsets, and offer an explanation for the biological and clinical peculiarities of PP-MS (TH1 vs. TH17/TH9).
Distinct macrophage subsets with divergent phenotypes and functional properties in murine brain abscess Richter Lydia ⁎ , Held Josephin, Döser Alexandra, Meisen Michael, Heppner Frank L., Stenzel Werner
Macrophages can be activated by so called classical and alternative pathways giving rise to at least two different phenotypes (M1 or M2) with different functional properties. Interferon gamma-induced M1 classically activated macrophages are main effector cells in acute infections with bactericidal functions via production of reactive oxygen species. These macrophages should primarily have host-protective properties but collateral damage within certain organs and in certain situations may also be detrimental. On the other hand, T helper 2 cells are able to induce an alternative pathway of macrophage activation (M2) via production of IL-4 and IL-13 that should theoretically reduce excessive inflammation. M2 macrophages have been studied intensely in chronic fungal, worm or parasite induced diseases, but have to our knowledge, never been studied in bacterial infections of the CNS.
We use a well-established model of murine brain abscess that has a prominent acute phase from d1 to d3, characterized by severe cerebritis and a sepsis-like illness, followed by a chronic healing phase from d14 to d28, with formation of a fibrous capsule delimiting the process against the bordering healthy CNS tissue.
In the early phase of the disease, Th1-induced CD11b + macrophages prominently co-express iNOS but no markers for alternative activation, while in the late phase Th-2-induced CD11b + macrophages co-express YM-1, CD206 and CD301 and are only weak iNOS producers, results which are confirmed on the mRNA level. Capsule formation subsequent to CCL-18 upregulation with evidence of collagen VI + fibroblasts is maximal at day 28.
We show that different macrophage subsets with distinct functional properties are active during brain abscess development and healing, and that there is probably a broad spectrum of macrophage diversity according to the immunological necessity.
Exploring the mechanisms of axonal and neuronal damage caused by CD8 T cells Chevalier Gregoire ⁎ , Suberbielle Elsa, Liblau Roland, Gonzalez-Dunia Daniel Inserm U563 Centre de Physiopathologie de Toulouse Purpan, Toulouse, France Cytotoxic CD8 T cells (CTLs) are increasingly recognized as key players in various neuroinflammatory or neurodegenerative diseases, such as multiple sclerosis, Rasmussen's encephalitis or Alzheimer's disease. CTLs are believed to contribute to neuronal damage in these diseases although their precise contribution remains unclear. Indeed, the possibility that neurons could represent a target for CTLs is still controversial, in part due to the paucity of major histocompatibility complex (MHC) class I expression by neurons. Therefore, a better understanding of the mechanisms underlying neuronal and axonal attack by CTLs is much wanted. Here, our objective was to bring new information about the mechanisms of neuronal injury caused by CTLs.
To address this question, we used the model of CD8 T cellmediated neuroinflammation induced by neurotropic Borna Disease Virus (BDV). CTLs ex vivo purified from brain of BDV-infected animals were co-incubated with primary cultures of cortical neurons infected by BDV. We then assessed the dynamics and consequences of CTL interaction with neurons, using live-cell imaging and time-lapse microscopy techniques.
We observed that BDV infection induced MHC class I expression in neurons, rendering them susceptible to CTL attack. Upon incubation with infected neurons, ex vivo CTL mobility was dramatically reduced in an MHC class I-dependent manner, suggesting specific interaction with neurons. Analysis using calcein staining revealed changes in neuronal permeability within minutes after CTL contact. Surprisingly, neurons appeared to resist quite well to this initial CTL contact, since caspase-3 and -7 activation was detected only 4 h after co-incubation. Moreover, recordings of electrical activity using MicroElectrode Array (MEA) revealed that the neuronal network remained functionally active during this period.
Taken together, these data suggest that the dynamics of CTL-neuron interaction may be quite different to that of a classical CTL target. The aim of this project is to characterize how vitamin D influences inflammatory cells mediating multiple sclerosis (MS), with emphasis on epigenetic regulation of genes in the HLA complex. Recent data indicate that high levels of sunlight and vitamin D protect from MS. However, it is not known how this protection functions on a molecular basis. Several data implicate that epigenetic changes mediate the influence of vitamin D on MS.
Rats were fed with a special diet without and with Vitamin D. Subsequently EAE, the animal model for MS, was induced. Lymphnodes were harvested and methylated DNA was purified using methylated DNA immunoprecipitation (MeDIP). Differences in the methylation pattern at the HLA complex genes and genome-wide have been assessed by hybridizing the DNA to promotor and CpG specific arrays. Data were confirmed using RT-PCR. Differentially methylated genes will subsequently be validated on human MS samples.
Epigenetic modifications have been shown to be reversible. For example, deficiency of folate leads to genomic hypomethylation causing open neural tube defects. However, folate therapy can restore normal methylation patterns. The reversibility of epigenetic modifications is very valuable for MS prevention and possible therapies for this severe disease.
If vitamin D can be established as a main environmental trigger for MS, supplementary administration of vitamin D could reduce the risk of developing MS, especially in countries with low levels of winter sunlight such as Sweden. Therefore, elucidation of the molecular mechanisms following epigenetic changes that occur under vitamin D deficiency in vitro and in animal models can lead to new therapeutical approaches for curing MS patients.
Genetically determined beta-chemokine expression regulates the neuroinflammation in rats MS is believed to be an autoimmune disease triggered by an unspecific environmental factor in genetically susceptible individuals. Therefore, an approach that aims in identification of naturally occurring genetic variants that predispose for disease, can pinpoint causal candidate genes.
We investigated basic inflammatory mechanisms in the early stages of neuroinflammation and perpetuation of the disease in the congenic rat strain (DA.PVG-CCLs), where naturally occurring genetic variants of CCL1, CCL2, CCL7, CCL11 and CCL12 predispose to and regulate neuroinflammation, and in the backcross population. This beta-chemokine cluster has been implicated as the underlying genetic cause of one of the major EAE-regulating QTLs, designated as Eae18b, initially identified in an (DAxACI)F2 cross. Furthermore, several MS studies showed evidence of association of CCL2 with this disease.
Initial results confirmed that the DA.PVG-Eae18b display a less severe disease in response to MOG immunization. An experiment on a minimal congenic, DA.PVG-CCLs, comprising only CCL1, CCL2, CCL7, CCL11 and CCL12, indicate that the effect maps to this chemokine cluster. Furthermore, expression of chemokines in lymph nodes 7 days after immunization correlates with the genotype at the locus, indicating that the genetic variants of these chemokines partially govern their expression levels. Association studies in Scandinavian material show evidence for association of CCL1, CCL2 and CCL13 with MS. Higher levels of CCL2 were demonstrated in CSF of MS patients compared to controls.
The classical hypothesis-driven research that focuses on investigation of a molecule cannot distinguish if differences regarding the molecule are the cause or the consequence of disease process. This, together with disease heterogeneity and complexity, could account for the slow progress of treatment development. Accordingly, an approach that aims in identification of disease-regulating genes can provide primary candidates and mechanisms that might be more suitable targets for drug development.
Genome-wide profiling of micro RNAS that associate with susceptibility to experimental neuroinflammation Bergman Petra ⁎ ,1 , Gillett Alan 1 , Goga Supic 1 , Larsson Kristina 2 , Kvist Anders 3 , Jagodic Maja 1 1 Karolinska Intitutet, Stockholm, Sweden; 2 Uppsala University, Uppsala, Sweden; 3 Lund University, Lund, Sweden Dysregulated expression of micro RNAs (miRNAs) in various tissues has been associated with a variety of diseases. We sought to profile miRNAs that associate with susceptibility to develop pathogenic neuroinflammation. In order to detect differences that are relevant for susceptibility and not only associated with induction of neuroinflammation we compared miRNA profiles in activated lymph nodes of susceptible and resistant inbred rat strains.
High-throughput deep-sequencing technology (Solexa, Illumina) was first applied to identify and quantify miRNAs. Furthermore, adapters were tagged with unique index sequences to allow multiplexing of samples. We identified more than 200 known miRNAs and several novel candidate miRNAs. TaqMan (rodent) micro RNA array (Applied Biosystems), comprising 226 unique rat miRNAs, confirmed a number of differentially expressed miRNAs detected by Solexa that passed 5% FDR. Since every miRNA potentially regulates hundreds of target genes, we next used data on differentially expressed mRNAs from the same strains and the same tissue (Affymetrix), to elucidate miRNAs and their mRNA targets and pathways that are dysregulated in the susceptible strain. Solexa sequencing also enabled identification of several novel miRNAs which were all up-regulated in the resistant strain.
Differential expression of miRNAs and their targets implicates pathways that associate with susceptibility to neuroinflammation, involving proliferation, innate immune response and TLR signaling, tumor suppression and neurodegeneration. Inflammation plays an important role in cerebral ischemia. Interleukin (IL)-10 is an anti-inflammatory cytokine in the immune system. Here we explored the change of IL-10 gene in a transient global cerebral ischemia model.
Global cerebral ischemia was induced by occluding both common carotid arteries and withdrawing 0.3 ml of blood from the tail vein in mice. Three days later, IL-10 gene expression in hippocampal tissue of cerebral ischemia mice was examined using real-time PCR. The results showed that IL-10 gene was upregulated at least two fold after transient global cerebral ischemia.
The results made an interesting observation that subjecting mice to ischemia significantly upregulates the IL-10 gene expression, and this is probably one of the self-neuroprotective mechanisms of the mice to against inflammation after cerebral ischemia. Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS). The imbalance of proinflammatory versus anti-inflammatory cytokines is key determinant to develop autoimmunity. The expression of pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, IFN-and TNF-a is correlated with an increase of disease susceptibility. On the other hand, Th2 cytokines such as IL-4, IL-5 and IL-13 have been shown to be important to prevent or ameliorating disease.
Delivery of anti-inflammatory molecules into CNS is one of the possible approaches to treat EAE. It has been demonstrated that the delivery of IL4-expressing helper-dependent adenoviral vector (IL-4-HD-Ad) induces clinical and functional recovery in EAE. IL-25 (IL-17E), a new member of the IL-17 family of cytokines, promotes Th2 response in an EAE chronic mouse model and regulates the development of autoimmune inflammation mediated by IL-17producing T cells (Th17).
In this study we show that IL-25 gene therapy does not modulate the relapsing-remitting (RR) EAE mouse model.
Briefly, to investigate the effects of mIL-25-HAgene therapy on RR-EAE we intrathecally injected RR-EAE-SJL mice with mIL-25-HAexpressing lentivirus, at the onset and during the first disease remission, and followed clinical outcome for 50 days. mIL-25-HA expressing lentivirus infected ependymal cells along the entire ventricular spaces, as shown by anti-HA immunostaining and cerebro spinal fluids and brain homogenates contained mIL-25-HA protein, as demonstrated by ELISA assay for mIL-25. Moreover, mIL-25-HA was also able to up-regulate mIL-5 and mIL-13 mRNA expression in CNS, as measured by real time-PCR. But despite these results, mIL-25-HAlentivirus gene therapy had no effect on clinical and pathological features of EAE, evaluated respectively as disease clinical score and number of inflammatory infiltrates, percentual of axonal and myelin damage in spinal cord sections. We are now analyzing by ELISA assay the amount of mIL-4, mIL-5 and mIL-13 proteins into brain homogenates of RR-EAE SJL mIL-25-HA-lentivirus injected mice vs RR-EAE SJL uninjected mice.
In conclusion, our data suggest that, unlike data in EAE chronic mouse model, IL-25 gene delivery in RR-EAE SJL mice has no effects on clinical and pathological features of the disease, even if IL-25 is able to exert its biologic functions.
Interleukin-18 alters expression profiles of amyloid precursor protein cleaving enzymes in SH-SY5Y cells Alzheimer's disease (AD) involves neuronal loss, amyloid-beta composed plaques and neurofibrillary tangles (NFT), containing Tau protein, in the various regions of neocortex, and the presence of inflammation. Inflammatory cytokines, e.g. interleukin 18 (IL-18), produced mainly by activated microglia in the brain, can have an important role in nerve cell degeneration e.g. by enhancing production of plaque. Our earlier studies showed that IL-18 colocalizes with plaques and NFT containing neurons, and IL-18 can induce expression of kinases involved in hyperphosphorylation of Tau. In this study, we examined the effects of IL-18 on amyloid precursor protein (APP) processing enzymes in neuron-like differentiated human SH-SY5Y neuroblastoma cells, and generally, evaluated the role of IL-18 in plaque formation.
The cells were exposed to bioactive recombinant IL-18, and the changes were detected with immunoblotting. In immunohistochemistry, we used paraffin sections from the brain samples of AD patients. The IL-18 treated neuron-like SH-SY5Y cells showed increased protein levels of Presenilin-1 (PS-1) and beta-Amyloid cleaving enzyme BACE-1 after the 24 h or 72 h treatments compared to the untreated controls. The increase was abolished by simultaneous treatment with neutralizing IL-18 binding protein (IL-18BP). Expression profile of the cleavage products of APP showed also alteration during the treatments. Further, in IHC, we detected IL-18, IL-18BP and surprisingly, also IL-18 receptor (IL-18R) in association with plaques. Thus, it seems that IL-18 can enhance plaque formation by inducing expression of BACE-1 and PS-1, and further, IL-18 may have a role in plaque clearance through its interactions with plaques. IL-18BP, localized in plaque, may function as binding site for IL-18, and this complex may inhibit the plaque uptake from the extracellular space to the glial cells by interacting with IL-18R on the cell membranes.
IL-18 can enhance progression of AD. It has been shown that A(beta) accumulation in the brain, a key feature of the Alzheimer's disease (AD), can be reversed by A(beta) immunotherapy and that this effect is mediated at least in part by phagocytosis by microglia. In our study we aim to improve our understanding of the role microglia play in AD pathology and in the outcome of the A(beta) immunisation.
We have neuropathology on 11 AD patients who were actively immunised with A(beta)42 peptide (iAD -Elan Pharmaceuticals). We are exploring the expression of microglial proteins in immunised and control AD subjects in order to define a microglial profile in relation to the pathological features of the disease (A(beta) and tau proteins). Immunohistochemistry for HLA-DR (antigen-presenting complex), CD68 (phagocytic activity), complement C1q, Fc(gamma)RI (CD64), Fc(gamma)RII (CD32) and IgG was performed in the frontal, temporal and parietal lobes.
A significantly higher load of CD68 was observed in immunised cases compared to non-immunised controls (P = 0.05). In addition, within the immunised group only the CD68 load showed a significant correlation with A(beta)42 load (P = 0.01). Quantification of HLA-DR load did not show a significant difference in iAD cases vs AD controls (P = 0.3). Preliminary results for other microglial markers suggest no significant changes in the level of IgG and complement C1q, and a decrease in the expression of both Fc(gamma)RI and II in iAD cases compared to AD control cases.
A(beta)42 immunisation has an effect on the function of microglia, increasing their capacity for A(beta) phagocytosis. The correlation between CD68 load and A(beta)42 load in the immunised cases and lack of such correlation in the control group, highlight the point that it is not just the presence of microglia, but rather their target and specific profile that determines their role in AD pathology. So far we have found no evidence for an increase in the overall humoural autoimmune activity.
Kruppel like factor 4, a novel transcription factor in microglial activation and subsequent neuroinflammation Kaushik Deepak, Basu Anirban ⁎ National Brain Research Center, Gurgaon, India Activation of microglia, the resident macrophages of the CNS, in several neurodegenerative diseases like Alzheimer's, Parkinson's, multiple sclerosis and other pathological conditions associated with CNS infection is often associated with neuronal death. Therefore, the understanding of inflammatory mechanisms leading to its activation becomes indispensable part of studies focusing on neurodegeneration. Nuclear factor-kappa B (NF-kappa B) is one of important transcription factors known to be associated with macrophage and microglial activation and is involved in mediating the upregulation of inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and other proinflammatory cytokines. However, inflammation is a complex event and co-activators and other transcription factors are known to play a key role in this process. Recently, Kruppel like factor 4 (Klf4), one of the zincfinger transcription factors was reported to play an important role in inflammation in peripheral macrophages where it was shown to regulate iNOS expression potentially by interacting with pNF-kappa B. We therefore investigated its role in neuroinflammation.
For our studies, BV-2 microglial cells and primary microglia were treated with Salmonella enterica lipopolysaccharide (LPS) and changes in Klf4 expression were observed. We found that Klf4 translocates to the nucleus and its expression increases in a dose and time-dependent manner upon LPS stimulation. Studies revealed that Klf4 is required for the up-regulation of both iNOS and COX-2 as their expression at both the mRNA and protein levels were significantly reduced within 12 h of LPS stimulation after Klf4 knockdown. It potentially binds to their promoter elements in microglial cells which was confirmed by promoter binding assays. This transcription factor has also been found to be important in the upregulation of key proinflammatory cytokines that include TNF-alpha, MCP-1 and IL-6 upon LPS treatment.
Our data suggests that Klf4 is responsible for the upregulation of proinflammatory mediators and cytokines in microglial cells upon endotoxin stimulation. The detailed mechanisms by which this transcription factor is involved in neuroinflammation are a subject of further studies. In Alzheimer's disease, microglial responses are thought to occur without significant leukocyte infiltration. We have evaluated microglial/leukocyte dynamics in the neocortex of young (4-months) and aged (18-months) wildtype (WT) and amyloid precursor protein (APP)695/presenilin-1 (PS1)E9 transgenic (Tg) mice.
Stereological estimation of iba1+ cells showed a 2.2-fold increase in microglial numbers in aged APP/PS1-Tg mice, in which 3.5% of the neocortex was occupied by amyloid plaques but no neuronal loss occurred. Microglial numbers were not significantly different between young WT mice and young APP/PS1-Tg mice, in which only few amyloid deposits were detected. Similar results were obtained by quantification of CD11b + CD45dim microglial cells using flow cytometry. Flow cytometric analyses also revealed significant infiltration by CD4+ and CD8+ T cells, and a 27-fold increase in numbers of CD11b + CD45 high macrophages in aged APP/PS1-Tg mice. Leukocyte recruitment coincided with emergence of microglial and macrophage subpopulations that expressed high levels of CD86 and MHC Class I in aged APP/PS1-Tg mice, and was consistent with immunohistochemical stainings showing CD45+ leukocyte-like cells in post-mortem brain tissue from patients with Alzheimer's disease. Despite increased numbers of myeloid cells, rates of microglial and macrophage proliferation measured over a 24 h period immediately prior to perfusion were unchanged by age or expression of APP/PS1. Instead, rates of microglial and macrophage apoptosis were increased in aged APP/PS1-Tg mice. Ex-vivo analysis showed that microglia and macrophages that had ingested amyloid in vivo were more likely to undergo apoptosis, and less likely to proliferate, than cells that had not.
Our data demonstrate that amyloid pathology elicits greater leukocyte infiltration than previously anticipated, and suggest that apoptosis of phagocytic cells leads to a failure in amyloid clearance. Plaque deposition may therefore increase when the rate of microglial/ macrophage cell death exceeds the rate of cellular recruitment.
Loss of astrocyte connexin 43 and 30 does not significantly alter susceptibility or severity of experimental autoimmune encephalomyelitis in mice Lutz Sarah E., ⁎ Raine Cedric S., Brosnan Celia F.
In the adult brain, astrocytes express connexin (Cx) 43 and Cx30, which form gap junction channels between astrocytes, between astrocytes and oligodendrocytes, and between astrocytes and myelin. This glial syncytium facilitates distribution of metabolic substrates such as glucose and lactate, and spatial buffering of ionic potassium and glutamate. Cx43 expression is altered in a number of pathological conditions, including at sites of inflammation in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Furthermore, recently published proteomic analyses indicate altered Cx43 expression in human MS lesions (Han et al., 2008 Nature 451:1076 . The goal of this study was to determine if astrocyte gap junctions affect inflammation in the central nervous system (CNS).
Mice with mGFAP-Cre astrocyte-targeted deletion of Cx43 (single knock-out, sKO) or Cx43del/del/Cx30 −/− (double knock-out, dKO) were sensitized for EAE using MOG35-55. In sKO but not dKO mice, clinical signs of disease were slightly increased compared to control genotypes (p b .05). However, lesion load and the susceptibility of different areas of the CNS to inflammation were similar in all genotypes. Moreover, no differences were noted in blood-brain barrier (BBB) permeability to Evans blue dye, tissue wet weight, axonal pathology, gliosis or demyelination.
To explore the potential relevance of these findings to human disease, we examined expression of Cx43 in MS lesions using immunohistochemistry and western blotting. In normal appearing white matter, Cx43 was widely expressed in association with myelin. In most chronic active MS lesions, Cx43 was restricted to perivascular endfeet and the Golgi compartment (co-localization with GM130), suggestive of altered trafficking of Cx43. Chronic silent lesions exhibited no detectable Cx43 protein.
In summary, we observed a slight exacerbation of EAE only in Cx43 sKO mice. In all other genotypes, we observed no differences between groups in measures of lesion load, inflammation, demyelination, or BBB permeability. Human MS lesions were associated with changes in the expression levels and subcellular distribution of Cx43. We conclude that genetic loss of Cx30 and Cx43 in astrocytes does not meaningfully change expression of EAE and does not contribute to BBB breakdown. The clinical relevance of altered regulation of Cx43 in MS remains unclear.
Microparticles released from reactive microglia: A potential biomarker for neuroinflammatory and neurodegenerative diseases Shedding vesicles are microparticles shed from plasma membranes in response to cell activation, injury, and/or apoptosis. In the CNS they are supposed to be a fundamental way of intercellular signalling and communication between the immune and neuronal system. Microglia, the immune cells of the CNS that mediates inflammatory immune response responding to neuronal damage, have been shown to release microparticles, containing cytokines such as IL-1, after ATP stimulation. The chronic activation of microglia has shown to be both detrimental and beneficial. Its role in neurodegenerative disease is under debate. Current clinical diagnostic criteria for Alzheimer disease (AD) require a patient to have dementia before a diagnosis can be made and no clinical method is available for identifying prodromal AD in patients with mild cognitive impairment (MCI).
Our aim is to characterize the presence of microglial shed vesicles in the CSF patients with different neurological diseases in order to detect possible alterations, in number or content, associated to neurodegeneration.
Forty-five patients were examined and recruited since September 2009 in the Department of Neurology of San Raffaele Scientific Institute. Patients underwent neuropsychological assessment, MRI, and CSF analysis. The cohort of patients included a total of 16 Alzheimer Disease patients aged 71 ± 12 (mean ± SD) years, 10 mild cognitive impairment (MCI) patients aged 67.7 ± 5.7 years, 9 relapsing remitting multiple sclerosis patients, 7 peripheral nerve disease patients and 3 cephalalgic patients with no underlying parenchymal disease. CSF samples were collected by lumbar puncture (performed in the L4-L5 vertebral interspace), and the first 15 to 20 ml of CSF was collected into sterile, screw top, leakproof tubes. The CSF samples were split into two parts: one directly analyzed by flow cytometry (stained for AnnexinV, IB4, and CD63) to detect microparticles, and the other centrifuged through several differential centrifugation obtaining pellets and stored for gene expression analysis. Results: We found an increased number of shed vesicles of microglial origin in the CSF from AD patients and MS patients, in comparison to MCI and control patients.
Shed vesicles of myeloid origin in the CSF may represent a novel biomarker of microglial activation and a useful tool to investigate the immunopathogenesis of neuroinflammation and neurodegeneration. Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive and massive loss of dopaminergic neurons (DNs) in the ventral part of the mesencephalon (i.e. the substantia nigra), resulting in motor disturbance. Accumulating evidence suggests that non-cell autonomous mechanisms are involved in neurodegeneration in PD. In line with this, we have recently shown that peripheral T CD4+ cells, which invade the brain in PD patients and in MPTP-treated mice (an experimental model of PD), actively participate to neuronal cell death suggesting that therapeutic strategies aimed at preventing lymphocyte extravasation may be of interest (Brochard et al., JCI, 2009 ). Yet, the molecular mechanisms underlying this infiltration are still largely unknown.
Our major aim is to unravel these mechanisms, in particular, the adhesion and chemotactic factors involved in these processes. Since CD4+ T lymphocyte infiltration reached a pick 2 and 7 days after MPTP, we intoxicated 40 mice and sacrificed half at each time point. From the dissected ventral mesencephalon of each mouse total mRNA was extracted and a panel of cellular markers (e.g. tyrosine hydroxylase (TH)-CD68-glial fibrillary acidic protein) was tested by real time RT-PCR. Individuals (n = 7 per time point) presenting the most pronounced inflammatory reaction and decrease in TH were further used in TaqMan® Low Density Arrays to study the changes in chemokine and adhesion molecule expression (192 genes in total). Then, the impact of corresponding chemokine receptor deficiency on T lymphocyte infiltration and dopaminergic cell death following MPTP challenge was tested using knock-out animals.
Our results indicate that chemokines known to stimulate T cell tactism, in particular CCL3, CCL4, CCL5 and CXCL10, are overexpressed in the ventral mesencephalon after MPTP treatment. Yet, mice deficient either for CCR5 or CXCR3, two major chemokine receptors involved in T cell trafficking, were found to display as much dopaminergic cell loss and brain infiltrated T cells as their wild-type littermates after MPTP. Although one cannot exclude the possibility that CCR5 and CXCR3 are not involved in T cell brain extravasation in the MPTP mouse model of PD, our data could also suggest that compensatory and/or redundant mechanisms may underline these negative results. Further investigations are therefore needed to test such hypothesis. All forms of Alzheimer's disease (AD) have increased levels of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein 1 (MCP-1) in brain tissue, with MCP-1 being the most reliable disease predictor. The present study was designed to determine whether these or an additional 14 inflammatory mediators significantly differ in their brain levels and cellular expression between AD patients with mutations in the presenilin (PS1) gene (N=6) versus the more common sporadic disease (N=6) and to determine any relationship with the two pathologies. Soluble brain extracts were analysed for the levels of 17 cytokines and chemokines simultaneously using multiplex analysis (BIO-RAD 17-plex). Immunohistochemistry was carried out using specific antibodies against tumour necrosis factor-a (TNF-a), IL-6, A-beta and tau. Quantitative analysis of the densities of immunoreactive glia (TNF-a and IL-6), neurofibrillary tangles (tau) and cored and noncored plaques (A-beta), and the proportion of neurons containing TNF-a, IL-6 and tau immunoreactivity was carried out. Stepwise logistic regression modelling showed that increased levels of soluble TNF-a and IL-6 reliably predicted a diagnosis of PS1 AD versus sporadic disease (R2 = 0.967, p b 0.001). Similar modelling of the tissue pathologies identified the proportion of TNF-a immunoreactive neurons as the most reliable factor differentiating sporadic from PS1 AD. Across groups, increased neuronal TNF-a immunoreactivity correlated with the densities of IL-6-and TNF-a-immunoreactive glia and neurofibrillary tangle stage. Importantly, these differences were unrelated to the density of A-beta plaque pathology.
Our data demonstrate that the mutant PS1 protein enhances specific inflammatory mediators in the AD brain beyond that expected due to an increase in A-beta plaque pathology. These findings have important implications for PS1 models of AD and the roles of PS1 in inflammatory processes. Neuromyelitis optica (NMO), also named Devic disease, is a demyelinating disorder of central nervous system. Though its involvement is confined to optic nerve and spinal cord by definition, asymptomatic brain lesion is occasionally found in magnetic resonance imaging. Symptomatic brain lesion as the first manifestation of NMO is not well informed, but has a clinical implication for the treatment issue.
A 22-year-old woman presented with dizziness, dysarthria and sensory disturbance.
Neurologic examination showed bilateral spontaneous upbeat nystagmus and left facial hyperalgesia with brisk deep tendon reflex more pronounced in the right side. Magnetic resonance imaging (MRI) revealed diffuse T2 high signal intensity lesion over the lower medulla and upper cervical spinal cord. Under the diagnosis of brainstem glioma, the patient underwent craniectomy and biopsy. The pathologic finding was diffuse demyelination with axonal injury and scattered macrophage, consistent with ischemic or demyelinating disease. After a month, new abnormal hyperintense lesions in brainstem were noted in the follow up MRI. We diagnosed to multiple sclerosis and started interferon-beta. Despite disease modifying treatment, she had several attacks of long segment transverse myelitis, and optic neuritis. We correct the diagnosis as a neuromyelitis optica (NMO). She has remained in a relapse-free state over the following 25 months of treatment with high-dose corticosteroid and azathioprine.
Our NMO patient was initially misdiagnosed to brainstem glioma due to diffuse and extensive brainstem lesion. The clinician should mind a possibility of demyelinating disease in diagnostic approach to diffuse brainstem lesion, because differential diagnosis depending on MR imaging is limited and early, accurate diagnosis is critical to proper treatment. Inflammatory diseases of the central nervous system (CNS) often lead to neurological deficits, such as paralysis and autonomic and cognitive dysfunctions. During the onset of inflammation the tissue is infiltrated by peripheral immune cells that may cause local tissue destruction. After the resolution of CNS inflammation, the ineffectiveness of inherent neuroprotective and repair mechanisms results in a wide-spread accumulation of degenerating axons. Later, in the disease course there may be loss of specific axonal tracts such as corticospinal tracts (CSTs) which control motor activity. At this point neurological disabilities become irreversible. Development of neuroprotective interventions that limit axonal loss is critical for a better prognosis and prevention of permanent neurological disability.
To recapitulate the pathology of CNS inflammation, we induced experimental autoimmune encephalomyelitis (EAE) in mice. IFNg is a cytokine involved in the establishment of inflammation in peripheral immune organs. Interestingly, our data and studies by others have shown that IFNg −/− and IFNgR −/− mice with EAE suffer increased neurological severity compared to wild type controls. Additionally, EAE induction in the absence of IFNg/IFNgR results in the induction of a set of neurological deficits, not typically observed in controls. These deficits include axial rotation, head tilting and balance impairments suggesting malfunctions in the vestibular system. This protective effect was previously attributed to inhibition in the generation of pathogenic Th17 cells by IFNg. However, our data show that the generation rates of Th17 cells in IFNg −/− mice with EAE are only transiently higher than those of controls. Taken together, these data suggest that the beneficial effects of IFN may be exerted in the CNS by promoting neuroprotective mechanisms. To examine if this is the case, we induced EAE in irradiation bone marrow chimeric mice in which IFNgR is absent in the CNS but present in peripheral immune cells and vice versa. The degree of acute axonopathy in the spinal cord was revealed by SMI32 staining loss of CSTs by counting SMI312+ small diameter axons in the dorsal funiculus. Our data show that the absence of IFNg signaling specifically in the CNS resident cells is sufficient for the onset of atypical neurological deficits and increased clinical severity and axonal loss.
We conclude that IFNg signaling in the inflamed CNS contributes to the preservation of axons and limits tissue atrophy.
Mausberg Anne K., ⁎ Dorok Mareike, Dehmel Thomas, Kieseier Bernd C.
Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune mediated disorder affecting the peripheral nerves, causing motor weakness and sensory symptoms and signs. The precise pathophysiology of CIDP remains uncertain although T cells recognizing self antigens are believed to trigger the inflammation in the peripheral nerves and nerve roots. Currently, neither the target antigens nor the specific cell population responsible for the pathogenesis of CIDP has been identified.
To elucidate the role of contributing T cells the T cell receptor (TCR) repertoire of CD4+ and CD8+ T cells in the peripheral blood of patients with CIDP was analyzed using CDR3 spectratyping. As controls healthy age-and sex-matched individuals and controls with non-inflammatory polyneuropathies were also included. While the CD4 TCR length distribution was almost not altered for most of the V beta elements, the CD8+ population of CIDP patients displayed an impressive spectrum of oligoclonal expansions in all analyzed 24 V beta elements. This vast proliferation of different T cell clones indicated a broad activation of T cells especially in the CD8 population. A public expansion of a distinct TCR length in one V beta element within a majority of affected patients was not detectable.
This striking heterogeneous response in activation can either be caused by epitope spreading during the proceeding disease and/or imply that multiple peptides are potent to induce this autoimmune mediated disease. Interestingly, the cytotoxic CD8+ T cells demonstrated a much broader activation than CD4+ helper and regulatory T cells. As such our findings point to a potentially crucial role of CD8+ T cells in the immunopathogenesis of CIDP, underlining the importance of a cellcontact directed destruction of the peripheral nerve in the pathogenesis of chronic inflammatory neuropathies. University of Glasgow, Glasgow, United Kingdom; 2 Nagoya University School of Medicine, Nagoya, Japan; 3 University of Edinburgh, Edinburgh, United Kingdom
In the axonal variant of Guillain-Barré syndrome (GBS) antibodies to complex gangliosides are frequently found. Evidence shows that these antibodies have pathogenic effects on the structure and function of the neuromuscular junction (NMJ) and nodes of Ranvier. Mouse models of this disease are confounded by the fact that wild type (WT) mice, expressing the normal complement of gangliosides, are immunologically tolerant when immunised with exogenous gangliosides. Conversely, N-acetylgalactosaminyl-transferase knockout (Gal-NAcT −/− ) mice, lacking all complex gangliosides, produce high affinity antibody responses to complex ganglioside immunisation, but do not develop neuropathology as they lack the target antigen. Our current model therefore relies on passive immunisation into WT mice of anti-ganglioside antibody generated in GalNAcT −/− mice.
To overcome this double step, and assess the tolerogenic effect of gangliosides solely expressed in axons, we crossed a transgenic mouse expressing GalNAcT driven by an axonal neurofilament-light (NFL) promoter onto a GalNAcT −/− background to create GalNAcT Tg/KO mice. Rescue of neuronal ganglioside expression was demonstrated by thin layer chromatography of brain glycolipid, and by immunofluorescence staining of neuromuscular tissue, using an anti-GT1b ganglioside antibody. The sensitivity of the transgenic NMJ to anti-GT1b antibody and complement mediated injury was first established using ex vivo preparations of hemidiaphragm. Antibody bound to the pre-synaptic membrane, fixed complement, and led to membrane attack complex deposition in GalNAcT Tg/KO and WT mice, but not in GalNAcT −/− mice. In in vivo passive transfer studies with intraperitoneal (IP) anti-GT1b antibody plus complement, GalNAcT Tg/KO mice developed respiratory compromise, whereas GalNAcT −/− mice did not. Furthermore, immunisation of transgenic animals with GT1bliposomes, or GM1:GD1a complex liposomes, produces a vigorous antibody response indistinguishable from that seen in GalNAcT −/− mice, compared with a blunted response in WT mice. When provided with a source of complement delivered IP, GT1b immunised transgenic mice develop diaphragmatic paralysis.
This model demonstrates that local axonal expression of ganglioside does not induce systemic immunological tolerance, and in doing so allows for the production of an active immunisation model of motor axonal GBS in a single mouse line, through the re-sensitisation of axons to antibody mediated injury. Many neurodegenerative diseases are underpinned by a dysregulated inflammatory response in the central nervous system (CNS). However, it is imperative that this process is tightly controlled. The inappropriate or chronic deployment of the inflammatory system can lead to a loss of its protective and reparative function and its emergence as a destructive force in pathogenic processes such as multiple sclerosis (MS) and Alzheimer's disease. In the CNS, the Notch pathway is implicated in many aspects of cellular development and functions. Recent studies have led to recognition of the role of Notch pathway in neurodegenerative diseases. We and the others showed that Notch plays a critical role in steering an immune response toward inflammation by regulating expression of various cytokines and proinflammatory compounds. The reexpression of Notch components in neurodegenerative diseases seems to create an environment that induces neuronal death.
The aim of our study is to understand the implication of the Notch pathway in the adult CNS during neurodegenerative processes. We have previously shown, in vitro, that the inhibition of Notch ligand, Jagged1, inhibits the inflammatory reaction on astrocytes. Using a mouse model we have validated a method to modulate in vivo Notch ligands expression and given our previous results we have decided to study the effect of Jagged1 silencing. In our model, we have induced gliosis in the forebrain of B6 mice by a mechanical lesion and we have injected simultaneously different types of siRNA Jagged1 (siRNAJ1) in the lesion. Crude siRNAJ1 and Cholesterol-labelled siRNAJ1 have been used in combination with or without transfection reagent and at various concentrations to determine the most efficient inhibition. A kinetic study has been realized to study the modulation of Jagged1 expression in the lesion until 1 month after stab wound injury. This in vivo strategy enabled us to observe major modulations of the inflammatory process in the CNS. Jagged1 inhibition modulates microglial activation and astrogliosis within the lesion.
Our results show that Jagged1 plays an important role in the control of inflammatory reactions and could thus allow to develop new therapeutic strategies helping to diminish the gliosis and the inflammatory response in the CNS. YKL-40 (chitinase 3-like 1) is expressed in a broad spectrum of inflammatory conditions and cancers. We have previously reported that YKL-40 levels were elevated in the cerebrospinal fluid (CSF) of macaques and humans with lentiviral encephalitis. In the current study YKL-40 transcription and expression were evaluated in a variety of acute and chronic human neurological conditions as well as animal models of traumatic brain injury and experimental autoimmune encephalomyelitis (EAE).
CSF analysis revealed significant YKL40 elevation in multiple sclerosis patients as well as patients after traumatic brain injury. In situ hybridization showed YKL-40 transcription mostly associated with reactive astrocytes, that was more pronounced in inflammatory conditions like simian virus encephalitis and multiple sclerosis. Comparison of YKL-40 expression in different stages of brain infarction showed that YKL40 was abundantly expressed in astrocytes during acute phases and diminished to low levels in chronic infarcts. Similarly, YKL-40 transcription is primarily associated with reactive astrocytes in pericontusional cortex after controlled cortical impact or areas of reactive gliosis in the mouse model of EAE.
Taken together, these findings demonstrate that YKL-40 is induced in astrocytes during neuroinflammatory conditions.
Adjunct minocycline therapy affects anti-Abeta antibody induced microhemorrhage progression in TG2576 mice Vasilevko Vitaly ⁎ , Quiring Daniel, Cribbs David
Cerebral vascular pathology in the elderly increases the risk of microhemorrhages and vasogenic edema in response to anti-Abeta immunotherapy in AD patients. We have investigated whether the anti-inflammatory drug minocycline can attenuate Abeta-antibody immune complex-induced microhemorrhages in passively immunized Tg2576 mice.
Twenty one months old APP Tg2576 mice with mild to moderate CAA in the meningeal and pial vessels and extensive parenchymal pathology were passively immunized (PI) with an anti-Abeta40 Cterminal specific monoclonal antibody of IgG1 isotype. Mice were dosed weekly i.p. at 10 mg/kg per mouse for 9 weeks. Minocycline supplemented (0.5 g/kg) or control diet (con) was given to mice ad libitum starting from the day of first passive injection. There were 13 mice per group at the beginning of experiment, and 10 mice in each group at the end of the experiment. AD-like amyloid pathology was analyzed for plaque load, microglial activation markers and microhemorrhages.
Comparison of neuropathology in the frontal cortex and hippocampus of Tg2576 mice showed a significant clearance of 4G8positive and anti-AbetaX-42-positive amyloid plaques after passive immunization. However, in the frontal cortex of the PI-Tg2576 only group there was significantly lower (11.6%) plaque load than the PI-Tg2576 + minocycline (17.9%), and similar results were observed in the hippocampus (3.3% versus 5.8%). Passive immunotherapy alone reduced the 5D4 positive microglial activation, while in combination with minocycline it demonstrated even more pronounced effect. While both PI groups had extensive vascular CAA and antibody induced microhemorrhages, the minocycline intervention did not change the number of microhemorrhages, but decreased the size of these pathological lesions.
Chronic anti-inflammatory therapy reduced immunotherapyinduced microhemorrhage progression in Tg2576 mice, however minocycline also attenuated plaque clearance. Our data suggest microglial involvement in antibody mediated plaque clearance in older mice and progression of microhemorrhage lesions. Pregabaline is a new agent used for the treatment of pheripheral neuropathic pain. In this study for 9 months period we aimed to investigate the adverse effects of pregabaline on the neuropathic pain patients.
After the examination of patients at a pain policlinic, adverse effects were asked on their first control.
Pregabalin's adverse effect has been determined 38 of … patients whom were admitted to a policlinic. These adverse effects are: (1), dizziness (6), vertigo (15), somnolance (2), nausea vomiting (3), hypotension (2), headache (2), weakness (3), hallucination (2), extremity edema, and constipation (1).
Pregabaline's adverse effects are importantly determined because these are used for a long time for chronic pain patients. In this study pregabaline's adverse effects were detected but there were those that were not very important so that pregabaline administration in some patients had to be ceased. Recently the proteasome inhibitor Bortezomib, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease (Neubert et al., 2008) . Bortezomib depletes both short and long-lived plasma cells, which normally survive the standard immunosuppressant treatments. These findings encouraged us to test the potential effects of Bortezomib in the experimental autoimmune myasthenia gravis (EAMG) model.
Lewis rats were immunized with saline (Control, n = 36) or Torpedo AChR (EAMG, n = 54) in complete Freund's adjuvant in the first week of the experimental period of eight weeks. After immunization rats received twice a week subcutaneous injections of saline solution ('Untreated'), Bortezomib (0.2 mg/kg in saline, 'Prevention' of EAMG) or a combination of saline for the first four weeks and then Bortezomib for the rest of the experiment ('Treatment' of EAMG).
The clinical condition in the EAMG model was significantly improved by Bortezomib in the Prevention group compared to the Untreated group (p= 0.03), and in the Treatment group also a slight amelioration was observed. This was correlated with the observation that Bortezomib efficiently reduced production of anti AChR antibody titers (by 67% in the Prevention group and by 65% in the Treatment group) and improved neuromuscular transmission in the prevention group. Finally, Bortezomib induced apoptosis in bone marrow cells (but not in spleen or thymus) and reduced the amount of plasma cells both in bone marrow (80% reduction) and in peripheral blood (50% reduction).
Our results indicate that Bortezomib can improve EAMG symptoms and significantly reduce autoantibody titers, therefore proteasome inhibition is a promising therapeutic strategy to target plasma cells in MG.
Celecoxib analogue lacking COX-2 inhibitory activity suppresses inflammatory disorders by inhibiting inflammatory cytokines Previously we reported that both celecoxib and a trifluoromethyl analogue of celecoxib (TFM-celecoxib) lacking COX-2-inhibitory activity induce cellular retention of IL-12 and IL-23. Because these cytokines are involved in autoimmune or inflammatory diseases, we asked whether TFM-celecoxib suppresses animal models of human autoimmune diseases. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunizing with MOG35-55-peptide (100 μg) in complete Freund's adjuvant (CFA) containing mycobacterium tuberculosis H37Ra followed by pertussis toxin administration shortly after the immunization and 48 h later. To induce collageninduced arthritis (CIA), DBA1/J mice were immunized with bovine type II collagen (CII) (150 g) in CFA on day 0 and CII (150 g) in IFA on day 21. Antibody-induced arthritis (AIA) was induced in C57BL/6 mice by injecting anti-CII antibodies followed by lipopolysaccharide (LPS) administration 2 days later. Mice received 10 μg/kg of TFM-celecoxib or celecoxib every other day from day 1 in EAE and AIA experiments, every day from day 21 in CIA experiments. The control animals were injected with the vehicle alone. Splenic dendritic cells (DCs) of mice treated with TFM-celecoxib or celecoxib 1 and 3 days earlier were stimulated by LPS in vitro. Results: Both TFM-celecoxib and celecoxib treatments suppressed the clinical features of EAE, and TFM-celecoxib treatment suppressed cell infiltration and demyelinization in the spinal cords compared to vehicle treatment. Although lymph node cells form TFM-celecoxib and celecoxib-treated mice produced less IFNγ and IL-17 when rechallenged by MOG in vitro, the inhibitory effects of TFM-celecoxib on recall responses were more potent than those of celecoxib. The severity of clinical and pathological features of CIA and AIA were reduced both in TFM-celecoxib and celecoxib groups. However, the TFM-celecoxib treatment showed stronger suppressive effects on arthritis, and this was more obvious in AIA. TFM-celecoxib treatment suppressed the LPS-induced production of inflammatory cytokines including IL-1α by DCs compared to celecoxib or vehicle testament. This may explain the more profound disease inhibitory effects by TFM-celecoxib in AIA model. TFM-celecoxib possesses the ability to inhibit inflammatory disease models not only by decreasing the production of IFNγ and IL-17 by T cells, but also by suppressing inflammatory cytokines by innate immune cells. We have reported that the glycogen synthase kinase-3 (GSK3) inhibitor, lithium, is therapeutic in acute and relapsing-remitting experimental autoimmune encephalomyelitis (EAE) and that relapsing-remitting multiple sclerosis (RRMS) can be segregated into a T helper type 1 (Th1) disease or a T helper type 2 (Th17) disease. The goal of this study is to determine if lithium differentially regulates Th1 versus Th17 disease.
In order to determine the therapeutic effect of lithium inTh1 EAE versus Th17 EAE we performed passive transfer studies where we transferred Th1 or Th17 encephalitogenic T-cells into lithium-treated or untreated naïve recipients. We determined that lithium treatment is effective in attenuating EAE induced by Th1 encephalitogenic Tcells but not Th17 cells. Ex-vivo analysis (3H-Thymidine proliferation assay and ELISA) of MOG33-55-reactive Th17 cells indicated that they hyperproliferate and secrete more IL-17 in the presence of lithium. We recently identified a novel cooperative relationship between type 1 and type 2 interferons (IFNs) in the immunopathogenesis of EAE. Therefore to further dissect the mechanism of lithium's protection on EAE, we evaluated the development and progression of EAE in mice lacking IFN-a/b signals (IFN-aR1 −/− mice), lacking IFN-g signals (IFN-gR1 −/− mice) or lacking both IFN-a/b and IFN-g signals (IFN-aR1gR1 −/− mice) treated with lithium at the onset of disease, compared to untreated mice. We found that lithium administered after onset of clinical disease equally suppressed EAE in IFN-aR1 −/− mice, IFN-aR1gR1 −/− mice and wild type mice. In contrast, in IFN-gR1 −/− mice, while lithium reduced partially the severity during the initiation phase of EAE, it was completely ineffective in the chronic/effector/ progressive phase of the disease.
The data suggests that cooperatively type 1 and type 2 IFNs are involved in lithium-dependent therapeutic effectiveness in EAE. Importantly, lithium, a selective inhibitor of GSK3, suppresses chronic/progressive EAE by IFN-g mediated signals, and is not able to suppress Th17 disease in mice. This finding has crucial implications for identifying what type of MS patients will mostly benefit from GSK3-targeted therapy. Encapsulation into polyethylene-glycol-coated long-circulating liposomes is a strategy to increase efficacy and alter the therapeutic profile of pharmaceutically active drugs. Recent work demonstrated that this approach is also promising for glucocorticoids (GCs), the mainstay in the treatment of acute relapses in multiple sclerosis (MS) patients. Here we have used experimental autoimmune encephalomyelitis (EAE) to explore the mechanisms that may account for the superior therapeutic efficacy of liposomal GCs as compared to free compounds.
Treatment of MOG35-55-induced EAE in C57Bl/6 mice by a single injection of liposomal prednisolone was as potent as an application of a ten-fold higher dose of free dexamethasone at three consecutive days. Nevertheless, both drug preparations similarly act through the classical cytosolic GC receptor (GR) as shown by the use of GR knockout mice. Interestingly, conditional mutagenesis of the GR demonstrated that modulation of T cell function is no stringent prerequisite for therapeutic efficacy of liposomal GCs. These findings contrast to our previous observations that peripheral T cells are essential targets of free dexamethasone. Further analysis revealed that major features of macrophage function such as cytokine and chemokine expression, NO production and MHC class II surface levels are strongly affected by liposomal GCs. Consequently, specific deletion of the GR in myeloid cells also partially impaired EAE therapy by liposome-encapsulated, but not by free GCs. Liposome-encapsulated GCs had similar side effects on certain parameters of liver metabolism as compared to higher doses of free GCs, indicating that the new formulation may not reduce all adverse effects of GC therapy.
Collectively, our findings demonstrate that liposomal encapsulation of GCs alters the therapeutic targets in the treatment of CNS autoimmunity and may allow a more focused and stringent GC therapy of MS patients. Statins (HMG coenzyme A reductase inhibitors) are known to reduce serum low-density lipoprotein and are widely used in the treatment of hypercholesterolemia. Associations between Myasthenia Gravis (MG) and statins have been described in case reports and small case series, and they concluded that stains were safe in most cases but they might aggravate MG.
We retrospectively investigated 247 MG patients (all Japanese) with elavated AchR antibody being treated at Chiba University Hospital, and among them 33 patients (13%) were using statins or had a history of statin using. As to the previous reports, we defined statin-induced MG worsening as it happened within 4 months after statin use.
In our investigation, only 1 patient had a history of MG worsening within 4 months after statin use. But her MG worsening occurred in the course of steroid reduction after high dose steroid taking therapy for 1 month during thymectomy. Then she had a re-increased dosage of steroid together with cyclosporin-A, continued statin using, her MG worsening ceased and she achieved steroid reduction again in the ordinal course. Thus we considered that her worsening was caused by steroid reduction, not by statin using.
Hypercholesterolemia caused by steroid therapy is still common among MG patients, and statins are very effective solution for that, we consider that statin could be used positively to MG patients with hypercholesterolemia, since statin using might have little relationship with MG worsening from our investigation. Glatiramer acetate (GA) therapy in multiple sclerosis (MS) is considered to lead to systemic alterations in circulating monocytes. It is the aim to infer accessory regulatory pathways of applied drug in human MS blood monocytes. Hence, genome wide interrogation of monocyte messenger RNA transcripts in time offers a substantial view on both the complexity of MS and potential impact of GA. To further elucidate the molecular interactions, specificities of transcription factor binding sites (TFBS) in the promoter regions of GA-responsive genes were aimed to be characterized.
Employing Affymetrix HGU133Plus 2.0 arrays, eight MS patients were monitored ex vivo receiving subcutaneous GA. Peripheral blood samples were processed before treatment and correspondingly before each upcoming injection after one day, one, four and eight weeks. Magnetic activated cell sorting (CD14) was applied to isolate the monocyte cell fractions. Transcriptome analyses included gene expression intensity corrected filtering (MAID), gene functional analysis (Gene Ontology-GO) and construction of molecular interaction networks using R software packages, Pathway Studio, and an algorithm for inferring gene regulatory networks that integrates TFBS (TILAR).
During two months of treatment 418 differentially expressed genes (DEGs) were attained, 48 of which were consistently detected to be significantly altered. Supporting previous evidence with respect to GA activation properties, CD163, a marker of monocyte activation, was initially downregulated in most patients. Further of note were upregulated genes such as IL-18, IL-21, and IL-27 which are considered essential in Th1 and Th17 cell differentiation. DNA binding motifs of transcription factors that function as molecular switches for antiviral activity were overrepresented in the genes' promoter regions, e.g. IRF-1/-2/-7/-9, which were linked to 35 DEGs (e.g. CD68, IDO, IL2RG, OASL, and PPM1K). Several other-natured TFs were also assigned.
Immune system related groups of functionally related genes (GO categories) significantly changed coordinatedly over time and gene interaction exploring analysis using DEGs only revealed time dynamic networks with high connectivity centering biologically important genes such as CD34, CXCL10, CYP2, NOS1, OXT, PAM, PRKCA, VCAM, and WARS.
Significant changes of monocyte RNA profiles in time revealed hitherto unrecognized GA-affected transcripts and key regulators that contribute to its mechanism of action in MS.
Muramoto Kenzo ⁎ ,1 , Daun Jane 2 , Zheng Wanjun 2 , Shirota Hiroshi 2 , Gusovskey Fabian 2 , Hishinuma Ieharu 2 , Kobayashi Seiichi 1 1 Eisai Co., LTD., Tsukuba-shi, Japan; 2 Eisai Inc., Boston, United States
IKKβ is a key protein kinase that regulates the activation of NFκB. NFκB is involved in cytokine production and up-regulation of adhesion molecules including ICAM-1 andVCAM-1. E6070 is a novel potent and selective IKKβ inhibitor (IC50 = 0.11 μM) and has shown good efficacy at inhibiting several inflammatory cytokines and adhesion molecules. Multiple sclerosis is a demyelinating disease that is exacerbated by the migration of leukocytes to central nervous system through adhesion molecules and chemokines. E6070 inhibited TNF-induced expression of ICAM-1 and VCAM-1 on human umbilical vein endothelial cells and activated T cells induced MCP-1 production from mice microglia.
To evaluate E6070 in a model of MS it was administered prophylactically in the MOG (myelin-oligodendrocyte-glycoprotein)-induced EAE model and significantly inhibited the development of EAE symptoms starting at doses of 1 mg/kg. Histological analysis demonstrated that E6070 inhibited the infiltration of leukocytes into the spinal cord. FACS analysis of the infiltrating leukocytes revealed that both T cell (CD3+) and dendritic cells (CD11c+) were reduced in the spinal cord after administration of E6070. Furthermore, therapeutic administration of E6070 in PLP (proteolipid protein)-induced EAE model showed decreased disease severity.
To determine if E6070 can affect the induction of Th1 and Th17 cells in MOG-EAE model, splenocytes from MOG-immunized mice were activated in vitro with MOG peptide. The secretion of both IFN-g and IL-17 was reduced in the splenocytes from the E6070-treated animals compared to vehicle-treated animals.
This result suggests E6070 inhibited antigen-dependent Th1 and Th17 differentiation in this model. E6070 has potential to be a promising therapeutic for the treatment of multiple sclerosis.
Early and aggressive treatment with IAPP and IVMP combination therapy for NMO-Clinical efficacy of combined therapy and serum IL-10
Ohji Satoru ⁎ , Kubota Akihiro, Kojima Miki, Isaki Shoko, Nomura Kyoichi Department of Neurology, Saitama Medical Center, Saitama Medical University, Saitama, Japan
Purpose: Plasmapheresis has recently become available as a therapy for neuromyelitis optica (NMO). We had reported the changes of serum cytokines before and after immunoadsorption plasmapheresis (IAPP) in patients with the active phase of NMO. Serum level of IL-10 increased immediately just after IAPP treatment compared with before IAPP. The removal of anti-AQP4 antibody with IAPP induced clinical improvement, however the increase of serum IL-10 level just after IAPP might promote the re-exacerbation with increased production of anti-AQP4 antibody.
In this present study, we performed intravenous methylpredonisolon (IVMP) treatment just after IAPP, in addition as an "Early and aggressive treatment". And we studied the clinical efficacy after the combination therapy and the changes of serum cytokines during the combination therapy compared with IAPP therapy only. Methods: Six of combination therapies were performed in 3 patients with the active phase of NMO. The clinical efficacy was estimated with neurological examination and symptoms. Serum levels of cytokines 2, 4, 5, 6, 8, 10, 12, were measured using the Beads Array method, just before treatment, during treatment, and after the combination therapy. Results and discussion: In the study of the clinical efficacy, neurological examination and symptoms were improved earlier the combination therapy than IAPP therapy only. In the study of the changes of serum cytokines, serum level of IL-10 has increased immediately just after IAPP therapy only, and then has decreased just after IVMP treatment. The earlier neurological improvement might be provided with not only the removal of anti AQP4 antibody with IAPP, but also the suppression of serum IL-10 with IVMP.
Conclusion: The early and aggressive treatment, the combination therapy with IAPP and IVMP might provide early neurological improvement compared with IAPP therapy only. The increase of serum IL-10 after IAPP was suppressed with combined IVMP treatment. Interferon beta (IFNβ) therapy has been accepted as the first-line treatment for patients with multiple sclerosis (MS). However, a proportion of IFNβ-treated patients show a poor clinical responses. On the other hand, ramatroban, which is known as a thromboxane A2 receptor antagonist, can suppress the expressions of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in endothelial cells. Further, ramatroban prevents exacerbation of inflammation by blocking the above mentioned molecules. We investigated whether administration of ramatroban could prevent relapse of MS via inhibitoin of lymphocytes and macrophage invasion through the blood brain barrier (BBB) in the central nervous system (CNS).
A total of 5 patients in whom MS was clinically confirmed (according to McDonald's criteria) were enrolled in an open-label study. All the patients were treated with administration of ramatroban (150 mg/day p.o.) in addition to the conventional MS therapy. The number of relapses was used as the primary outcome measure. The patients were monitored for the development of any adverse effects.
In 1 patient, ramatroban therapy was discontinued because of acute eruptions resulting from drug allergy. However, in 4 of the 5 patients, ramatroban treatment was continued without the development of any adverse effects. In the case of the 4 patients who completed 2 years of the therapy, a significant difference in the mean (standard deviation [SD]) annual relapse rates was noted before and 2 years after the treatment (4.0[2.8] and 0.8[0.5], respectively).
Ramatroban might be useful as an additional therapy to decrease clinical and laboratory exacerbations; further, it has been found to be reasonably safe for Japanese patients with MS. ELND002 is a potent and selective pegylated small molecule alpha-4 integrin inhibitor currently being evaluated for the treatment of multiple sclerosis. ELND002 is expected to inhibit leukocyte entry into the inflamed central nervous system through the blockade of alpha4beta1-mediated transendothelial migration. ELND002 binds to guinea pig and human leukocytes with similar affinity, thus we sought to determine the efficacy of ELND002 in a guinea pig model of experimental autoimmune encephalomyelitis (EAE), which mimics several pathological processes associated with multiple sclerosis.
Guinea pig EAE was induced by injection of spinal cord homogenate in complete Freund's adjuvant. Weekly subcutaneous injections of ELND002 at 1 and 10 mg/kg administered during the chronic phase of guinea pig EAE resulted in rapid and significant reductions in clinical scores when compared to the vehicle-treated group. In the interim period between weekly doses, clinical score reductions were sustained in the 10 mg/kg group but lasted only 4-5 days in the 1 mg/kg group. This is consistent with the low drug plasma levels observed at trough in the low dose animals. Histopathological examination of the spinal cords at the end of the treatment period revealed that both inflammatory cell infiltration and microglial activation were significantly reduced in ELND002-treated groups compared to the vehicle group.
We conclude that ELND002 is efficacious in the guinea pig EAE model.
ELND002 is a potent inhibitor of a4 integrin-mediated human leukocyte adhesion in vitro Garrido Caroline, Mao Jennifer, Xu Yi-Zheng, Nguyen Van, Yednock Ted, Bard Frédérique Elan Pharmaceuticals, South San Francisco, United States ELND002 is a potent and selective pegylated small molecule alpha-4 integrin inhibitor currently being evaluated for the treatment of multiple sclerosis. Entry of inflammatory leukocytes into the central nervous system through vascular transmigration is highly dependent on the interaction of a4β1 integrin-expressing leukocytes with VCAM-1expressing endothelial cells. a4β7 integrin-mediated leukocyte adhesion is not thought to play a major role in this pathological process.
We observe preferential binding of ELND002 to a4β1 integrin over a4β7 integrin on a4 integrin-expressing human whole blood lymphocytes. In addition, ELND002 inhibits the adhesion of a4β1positive Jurkat cells to immobilized human VCAM-1-Fc with an IC50 of 0.5 nM, while ELND002 inhibits the adhesion of a4β7-positive 8866 cells to immobilized human MadCAM-Fc with an IC50 of 38 nM. ELND002 did not inhibit the adhesion of aLβ2-expressing 8866 cells to human ICAM-1-Ig or the adhesion of a5β1-positive THP-1 cells to human fibronectin.
Thus ELND002 is a specific and potent inhibitor of a4 integrinmediated leukocyte adhesion characterized by a 30 to 80 fold greater potency towards a4β1 over a4β7 integrin. These properties may provide a novel means to treat MS patients by specifically inhibiting a4β1-mediated leukocyte trafficking into the CNS while preserving a4β7-mediated trafficking and function. Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Current treatments for MS are nonspecific in their action and have significant side effects on normal immune function. Antigenspecific immunotherapeutic agents that change the response of pathogenic CD4+ T cells could help to reduce these side effects. The best way to target these cells specifically is to use modified peptides to target the interaction between the T cell receptor and the MHC; however, peptide-based reagents typically are poorly immunogenic. Our work aims to enhance the bioavailability and effectiveness of immunomodulatory peptides that modulate CD4+ T cells and have potential therapeutic application in MS. We have used a well established altered peptide ligand (APL) model that has been shown to be therapeutically effective in experimental autoimmune encephalomyelitis (EAE) to test the efficacy of our approach.
Myelin proteolipid protein (PLP)-specific APL was modified by attaching palmitic acid via a thioester bond to a cysteine residue of the peptide. The ability of these thiopalmitoylated (S-palm) APL to modulate pathogenic immune responses in vitro and show clinical efficacy in an SJL/J mouse model of MS was then compared with that of nonacylated APL and controls.
The serum half-life of S-palm APL was approximately 20-fold longer than that of APL alone. Furthermore S-palmAPL induced stronger proliferative responses than APL, induced greater numbers of regulatory T cells (N15% difference), and induced a 10-fold increase in the production of the anti-inflammatory cytokine interleukin 10 (IL-10) compared to APL alone. Therapeutically, S-palmAPL used at a 50-fold lower concentration was as effective as APL in preventing disease in the animal model. In addition, S-palmAPL was also more effective at treating established disease, resulting in full recovery 3-5 days sooner than APL treated mice.
S-palm APL retain and improve on the beneficial effects of APL, both in vitro and in vivo, and have good potential as a method to enhance the therapeutic effects of specific peptide-based agents in diseases such as MS.
Genetic engineering of a soluble tumor necrosis factor receptor 2 selective TNF with membrane TNF mimetic activities Multiple sclerosis is characterized by an autoimmune response against oligodendrocytes, the myelinating cells of the central nervous system (CNS), resulting in demyelination and consecutive neurodegeneration. Repair of demyelinated tissue requires de novo differentiation of oligodendrocyte progenitor cells, which form new myelin sheets when they access an axon. A regenerative role in the CNS has been postulated for tumor necrosis factor receptor 2 (TNFR2) by inducing oligodendrocyte precursor proliferation. Accordingly TNF variants selectively activating TNFR2 could potentially be useful as therapeutic regimen in neurodegenerative diseases.
Soluble recombinant TNF is a strong mediator of inflammation, predominantly through TNFR1 activation, as soluble TNF is not sufficient to activate TNFR2. In contrast, the membrane bound form of TNF (memTNF) fully activates both, TNFR1 and TNFR2. Therefore, TNFR2 specific therapeutics need to comply with two basic requirements, mimicry of memTNF and receptor selectivity in order to avoid dose limiting severe inflammatory responses.
Trimer stability and oligomerization status are crucial determinants of TNF receptor activation. As a basis for construction of a membrane mimetic, TNFR2 selective TNF variant, we used a singlechain TNF (scTNF) molecule, consisting of three TNF monomers fused by short peptide linkers. We have previously shown that scTNF possesses increased stability in vitro and in vivo in comparison to wild-type TNF. Introducing two amino acid exchanges (D143N/ A145R) into a scTNF variant results in loss of TNFR1 affinity under retension of TNFR2 binding. To mimic memTNF we linked such a receptor selective single chain TNF (scTNFR2) to the tenascin-C (TNC) trimerization domain, resulting in stabilized TNC-scTNFR2 nonamers with respect to TNF domains.
In vitro we could demonstrate that TNC-scTNFR2 mimics memTNF and exclusively activates TNFR2, evident from TNFR2 complex formation, TRAF2 recruitment and NFkB activation. In ongoing studies the in vivo properties of TNC-scTNFR2 are presently investigated. In particular, appropriate disease models in TNFR2 transgenic mice will reveal the usefulness of TNC-scTNFR2 as a potential therapeutic of neurodegenerative diseases.
Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies Perez-Garmendia Roxanna ⁎ ,2 , Ibarra-Bracamontes Vanessa 2 , Vasilevko Vitaly 1 , Luna-Muñoz Jose 3 , Mena Raul 3 , Govezensky Tzipe 2 , Manoutcharian Karen 2 , Cribbs David 1 , Gevorkian Goar 2 1 The Institute for Memory Impairments and Neurological Disorders, University of California Irvine, Irvine, United States; 2 Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico, Mexico; 3 Department of Neurosciences, CINVESTAV-IPN, Mexico, Mexico N-truncated/modified forms of amyloid beta (Aβ) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full-length peptides. Thus, N-terminally truncated/modified Aβ peptides represent highly desirable and abundant therapeutic targets. In the present study we have focused on N-truncated/modified Aβ peptide bearing aminoterminal pyroglutamate at position 11 (AβN11(pE)).
We produced AβN11(pE)-specific polyclonal antibodies in rabbits, and identified epitopes recognized by these antibodies using the library of random heptapeptides displayed as a fusion to the minor coat protein of M13 phage. Two B-cell epitopes recognized by anti-AβN11(pE) antibodies were determined: the first one located in the amino-terminal part and the second one in the central part of the peptide. Peptide inserts of selected positive phage clones are mimicking antigenic properties of these epitopes. In addition, we demonstrated that anti-AβN11(pE) antibodies inhibited AβN11(pE)-induced cytotoxicity in IMR-32 differentiated neuroblastoma cells, and recognized naturally occurring amyloid aggregates present in brain samples from AD patients. Importantly, anti-AβN11(pE) rabbit polyclonal antibodies bound also to full-len7th AβN1-42 and N-truncated/ modified AβN3(pE), suggesting that the three peptides may share a common B-cell epitope, and immunization with selected mimotopes may induce cross-reacting antibodies binding to all three major forms of Aβ.
Understanding the antigenic and immunogenic properties of mimotopes selected in this study may help to elicit B-cell response mimicking anti-AβN11(pE) one, which will represent a promising immunotherapeutic approach for the disease treatment and/or prevention. We believe our results are potentially important for developing novel immunogens targeting N-truncated/modified Aβ aggregates, since the most commonly used immunogens in the majority of vaccine studies for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. Antigen-specific therapy in multiple sclerosis holds promise for inhibiting autoimmune responses without affecting global immune function. The main autoantigens in MS include myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocytes protein (MOG). In experimental models of MS myelin peptides applied epicutaneously induced antigen-specific immune tolerance. In this 12month study we have treated 30 relapsing-remitting MS patients with a mixture of three myelin peptides (MBP85-99, PLP139-151, and MOG35-55), or placebo, applied once a week to the skin. Throughout the study in all patients we have assessed immune responses in the skin, lymph nodes and peripheral blood immune cells.
We have found using immunocytochemistry that myelin peptides activated dendritic Langerhans cells in the skin at the site of immunization. In local lymph nodes epicutaneous treatment with myelin peptides induced a unique population of granular dendritic cells. In the periphery epicutaneous immunization with myelin peptides led to the generation of IL-10 producing, type 1 regulatory T cells, suppression of myelin-specific CD4+ T cell proliferation and suppression of IFN-gamma and TGF-beta production.
We show for the first time that epicutaneous immunization with myelin peptides induces immune tolerance and inhibits autoimmunity in MS patients.
Neuroprotective autoimmunity after alemtuzumab treatment of multiple sclerosis
Mosely Suzanne ⁎ , Jones Joanne, Anderson Jane, Phuah Chia-Ling, Webber Daniel, Compston Alastair, Coles Alasdair
A phase 2 clinical trial (CAMMS-223) has shown the lymphocytedepleting humanised monoclonal antibody alemtuzumab to be highly effective in the treatment of early relapsing-remitting multiple sclerosis; reducing the risk of relapse and accumulation of disability by N70% compared to interferon beta-1a. Furthermore, those treated with alemtuzumab lost disability over the course of the trial. This was associated with increased brain volume on imaging, suggesting tissue restoration. This unprecedented observation led us to hypothesise that alemtuzumab promotes brain repair by enhancing neuroprotective autoimmunity; that is by the production of neurotrophins by lymphocytes.
We interrogated this hypothesis with ex-vivo peripheral blood mononuclear cell (PBMC) cultures, from patients before and after alemtuzumab. We analysed (i) neurotrophin production by ELISA of supernatants; (ii) brain-derived neurotrophic factor (BDNF) secretion by intracellular flow cytometry; (iii) the expression of growth factor mRNA by semi-quantitative RT-PCR of separated cell populations and (iv) the functional effect of PBMC conditioned media on in vitro rodent neuronal and oligodendrocyte precursor cell (OPC) cultures.
PBMCs regenerating after alemtuzumab secreted increased levels of BDNF (623.5 pg/mL vs. 202.1 at baseline; p = 0.004), plateletderived growth factor (136.3 pg/mL vs. 22.6 at baseline; p = 0.003) and ciliary neurotrophic factor (CNTF: 14.8 pg/mL vs. undetectable at baseline; p = 0.004) when stimulated with myelin basic protein (MBP). Intracellular flow cytometry identified CD4+ T cells as the main source of BDNF. BDNF was also found to be produced, to a lesser extent, by CD8+ T cells, B cells and monocytes. T cell, but not B cell or monocyte, BDNF mRNA expression increased after alemtuzumab, particularly in response to MBP. CNTF was only produced by T cells. Post-treatment derived PBMC-conditioned media increased the survival of rat neurones (by N3 fold), and the survival (by N1.5 fold) and maturation of oligodendrocyte precursor cells in culture, compared to pre-treatment derived media (all p b 0.001). These effects were partially reversed by blocking antibodies against BDNF and CNTF.
These findings support the hypothesis that alemtuzumab promotes neuroprotective autoimmunity through the increased secretion of neurotrophins. Thus the improvement in disability after alemtuzumab may, in part, be explained by increased T cell delivery of neurotrophins to the central nervous system. Proper treatment for the protection of axonal loss associated with neuroinflammatory diseases such as multiple sclerosis (MS) still remains a mystery. Survivin is a member of IAP family proteins whose neuro-protective effects have not been explored yet.
In this paper, we apply the dnSurR9-C84A, a survivin BIR motif mutant to demonstrate its neuro-protective role against cytotoxicity effects of GranzymeB derived from activated T-cells. First, we show that Granzyme B is responsible for the toxicity of activated T-cells by activating the apoptosis pathway. Next, we found that pre-treatment with dnSurR9-C84A protects neuronal cells, and leads to reducing the population of dead cells, decreasing the level of mitochondrial depolarization, expressions of CyclinD1 and Caspase3, and also balancing the cytosolic Ca2+ homeostasis.
Our findings suggest dnSurR9-C84A as a promising treatment for protecting neuronal cells against Granzyme B cytotoxicity, and open a new window for the treatment of neuroinflammatory diseases. Besides its strong antioxidative properties it has a broad range of other biological effects that are beneficial to human health, e.g. antiinflammatory, antithrombotic and anticarcinogenic properties. The development of neuroprotective therapies for multiple sclerosis (MS) represents one of the most important goals in MS treatment research.
In this project, we examined the neuroprotective effects of hydroxytyrosol in primary cortical cultures of mice with a neuronspecific viability assay, the MAP2-ELISA. We could show that preincubation with 10 μM HYT for 24 h significantly promotes neuronal survival under oxidative stress conditions (H2O2) and in a model of neuroinflammatory stress (i.e. in a co-culture with activated human neutrophils). On the basis of these results we performed a genomewide expression analysis (Affymetrix® whole-genome microarray) of HYT-treated cortical cells under oxidative stress conditions and in control cells. The analysis revealed that pre-incubation with HYT activates several components of the antioxidative defense system, i.a. enzymes of the glutathione metabolism (Gclm and Gsta4), antioxidative enzymes (Srxn1 and Prdx6) and Hmox1, thus "priming" cells for the following oxidative stress treatment. On the other hand, HYT leads to a downregulation of important cell cycle factors (e.g. Aspm, Cenpf, Bub1, and Tpx2), thereby presumably enhancing "pro-survival" repair mechanisms rather than "pro-apoptotic" damage-response.
Based on the above data and since HYT is orally available, crosses the blood-brain-barrier and appears to have a very favorable safety profile, we consider it an interesting candidate for future neuroprotective therapy trials.
Optimising regulatory macrophages activation phenotype for immunotherapy in autoimmune diseases Parsa Roham, Andresen Pernilla ⁎ , Harris Robert A.
Macrophages are important antigen-presenting cells with the ability to surface express or secrete a variety of effector molecules that either drive or modulate pathogenesis. In more recent years non-classical regulatory phenotypes have been described and these represent a new immunotherapeutic opportunity for treatment of chronic autoimmune diseases. We thus investigated the induction and regulatory properties of these non-classical states using cytokines such as IL-4, IL-10, IL-13, TGF-β or modulatory agents such as Dexamethasone and Vitamin D, either alone or in combination, in order to discern the optimal regulatory macrophage activation regimen.
Readouts included surface receptor expression, cytokine release, enzymatic and T cell suppressive activities. We determined a combination of IL-4, IL-10 and TGF-β to yield the optimal regulatory macrophage phenotype. Interestingly, the stability of this phenotype was enhanced when an additional Toll receptor agonist was subsequently applied, with consequent significant increase in IL-10 secretion. TGF-β stimulation was necessary in order to induce significant secretion of activated TGF-β. These cells had distinct profiles of CD24, PD-L1/2 surface expression and could efficiently suppress antigen-specific T cell recall proliferation. We have also investigated the immunotherapeutic use by performing adoptive transfer of regulatory macrophages in experimental models of autoimmune diseases.
We have found a new way to induce a regulatory macrophage phenotype. Further research will keep elucidating its possible immunotherapeutic use in therapies of autoimmune diseases. Universty of Foggia, Foggia, Italy; 2 University of Bari, Bari, Italy Background: ATP and its cognate receptors are involved in the inflammatory process. In particular, the purinergic P2X7 receptor (P2X7R) is thought to play an important role in macrophage/ microglial function by regulating cytokine production and apoptosis. P2X7R is known to be upregulated during inflammation and antagonists of this receptor may serve as novel anti-inflammatory agents. Experimental evidences show that P2X7R is certainly involved in Multiple Sclerosis (MS) pathology. Aims: To investigate the expression of P2X7R, IL-1beta and ectoapyrase CD39 on peripheral blood monocytes of MS patients and to see whether Glatiramer Acetate (GA) therapeutic effects in MS may be possibly mediated by mechanisms interfering with the ATP/P2X7R interaction and hence with the induction of IL-1beta or with the ATP activity modulating CD39. Patients and methods: Twelve RR and treatment-free MS patients have been selected and peripheral blood monocytes have been obtained. The expression of P2X7R, IL-1beta and CD39 on monocytes has been investigated by qrt-PCR. Thereafter, the in vitro effects of GA on the expression of P2X7R, IL-1beta and CD39 of BzATP (the most potent P2X7R agonist) stimulated-monocytes have been evaluated. Monocytes, without and with GA conditioning, were finally checked for Bax and Bcl-2 expression (qrt-PCR) in order to rule out apoptosis processes. Ten healthy donors (HDs) were similarly investigated. Results: No actual differences were found in P2X7R, IL-1beta and CD39 expression between patients and controls. In MS Bz-ATP stimulatedmonocytes, GA conditioning was clearly able to downregulate (p = 0.003) P2X7R expression but also IL-1beta expression even if the difference did not reach a statistical significance. Conversely, CD39 expression showed a trend (p = 0.06) to increase. Similarly, in HDs P2X7R (p = 0.01) and IL-1beta (p = 0.03) were downregulated by GA, whereas CD39 showed an upregulation even if not statistically significant. Moreover pro-apoptotic Bax vs. anti-apoptotic Bcl-2 ratio expression on monocytes was not modified by GA conditioning either in MS or HDs. Conclusions: Monocytes either from MS or controls express P2X7R, together with IL-1beta and CD39. GA immune modulation seems to interfere with the P2X7R but also with IL-1beta and CD39 expression on monocytes of both patients and controls. No apoptotic phenomena are induced on cultured monocytes by GA conditioning, therefore the observed changes seem to represent a GA net effect. Preclinical studies in animal models demonstrate cholesterollowering statins promote immune modulation, and may be beneficial in treatment of autoimmune diseases. Clinical trial data suggest that treatment with a statin can reduce the risk for developing new brain lesions in patients with CNS demyelinating disease. While all statins inhibit HMG-CoA reductase, it is known that their binding affinities and pharmacokinetic properties differ, which influences their potency in treatment of hypercholesterolemia. Therefore, it is possible that the immunodulatory potential of statins differ, and only certain statins may be beneficial in treatment of multiple sclerosis (MS). Our goal was to examine the relative potency of individual statins in EAE treatment and compare their capability to modulate T cell signaling and differentiation.
Oral simvastatin, lovastatin, atorvastatin and rosuvastatin were tested in PLP p139-151-induced EAE in SJL/J mice. Mice were treated for at least 60 days. Rosuvastatin and atorvastatin prevented EAE development, while lovastatin and simvastatin only delayed the clinical onset. All four statins suppressed proliferation of encephalitogenic T cells. Rosuvastatin and atorvastatin treatment also promoted significant T cell secretion of IL-4 and IL-10, whereas only low levels of these anti-inflammatory cytokines were detected in T cells after in vivo treatment with lovastatin or simvastatin. In concordance with the Th2 polarization by rosuvastatin and atorvastatin, the Th1 cytokine IFN^g was reduced more by treatment with these two statins than with lovastatin or simvastatin. In vivo treatment with rosuvastatin and atorvastatin was associated with greater redistribution of farnesylated Ras and geranylgeranylated RhoA GTPases from the membrane into the cytosol of T cells. Accordingly, this differential Ras and Rho inhibition blocked ERK and p38 MAPK activity, which are required for transactivation of IFN-γ and for repression of IL-4.
These data demonstrate that oral administration of individual statins vary markedly in their clinical and immunodulatory potential. Our results support further evaluation of atorvastatin and rosuvastatin in MS therapy.
Predictor of the efficacy of plasmapheresis/immunoadsorption on myasthenia gravis
Kitazono Hisao ⁎ , Konno Shingo, Murata Mayumi, Nakazora Hiroshi, Nomoto Nobuatsu, Sugimoto Hideki, Nemoto Hiroshi, Fujioka Toshiki Toho University Ohashi Medical Centre, Tokyo, Japan
Anti-acetyl choline receptor antibody (AChR-ab) is profoundly involved in the development of myasthenia gravis (MG). Thus immunomodulatory therapy that could suppress the antibody production is a mainstay of the treatment strategy. Plasmapheresis (PP) or immunoadsorption (IA) directly deprive such autoantibodies. PP/IA are employed mainly in case of crisis in Japan, however some MG patients do not respond. The goal of the present study was to evaluate the predictive parameters of successful treatment of PP/IA for MG.
We reviewed medical records of 16 patients with MG who underwent PP/IA in our institution during 20 years. One patient was seronegative MG. The indication for PP/IA was myasthenic crisis in all but two patients in whom generalized severe weakness without respiratory failure was present. The patients who received immunoglobulin because of development of hypo-IgGemia during a series of PP/IA were excluded in this study. In Japan, PP/IA is approved to be performed 3-5 times per 1-2 weeks, no more than 7 times per month. In our study PP/IA were carried out according to the approved program. MG-ADL score, AChR-ab titer, IgG values before and after each sequences of PP/IA were evaluated. Resveratrol is a natural polyphenolic compound in red wine. Resveratrol has been reported to be effective for axonal protection, although this has become controversial recently. Resveratrol also exhibits anti-inflammatory and anti-viral activities, and modulates the production of interleukin (IL)-17A, which has been proposed to be critical for the development of MS. Resveratrol has been suggested to be beneficial in immune-mediated diseases as well as neurological diseases, including stroke and Alzheimer's disease, without marked adverse effects. We investigated whether resveratrol could be therapeutic for multiple sclerosis (MS) using animal models for MS, experimental autoimmune encephalomyelitis (EAE) and Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD).
C57BL/6 mice were sensitized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Mice were fed a control diet (control) or a diet containing 0.04% resveratrol (20 mg/kg/day) prior to the disease onset (early, days − 1 to 8), during the disease progression (late, days 14 to 23) or whole course (all, days − 1 to 63). SJL/J mice were infected with the DA strain of TMEV. Mice were fed a diet containing resveratrol during the acute stage (days 0 to 14) or during the late stage (days 21 to 35) of TMEV infection.
All resveratrol treatments exacerbated clinical signs of MOGinduced EAE. The early resveratrol treatment resulted in the most severe EAE (mean clinical score on day 63: control, 0.3 ± 0.3; early, 2.4 ± 0.3, P b 0.01). Resveratrol did not alter cytokine production or anti-MOG immune responses. Similarly, during the chronic phase of TMEV infection, the levels of clinical signs and demyelination of mice treated with resveratrol during the late stage were greater than control, despite reduced IL-17A production resulting from resveratrol treatment. Axonal protection was not seen in resveratrol-treated mice. On the other hand, mice treated with resveratrol during the acute stage of TMEV infection recovered from the acute encephalomyelitis more rapidly than control mice.
Since resveratrol treatment resulted in detrimental effects on EAE and TMEV-IDD, this red wine polyphenol component may potentially detrimental effects in immune-mediated demyelinating disease, including MS.
Supported by the NIH (R21NS059724, P20-RR018724). We previously reported that infusion of murine bone marrow cells (BMC) transduced with an autoantigen (MOG40-55) into nonmyeloablated recipients induced tolerance to the autoantigen that prevented and improved experimental autoimmune encephalomyelitis (EAE), even in the absence of engraftment. We hypothesized that the tolerogenic effect was mediated by a subpopulation of transduced cells generated during ex vivo BMC culture. To this end, we have investigated the phenotype of BMC undergoing retroviral transduction, we sorted candidate cell subpopulations and analyzed their suppressive effects in vitro.
Murine BMC were transduced using our standard conditions (medium with 20% FCS, conditioned media containing murine stem cell factor and mIL-3, and exposure to supernatants containing retroviral vectors). At day 5 the vast majority of the transduced cells were either Mac-1+ Gr1low (31.910.2%), Mac-1+ Gr1+ (263.3%) or CD45-Lin-(13.54.2%). The phenotypes of the two myeloid (Mac-1+ Gr1low and Mac-1+ Gr1+) cell subpopulations correspond to those of the two (monocyte-like and granulocyte-like, respectively) subtypes of myeloid-derived suppressor cells (MDSC) that have been reported. To further characterize their functionality we analyzed the ability of transduced total BMC and the sorted candidate cell subpopulations to suppress the proliferative response of splenocytes from mice with EAE to autoantigen challenge. After transduction, total BMC and both myeloid cell subpopulations suppressed the proliferative T cell response in a dose-dependent manner. In addition, we found that the monocyte-like cells were significantly more suppressive than their granulocyte-like counterparts, and that suppression was also significantly higher if the cells were transduced with the autoantigen than with a control vector (in all cell subpopulations), which suggest that both non-specific and antigen-specific mechanisms contribute to the suppressive effect observed in vitro. Moreover, Mac-1+ Gr1low cell population displayed higher levels of arginase-1 and nitric oxide synthase activities upon stimulation than the other cell subpopulations tested, as it has been reported for MDSC.
To the best of our knowledge, this is the first report demonstrating the generation of MDSC in standard hematopoietic transduction cultures. These results also suggest the participation of antigenspecific MDSC in our model of tolerance induction in EAE and their potential therapeutic use in autoimmune diseases. Th17 helper cells appear to be of central importance in the development of autoimmune inflammation. Neuromyelitis optica (NMO) has recently been shown to be associated with increased levels of Th17 cells. Rituximab, a monoclonal antibody directed against CD20 is used as treatment for severe cases of NMO, leading to B-cell depletion. Decrease of Th17 cells along with clinical improvement after treatment with rituximab has been reported in a patient suffering from rheumatoid arthritis. We aimed to elucidate the effect of rituximab in Th17 T-helper cells in NMO.
Patient: A 42 year old caucasian woman developed severe bilateral optical nerve neuritis with loss of vision in 01/09. MRI showed no lesions within the brain or the spine but bilateral Gd-enhancement of the optical nerves. Lumbar puncture revealed 4/3 cells, normal protein level, no intrathecal synthesis of IgG and negative oligoclonal bands. Serum was positive for antibodies against aquaporin-4, supporting the diagnosis of aquaporin antibody-positive NMO. The patient did not respond to treatment with steroids. Only a slight improvement was achieved after 8 cycles of plasma exchange. Therefore we decided to initiate treatment with rituximab administered at a dosage of 1000 mg twice on days 0 and 14. For purposes of treatment monitoring we followed B cell levels and Th17 responses in vitro.
Peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 and cultured for 4 days. Cytokine analysis of T-h 17 type T cells was done by flow cytometry after intracellular staining with Interleukin-17(IL-17) before and shortly after therapy with rituximab.
Th17 cells were identified in the lymphocyte gate as CD4+ CD45+ Il-17+ cells. B-cells levels were monitored by routine diagnostic methods.
The patient's visual acuity improved to normal 7 months after the first cycle with rituximab. Numbers of CD-19+ B-cells were checked in regular intervals, showing the expected depletion of B-cells. The Th17 T cell response was elevated before therapy. Remarkably after rituximab administration we observed a significant decline in Il-17 producing Th17 cells with a decrease from 5.77% to 0.32%. Interestingly the timing coincided with the patient's clinical improvement.
This observation suggests an important role of Th17 cells in the Bcell-T-cell axis and in the pathogenesis of NMO and may provide useful insights into the mechanism of action of rituximab.
Short-and long-term differential gene expression changes in multiple sclerosis patients treated with subcutaneous interferon-beta-1a
Hecker Michael ⁎ ,1 , Goertsches Robert H. 1 The purpose of this study was to characterize the genome-wide transcriptional effects of subcutaneous interferon-beta-1a (IFN-beta) treatment in patients with relapsing-remitting multiple sclerosis (MS), and to identify expression signatures that correlate with individual clinical outcomes. Beyond, we aimed to analyze the transcription factor binding sites (TFBS) in the promoter regions of IFNbeta-responsive genes to elucidate the molecular interactions affected by the therapy.
By using Affymetrix HG-U133 Plus 2.0 DNA microarrays, we obtained for 12 MS patients expression levels of peripheral blood mononuclear cells before (baseline), as well as two days, one month, one year and two years after start of IFN-beta therapy (Rebif, Merck Serono). Up-and down-regulated genes were ascertained using MAID-scores, and the regulatory regions of theses genes were analyzed using TFBS predictions provided by three different databases: UCSC tfbsConsSites, cisRED Human 9 and SwissRegulon. Using the expression data and TFBS information, we constructed a gene interaction model using TILAR, a novel integrative network inference algorithm.
We identified a set of 96 genes that was significantly modulated during the observation period and that mainly demonstrates an up-regulated type I IFN signature (comprising e.g. MX1, OAS1, IFI44, STAT1 and IRF7), but also contains a few known down-regulated genes (e.g. FCER1A). However, there are remarkable expression differences between the patients: I) After two days and one month into therapy, only eight patients showed an increase of IFN-beta-responsive genes. II) CD163, a marker of monocyte activation, was only up-regulated in patients with higher baseline disease activity.
TFBS for IFN regulatory factors were significantly overrepresented in the promoters of up-regulated genes. Binding sites for PRDM1, a repressor of IFN-beta gene expression, were enriched in the regulatory regions of both up-and down-regulated genes.
To comprise, MS patients show different expression dynamics during IFN-beta therapy, which may explain individual adverse and beneficial therapeutic outcomes. The results of the promoter and network analyses indicate positive and negative feedback mechanisms that affect the expression of known and novel IFN-betaresponsive genes.
Structural insights into the molecular chaperone dependent therapeutic function of small heat shock proteins in autoimmune demyelination Brownell Sara ⁎ , Rothbard Jonathan, Steinman Lawrence
Small heat shock proteins (sHSPs) are highly dynamic 12 kDa-43 kDa oligomers most widely recognized for their intracellular molecular chaperone function. There are ten human small heat shock proteins and although they have diverse expression patterns, they all share the ability to be a molecular chaperone. Although their most commonly recognized function is to bind unfolded proteins, some have been shown to have anti-inflammatory and anti-apoptotic functions, suggesting that they might be therapeutic in treating autoimmune diseases (Arrigo et al., 2007 and Steinman, 2008) . Recently, we have shown that one of these small heat shock proteins, alpha B crystallin (HSPB5) ameliorated experimental autoimmune encephalomyelitis (EAE), the predominant mouse model for multiple sclerosis (Ousman et al., 2007) . The primary aim of this study is to determine how small heat shock proteins are therapeutic in EAE.
In this present study we have shown that each member of the whole family of human small heat shock proteins is therapeutic when EAE mice are treated at the peak of disease. This amelioration of clinical scores is accompanied by a decrease in peripheral immune activation and fewer immune cells present in the brain and spinal cord. Significantly, we have discovered that the therapeutic efficacy of these sHSPs is due to their common molecular chaperone function. By using a naturally occurring point mutation (Arg120Gly) of HSPB5 that impairs proper folding and thus molecular chaperone activity, we have shown that this mutation also disrupts the therapeutic efficacy of HSPB5 in EAE. Finally, we have taken a structure-function approach in investigating particular regions of HSPB5 and have identified specific peptide fragments of HSPB5 that are also therapeutic in EAE. These peptide fragments have previously been established in literature as being capable of molecular chaperone activity, providing further evidence that small heat shock proteins are dependent on their molecular chaperone activity for their therapeutic effects in EAE.
Small heat shock proteins are therapeutic in autoimmune demyelination. Using a peptide-based structure-function approach, we have found that this therapeutic function is dependent on the molecular chaperone ability of sHSPs. Alzheimer's disease (AD) is accompanied by β-amyloid (Aβ) accumulation and neuronal cell death in the brain of patients. Current AD therapeutics provides mainly symptomatic short-term benefit, rather than targeting disease mechanisms. Anti-Aβ immunotherapy did not advance to clinical trials because a subset of patients developed meningoencephalitis. Thus, development of new effective ways for AD therapy is required.
We propose new approach to AD therapy using antibodies not to Aβ, but to its receptor. One of the hypotheses of AD neuropathology involves high affinity binding of Aβ to a7-type acetylcholine receptor (AChR), cell lysis and amyloid plaque formation. Recent findings also identify the cellular prion protein as one of the Aβ receptors. We considered that antibodies to either a7-type AChR or the prion protein would prevent them from binding to Aβ and would protect the neurons from development of neurodegenerative processes.
To investigate a therapeutic effect of antibodies to the neuronal receptors of Aβ, we chose olfactory bulbectomized (OBX) NMRI mice which show impairments of learning and memory, loss of neurons in the cortex and hippocampus and have an increased level of the brain amyloid precursor protein and Aβ. To induce antibody formation in the mice we selected fragments of a7-subunit AChR and the prion protein. In case of a7-subunit AChR, synthetic fragment 173-193 was revealed to be immunogenic in a free non-conjugated form in NMRI mice. We have demonstrated that vaccination with fragment 173-193 restored spatial memory in OBX mice and four-fold decreased the level of Aβ in their brain compared to non-immunized OBX mice. Passive immunization with either mouse blood sera containing antibodies to the peptide 173-193 or affinity purified antibodies to the fragment also restored memory in OBX animals.
To induce antibodies to the prion protein synthetic fragment 95-123 from it has been chosen and synthesized. We have shown this fragment was low immunogenic in a non-conjugated form. Therefore, we investigated the effect of immunization with a protein conjugate of the fragment in OBX mice. We showed the immunization with fragment 95-123 conjugated with the carrier protein restored the spatial memory in OBX animals and decreased the level of Aβ in their brain.
The results obtained provide new immunotherapeutic approach to treatment of AD based on vaccination with fragments of either a7subunit of the AChR or the prion protein. Recently, the molecular mechanism of action of several specific BAs has been defined as inhibition of either cathepsin G, microsomal prostaglandin synthase (mPEGS)-1, and 5-lipoxygenase (5-LO) or dual inhibition of 5-LO and mPGES-1. Cathepsin G is one of the main enzymes of neutrophils and involved in their migration into inflamed lesions, and 5-LO ranks among the most highly overexpressed genes in inflammatory multiple sclerosis (MS) lesions. PGE2, the product of mPGES-1, is one of the major key players in inflammation and neurodegenerative diseases. We consider the triple inhibition of cathepsin G, mPEGS-1 and 5-LO a promising and novel anti-inflammatory treatment approach for relapsing-remitting MS that has not been studied so far.
We have generated both mechanistic data and a study design to test the safety, tolerability, efficacy and mechanism/s of action of BA in a Phase IIa proof-of-concept baseline-to-treatment study in relapsing-remitting MS patients with a standardized frankincense extract. The mechanism of action of BAs on several functions of neutrophils has been tested. Biomarkers that shall aid in dose-finding and in vivo assessment of pharmacodynamics of BAs have been identified. Dose finding, assessment of tolerability and efficacy on contrast-enhancing MRI lesions during the trial are accompanied by mechanistic studies that will address by ex vivo and in vitro experiments if treatment with the frankincense extract only affects the above enzymes or has further pharmacodynamic effects.
Several lines of evidence suggest an involvement of neutrophils in early steps of MS lesion evolution, and therefore, blocking neutrophil activity by BAs, that are orally available and have been successfully tested in clinical trials of several other autoimmune diseases showing very good tolerability, appears an interesting opportunity. Two clinical trials showed that Dimethyl Fumarate (DMF), a novel treatment for Multiple Sclerosis (MS), significantly reduced gadolinium enhancing lesions in relapsing-remitting Multiple Sclerosis (RRMS). However, neither the molecular mechanisms nor its possible neuroprotective properties have been fully investigated. Our hypothesis is that DMF exerts its therapeutic effects in both the immune system and Central Nervous System (CNS) through Heme Oxygenase 1 (HO-1) induction.
Bone-marrow derived dendritic cells (BMDCs) were stimulated by LPS/IFN-g (10 ng/ml each) in the presence or absence of DMF (70 μM) and cytokines were analyzed 24 h after stimulation. Our results suggest that DMF significantly reduces pro-inflammatory cytokine production (e.g. . In order to determine if the change in DC phenotype can lead to altered antigen presentation and subsequent T cell activation, a co-culture system was utilized. Stimulatory DCs were treated with DMF (70 μM) or vehicle for 24 h and supernatants were washed out. DCs were then co-cultured with naïve myelinspecific T cell receptor (TCR) transgenic T cells in the presence of MBP Ac1-11 (2 μg/ml) for 72 h. Our co-culture results show that T cells cocultured with DMF treated DCs express lower level of encephalitogenic markers, namely IFN-g and T-bet. In addition, [H]-thymidine incorporation assays demonstrate that T cells incubated with DMF treated DCs proliferate less compared to those from non-treated DCs. In addition to the immunoregulatory properties of DMF, possible neuroprotective effects were also investigated using an excitotoxic model induced by AMPA microinjection. Mice were pre-treated with DMF (200 mg/kg) or vehicle for 3 days followed by AMPA injection into the spinal cord. Our results demonstrate that DMF pre-treatment reduced neuronal damage by 30% and improved behavioral outcomes as compared to the vehicle-treated mice, indicating possible neuroprotective effects of DMF. Our study showed DMF upregulates HO-1 expression in both DCs and the CNS.
Our data suggest that DMF inhibits T cell encephalitogenicity and protects neuronal damage caused by excitotoxicity, potentially through HO-1 induction.
The effects of dietary antioxidants on gelatinase activity in cultured rat astrocytes. Implications for the complementary treatment of Multiple Sclerosis We investigated whether natural antioxidant compounds, which might represent a complementary therapy for the patients with multiple sclerosis (MS), are able to modulate matrix metalloproteinase (MMP) activity and expression in primary astrocyte cultures. MMPs are key mediator of tissue damage in the course of MS.
Primary cultures of rat astrocytes were activated by exposure to LPS and simultaneously treated with different doses of the antioxidants: resveratrol (RSV), N-acetylcysteine (NAC), quercetin (QRC), a preparation of tyrosol and hydroxytyrosol (Oliplus), green tea extract (GTE) and lycopene (LYC). Culture supernatants collected from astrocytes after 24 h incubation were subjected to gelatin zymography for the assessment of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) levels. Total RNA was extracted from astrocytes and and subjected to RT-PCR for the assessment of mRNA expression.
RSV, NAC, QRC, and Oliplus, but not LYC and GTE, dose-dependently inhibited the LPS-induced levels of MMP-2 and MMP-9 in cell culture supernatants. As assessed by RT-PCR, only RSV and Oliplus significantly inhibited the expression of both MMP-2 and MMP-9. The antioxidants were also tested for their ability to inhibit directly MMPs activity in zymographic runs (in-gel experiments). As a result, QRC, GTE and NAC were potent inhibitors of in-gel zymographic activity of MMP-2 and MMP-9, whereas oliplus was only partially effective and LYC and RSV failed to inhibit the in-gel gelatinase activity.
Our results suggest that the bioactive dietary molecules tested in this study exert their inhibitory effect on MMP displaying different mechanisms of action. Therefore, the combined use of low concentrations of these molecules may be useful and effective as complementary dietary supplement for the well-being of MS patients under conventional therapy.
Supported by the MS Project Grant n.2007/R/15 from the Italian Multiple Sclerosis Foundation (FISM).
The potential for neuroprotection and neurogenesis by immunomodulatory treatment with Glatiramer acetat Aharoni Rina ⁎ , Eilam Raya, Arnon Ruth
The Weizmann Institute pf Science, Rehovot, Israel Background: Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are complex diseases involving inflammation as well as oligodendrocyte and neuronal pathology. Treatment strategies aim to affect the CNS by reducing the inflammation and inducing neuroprotective repair processes. T-cells that produce IL-17 (Th17) are major contributors to the pathogenesis, whereas regulatory T-cells (Tregs) play a role in the suppression of autoimmunity. The therapeutic effect of Glatiramer acetate (GA, Copaxoneâ) has been attributed to immunomodulation from inflammatory towards anti-inflammatory pathway. Objective: to identify cell populations relevant to pathogenesis and repair within the injured CNS, and explore whether immunomodulatory treatment can lead to neuroprotection in situ. Methods: Daily injection of GA was initiated following disease induction or after disease development, in two EAE models: relapsing-remitting PLP-and chronic MOG-induced EAE. Th17 and Tregs were identified by their IL-17 and Foxp3 expression, respectively. Their incidence within the CNS and localization in injury sites was analyzed. The proliferation marker BrdU was injected concurrently with GA and its incorporation into progenitor cells expressing various developmental markers was detected. Neuronal and myelin damages were identified using electron microscopy and immunohistochemistry. Results: In the CNS of mice with either relapsing-remitting or chronic EAE, T-cells and activated microglia expressed IL-17, with apparent localization within regions of myelin loss. In GA treated mice CNS inflammation and Th-17 occurrence were drastically reduced, with parallel elevation in Tregs, indicating the immunomodulatory consequences of GA treatment in situ. Furthermore, GA treatment resulted in reduction of neuronal and myelin damages and augmented the three processes characteristic of neurogenesis-neuronal proliferation, migration and differentiation. The newborn neurons migrated through existing and dormant pathways, into injury sites in brain regions which do not normally undergo neurogenesis, and differentiated to mature neuronal phenotype. GA treatment induced also proliferation and differentiation towards the oligodendrocyte lineage.
Immunomodulatory treatment may counteract the neurodegenerative disease course, pointing to its direct linkage to neuroprotection and therapeutic activity in the CNS.
The use of A-beta-HSP conjugate as a novel vaccination approach for Alzheimer's disease Active vaccination with amyloid beta peptide (A-beta) has been shown to be remarkably effective in clearing cerebral A-beta and improving cognitive functions in mouse models of Alzheimer's disease (AD). However, when emulsified in the QS21 adjuvant, the A-beta vaccine induced meningoencephalitis in about 6% of AD patients during a phase I clinical trial; presumably resulting from a pathogenic activation of the immune system against A-beta. In this study, we generated a novel heat shock protein (HSP)-based vaccine, which contains the A-beta B-cell epitope (A-beta 1-15) and the self-HSP60 peptide p458, for A-beta vaccination.
We demonstrate that whereas the conjugated A-beta-HSP vaccine promoted a very mild T-cell response in a humanized mouse model of AD carrying the DR-DRB1*1501 allele, the humoral response was sufficient to promote a significant reduction of cerebral amyloid burden as well as of the associated inflammatory response. Analyzing the antibody isotypes evoked following A-beta-HSP vaccination revealed IgG1 and IgG2b as the predominant isotypes with relatively low titers of IgG2c, indicating the activation of a Th2-type immune response.
This type of vaccination, combining a gradual increase in antibody production accompanied by a mild T-cell response is likely due to the unique adjuvant properties of the self HSP peptide used, being both a TLR ligand and T-cell epitope, possibly providing a safer and efficacious approach for AD vaccination. Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease of the Central Nervous System. As current MS therapies decrease the frequency of relapses and modestly the accumulation of disability, but do not prevent progression of the disease, definition of alternative approaches for MS treatment is needed. Tolerance induction to selfantigens is a promising therapeutic strategy in autoimmunity. Dendritic cells (DC) are the main antigen presenting cells. DC therapy in tumour and infection immunology has been longly investigated as a potential tool to induce the immunoresponse in those situations. Our aim is to induce tolerance in MS patients using tolerogenic DC loaded with myelin derived peptides as a source of specific antigen.
Peripheral blood cells were obtained from donors (n= 8) and patients with relapsing-remitting MS (n = 8). Monocyte-derived DCs (MDDCs) were obtained following conventional protocols in presence of GM-CSF and IL-4; maturation was induced with a proinflamatory cytokine cocktail, in the absence [matDC] or presence of 1a,25dihydroxyvitamin D3 [tolDC] as tolerogenic inducing agent. The differentiation of monocytes to MDDCs was evaluated by morphology, viability, phenotype, cytokine production and the induction of T cell proliferation.
MDDCs differentiation from both healthy subjects and MS patients exhibited similar yield, viability and functionality. In particular, tolDC expressed reduced levels of CD83, CD86 and HLA-DR compared to matDC, and produced lower levels of IL-12. In addittion, proliferation of allogeneic T cells induced by tolDC was also lower, and analysis of cytokines found in these co-culture supernatants revealed a reduced secretion of proinflammatory mediators (IL-6, TNF-alfa, and IFNgamma). In addition, tolDC were resistant to a subsequent maturation stimulus induced by LPS. Finally, binding of myelin peptides to MDDC was shown by immunofluorescence and competition assays. Myelin peptide-loaded matDC induced proliferation of autologous T cells obtained from MS patients, thus confirming the specificity of the assay.
In conclusion, we have demonstrated that tolDC may be generated from monocytes of MS patients. These tolDC induce hyporesponsiveness in allogeneic T cells, thus suggesting their tolerogenic potential. Consequently, loading tolDC with myelin peptides might be an effective tool to induce antigen-specific hyporesponsiveness in MS patients, this leading to tolerance induction. Following spinal cord injury (SCI), neuropathic, chronic pain is a major cause of disability. Recent evidence suggested that cyclooxygenase-2 (COX-2) expression after SCI might be involved in the development of neuropathic pain. The aim of the present study was to test the effect of GW406381, a novel COX-2 inhibitor, on the evoked field potentials (EP) and long-term potentiation (LTP) of dorsal horn area of the spinal cord in the rats suffering from SCI.
Spinal cord was transacted at the level of T12 in adult rats. Behavioural data were collected from the SCI rats revealed both sensory and motoric deficits. After 4 weeks, spinal cord slices were obtained from SCI as well as control rats. Bioelectrical activities were recorded in the dorsal horn (lamina I-III) and electrical stimulation was applied through a bipolar platinum electrode attached to the dorsal root. A conditioning tetanic stimulation was delivered to the dorsal roots of spinal cord slices followed by pulses with stimulation parameters identical to controls.
The amplitude of the evoked potentials in the DH of spinal cord slices decreased to 41 ± 2% (P b 0.001) and 65 ± 4% (P b 0.001) of the initial values within 10-15 min after addition of GW406381 to the bath medium at concentrations of 5 and 10 μmol/l, respectively. Application of GW406381 at concentration of 10 μmol/l, 60 min before tetanic stimulation significantly inhibited LTP induction in 27 of 30 slices (149 ± 4.4 % control, P = 0.002).
The results suggest a possible analgesic effect of GW406381 on neuropathic pain in SCI.
Keywords: Cyclooxygenase-2 inhibitor, GW406381X, Nneuropathic pain, Injured spinal cord Recent evidence has indicated that proinflammatory cytokine IL-1beta plays an important role in inflammation-induced cachexia via mechanisms that interact with hypothalamic neuronal circuits regulating appetite. Adjuvant arthritis (AA) in rats is a useful model of inflammatory cachexia characterized by anorexia, increased metabolic rate, and body mass loss that mimics the pathophysiology of human rheumatoid arthritis. The aim of this study was to investigate whether chronic inflammatory process during AA affects mRNA expression of IL-1beta and neuropeptides involved in the control of appetite, along with circulating levels of leptin, ghrelin, insulin, corticosterone and adiponectin in conditions of over-/underand normo-feeding.
In the hypothalamic arcuate nucleus (ARC), mRNA expressions for orexigenic neuropeptide Y (NPY) and agouti-related protein (AgRP), and anorexigenic proopiomelanocortin (POMC) and cocaine-and amphetamine-related transcript (CART), and IL-1beta were quantified using quantitative real-time PCR. Plasma hormone levels were determined by RIA or ELISA. Body mass, leptin and adiponectin were decreased while ghrelin and corticosterone were increased by AA in all dietary regimes. Insulin was reduced by AA only in underfed rats. The expressions for NPY, AgRP and IL-1beta were enhanced, CART reduced, and POMC unchanged by AA in all dietary modulated groups. However, underfeeding during AA led to more profound body mass loss, reduced leptin and insulin, increased ghrelin and corticosterone levels, as well as enhanced expressions for NPY and AgRP, and reduced expressions for POMC, CART and IL-1beta compared to over-and normo-feeding in AA.
These results demonstrate that AA, independently on over-/ under-and normo-feeding, is associated with overexpression of IL-1beta in ARC which may be involved in inflammatory cachexia rather than anorexigenic neuropeptides CART and POMC whose expression was reduced or unaffected by AA in all dietary regimes. Moreover, the overexpression of orexigenic NPY and AgRP by AA in all dietary regimes indicates their importance in the maintaining of energy homeostasis during negative energy balance. In underfed AA rats, stronger response in orexigenic neuropeptides and weaker response in anorexigenic neuropeptides is in line with their previously attributed roles in body weight homeostasis. A lowered IL-1beta expression may reflect the milder inflammation in underfed rats. Supported by: 260703/SVV/2010,GACR303/10/ 1338.
Anti-NMDAR encephalitis: Complement activation in the tumor but not in the brain
Anti N-methyl-4-aspartate receptor (NMDAR) encephalitis is a severe, potentially lethal, but treatment responsive disorder with a well defined set of clinical features. The presence of an underlying tumor, mostly a teratoma, varies according to sex and gender.
Recently reported experiments demonstrated that NMDAR autoantibodies have a pathogenic effect, causing a selective and reversible decrease in NMDAR surface density and synaptic localization by a mechanism of crosslinking and internalization. As NMDAR antibodies are IgG1 and IgG3 subtypes, in this work we explore if complement cytotoxicity could be a second pathogenic mechanism.
We examined the activation of complement in cultures of embryonic rat hippocampal neurons exposed to patients' CSF and determined whether there is complement deposition in tumors (21 ovarian teratomas, 1 pancreatic, 1 testicular and 1 breast tumor) and brain (2 biopsies and 3 autopsies) of patients with anti-NMDAR encephalitis, and in the hippocampus of rats previously infused with patients' antibodies for 2 weeks.
Complement components C3 and membrane attack complex (MAC) were detected as a linear deposition on the cell membrane of neurons after incubation with fresh frozen serum from a healthy donor. Complement deposits were found in 23 of 32 tumors (18 of 24 patients' tumors and 5 of 8 control teratomas) with a linear or vascular pattern. In contrast, no complement deposits were seen in 2 control hippocampus and in 5 of 6 patient brain samples examined. MAC staining, in a granular pattern, was seen only in one patient's biopsy taken from a necrotic cystic lesion of the temporal lobe visible on the brain MRI. Similar radiological or pathological findings were not found in 340 patients with anti-NMDAR encephalitis studied in our laboratory. MAC or C3 immunostaining was absent in the hippocampus of rats infused with patients' CSF.
This study shows that antibodies from patients with anti-NMDAR encephalitis activate complement, and that deposits of complement are detectable in the tumor but not in the brain. The current data, coupled with previous studies showing the reversible loss of NMDARmediated synaptic function, suggest that the main pathogenic mechanism is antibody-mediated neuronal dysfunction and not complement-mediated neuronal lysis. The purpose of this study is to determine the associations between TLR9 rs352140 polymorphism and the clinical characteristics of MG in Chinese Han nationality.
TLR9 rs352140 polymorphism was determined by PCR-RFLP method in 171 sporadic MG patients and 198 unrelated healthy controls without any evidence of common autoimmune diseases. The frequencies of genotypes and alleles were compared between the MG group and the control group, and among different subgroups (classified by gender, age of onset, Osserman types at the maximal severity, and pathology of thymus) of MG patients. The relationship between the genotypes and alleles and MGFA scores at the maximal severity during the follow-up and short-term efficiency of glucocorticoid were also explored. All patients who required immunologic treatment were included in this study and treated with medium-dose glucocorticoid initially. The treatment efficiency is determined with a relative score, which is the proportion of the difference between the absolute score before treatment and the absolute score after treatment to the absolute score before treatment. Satisfactory short-term efficiency was defined as a relative score N50% within 3 months after regular treatment with glucocorticoid in a dose equivalent to prednisone 1/mg/kg/day or less. There was no significant difference in frequencies of genotypes and alleles between MG group and controls and among subgroups (gender, age of onset, ocular or generalized MG, and pathology of thymus) of MG (pN 0.05). There was no difference in mean ages of onset and mean maximal MGFA scores among different genotypes and alleles groups (p N 0.05). In those with unsatisfactory short-term efficiency of glucocorticoid, generalized MG patients were more prevalent than ocular MG patients (p= 0.03, OR= 3.592); patients with thymoma were more prevalent than patients without thymoma (P= 0.016, OR= 4.350). The mean pretreatment MGFA score was higher in patients with unsatisfactory efficiency of glucocorticoid (p = 0.026). The efficiency was not in relation to ages of onset or pretreatment duration of disease (pN 0.05). Logistic regression analysis revealed that the higher pretreatment MGFA scores, the worse efficiency of glucocorticoid was in MG patients (p= 0.043, OR= 1.154).
There were no significant correlations between TLR9 rs352140 polymorphisms and susceptibility to MG, maximal severity and shortterm glucocorticoid efficiency. Catholic University, Roma, Italy B lymphocyte induced maturation protein-1, Blimp-1, is a transcriptional repressor that plays crucial roles in the differentiated function of both T and B lymphocytes. In B lymphocytes, it is required for antigen-dependent terminal differentiation of B cells, into immunoglobulin-secreting plasmablasts and plasma cells. In T lymphocytes Blimp-1 is induced by IL-2, its expression represses Il2 gene transcription, inhibits cell proliferation, and influences the generation of Memory Precursor effector cells and Central Memory cells. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system and it is usually considered a CD4+ Th1/Th17-mediated autoimmune disease even if recent studies underline a new crucial role of CD8+ T cells and also B cells.
In this study we evaluated by Flow Cytometry the mean fluorescence intensity (MFI) of Blimp-1 in CD4+, CD8+ T cells and CD19+ B cells and the percentage of CD4+ Blimp-1+, CD8+ Blimp-1+ T cells, CD19+ Blimp-1+ B cells from the peripheral blood of 50 untreated Relapsing Remitting Multiple Sclerosis patients (RRMS; 23 in relapse and 27 in remission) and 25 healthy subjects (HS). We also stimulated T cells, in a 5 day cell-culture experiment, with different doses of IL-2 with and without IL-12. We observed a higher percentage of circulating CD19+ Blimp-1+ B cells in RRMS patients during relapse than during remission. We also found that Blimp-1 MFI is significantly lower in both CD4+ and CD8+ T cells from MS patients as compared to HS. The Blimp-1 MFI was significantly lower in both CD4+ and CD8+ T cells from relapsing MS patients than from HS. Furthermore the percentage of CD4+ Blimp-1+ and CD8+ Blimp-1+ T cells was higher in relapsing patients than HS. When we stimulated T cells with different amount of IL-2 we observed that high dose of IL-2 can induce Blimp-1 expression in T cells just after 1 day and its expression remains stable until day 5 while low dose of IL-2 induce Blimp-1 expression only after 3 days of culture. When IL-12 was added to the cultures this proinflammatory cytokine clearly inhibited Blimp-1 expression both in CD4+ and in CD8+ T cells, in RRMS patients and in HS, even in presence of high dose of IL2.
We demonstrated that plasma cells and plasmablasts may be involved in the acute phase of RRMS and that a dysregulation of Blimp-1 expression is present in T cells from relapsing MS patients. Our data suggests that Blimp-1 can be considered a potential new target of MS therapy. Potent tools are used to identify and to characterize these receptors. Among these tools, neurotoxins have been used to study the functional role of ionic channels, mainly N+ and K+ channels. Binding of neurotoxins to their receptors induces specific pharmacological properties for each type of channel.
Neurotoxins acting on K+ channels such as Kaliotoxin (KTX), a purified neurotoxin from Androctonus australis hector venom scorpion, exhibits a high affinity for K+ channels of mammalian nervous system. Their toxic action differs mainly on the chosen route of injection. It is well known that specific targets of KTX are located in central nervous system. The mode of inoculation could be essential in their fixation on specific target with neuro-pathological effects.
The aim of this study is to investigate the involvement of potassium channels in the regulation of various biological systems. Analysis of tissue damage, biochemical modifications and inflammatory response was carried out into mice after experimental envenomation by using an intra-cerebroventricular (i.c.v) or subcutaneous (s.c.) route.
The results showed that the inoculation of KTX (i.c.v) induces severe disturbances in the cerebral cortex (oedema, hemorrhagic, and necrosis). This toxin induced also an inflammatory response marked by an increase of inflammatory cells in animal. In brain, this response is accompanied by significant increase of protein markers of inflammation (EPO, MPO, albumin, globulin fractions Alpha 1, Alpha 2 globulin, beta and gamma globulin) indicating the infiltration of eosinophils and neutrophils in this organ. Other animal systems (cardio-respiratory and endocrine) are also altered. These dysfunctions result on tissue damage correlated with by high levels of biochemical parameters. The inoculation of KTX by the subcutaneous (s.c.) route did not provoke any effect.
These results reported in the present study suggest the involvement of K+ channels in various regulations (nervous, cardio-respiratory and hormonal systems). Their dysfunction could lead to neurological diseases.
Keywords: K+ channels, Kaliotoxin, Nervous system, Inflammation, Endocrine function Multiple sclerosis (MS) is an immune-mediated, demyelinating disease of the central nervous system (CNS), with a well-characterized animal model, experimental autoimmune encephalomyelitis (EAE). While the major focus of MS/EAE immunology has been CD4+ T cells, several reports from others and us show that CD8+ T cells play an important role in these diseases. The role of CNS-specific, autoreactive CD8+ T cells in MS and EAE pathogenesis is still unclear and was the focus of this study.
Using sensitive flow cytometry-based suppression assays, we saw that CNS autoantigen-specific CD8+ T cells, but not those specific for control antigens, exhibited regulatory ability, suppressing CD4+ CD25(−) T cell proliferation. Antigen-specific T-cell lines confirmed that CNS-specific CD8+ T cells suppressed CD4+ T cells, while control antigen-specific CD8+ T cells did not. This suppressive ability was dependent on HLA class I presentation, but not TNF-a secretion. Of note, CD8+ T cells from MS patients during acute exacerbations were deficient at suppressing CNS-specific CD4+ T cell responses, compared to healthy subjects or untreated, quiescent MS patients.
In mice, MOG35-55-specific CD8+ T cells were able to suppress EAE. This novel and unexpected function required in vivo Class Imediated presentation of cognate antigen and was dependent on IFNand perforin production by the CD8+ T cells. These regulatory CD8+ T cells were able to kill MOG-loaded CD4+ T cells as well as CD4depleted APC in vivo, suggesting a cytotoxic/suppressor mechanism. Regulation of disease was associated with modulation of APC function as well as decreased MOG-specific CD4+ T cell responses.
Our results demonstrate that CNS-specific CD8+ T cells have an important regulatory role in MS, which is a disease of perturbed immune regulation. Understanding this arm of the adaptive immune system offers a promising strategy for immunotherapeutic intervention in the future.
These studies were supported by grant awards from the NIH and National MS Society, including the Harry Weaver Neuroscience Scholar Award from the NMSS. Local tissue-draining lymph nodes are vital in generating immune responses during infections and autoimmune disease, but also in maintaining immune tolerance against self antigens. In most tissues, antigens reach the draining lymph nodes through the lymphatics.
However, lymphatic vessels are lacking in the CNS parenchyma. Despite this unique anatomical feature, CNS antigens can reach the CNS-draining cervical lymph nodes (CLN) and lumbar lymph nodes (LLN). Hence, CLN and LLN are likely to control CNS inflammation.
Therefore, we investigate access of CNS compounds to CLN and functional outcome using human tissue and animal models. We show that myelin and neuronal antigens are transferred to the CLN during MS and EAE in mice as well as rhesus monkeys and marmoset monkey. These self antigens are present in functionally distinct antigen presenting cells. These cells have characteristics of macrophages/microglia and of dendritic cells. Myelin-specific T cell responses occur in CLN of EAE-affected animals. We hypothesized that CNS-draining lymph nodes are involved in EAE disease progression during EAE. To test this, we surgically removed the superficial CLN, deep CLN and LLN in mice before EAE induction. Using three models, with a clinically different EAE course, we demonstrate that removal of CNS draining lymph nodes does not affect the first acute phase of disease. However, animals in which the CLN and LLN are surgically removed develop less severe relapses compared to sham-operation, indicating that CNS-draining lymph nodes are pivotal for fully developed relapses.
Collectively, our data demonstrate that 1. CLN during EAE are not involved in immune tolerance and control of the autoaggressive response. 2. CLN function supports relapse development, and 3. further support the concept that CNS-draining lymph nodes may be an interesting target for therapeutic interventions during MS. To further clarify the functional significance of IL-17A in the CNS, we generated a transgenic mouse line with astrocyte-restricted expression of the IL-17A gene.
GFAP/IL-17A transgenic mice develop normally and do not show any signs of neurological dysfunction. However, histological characterization revealed a substantial astrocytosis and a mild activation of microglia by morphological criteria. We did not observe signs of demyelination, neurodegeneration or prominent tissue damage but scattered calcifications of small intraparenchymatous vessels. Histology and FACS-analysis showed the absence of parenchymal infiltration of immune cells into the CNS of GFAP/IL-17A transgenic mice.
To investigate if IL-17A acts synergistically with other proinflammatory stimuli, we systemically treated GFAP/IL-17A mice and wild type controls with LPS and observed by flow cytometry a pronounced microglial activation with an increased amount of CD45high/ CD11bhigh microglia in GFAP/IL-17A mice compared with controls. Furthermore, quantification of mRNA levels revealed an upregulation of proinflammatory cytokine genes like TNFa and IL1b, which was substantially higher in GFAP/IL-17A mice compared with wild type controls.
These results demonstrate that chronic astroglial IL-17A synthesis in the CNS induces astrocytosis and microglial activation but not severe tissue damage nor directly promotes the infiltration of hematogenous cells. Our data argues against a key role of IL-17A in mediating tissue damage during neuroinflammation. More likely IL17A acts as a modulating factor in the network of induced cytokines. This novel mouse model will be a very useful tool to further characterize the role of IL-17A in neuroinflammatory disease models as demonstrated by other transgenic mouse models with a CNStargeted production of other cytokines.
Cytokines and peripheral blood natural killer cell cytotoxicity crosstalk during restraint stress can be influenced by stress genotype in pig Ciepielewski Ziemowit ⁎ , Stojek Wojciech, Glac Wojciech, Myslinska Dorota, Lewandowska Danuta
The aim of the study was to evaluate the peripheral blood natural killer cell cytotoxicity (NKCC) and plasma levels of two cytokines playing a crucial role in regulatory mechanisms of NKCC: interleukin 2 (IL-2) and interleukin 12 (IL-12) during stress in pigs of different, genetically based, stress vulnerability.
A 4-hour restraint (in specially constructed hammocks) was used as a stress model. The experiments were performed on 15 chronically catheterised Pietrain crossbred male pigs divided by the molecular analysis screening single amino-acid mutation of the ryanodine receptor RyR1 gene into 3 groups: stress susceptible homozygotes-nn, and stress resistant: heterozygotes-Nn and homozygotes-NN. It was found that mean baseline plasma IL-2 and IL-12 (ELISA) levels as well as NKCC (51Cr-release essay) differed significantly between RyR 1 genotypes, the highest baseline levels of both cytokines were observed in stress gene carriers (Nn animals). Different patterns of changes have emerged for particular cytokine during the stress period and the most significant amplitude of changes was observed in nn stress susceptible animals as compared to stress resistant Nn and NN pigs. In nn group IL-2 plasma level increased during stress reaching two separated peaks: the first one at 15 min (maximal increase of 207%) and the second at 120 min (of 78%). Plasma level of IL-12 increased at an early phase of stress peaking at 60 min (maximal increase of 185% in nn animals) and then returned to baseline values at 240 min in all genotype groups. Similarly, the highest level of NKCC was observed in stress susceptible nn pigs during stress period. Restraint also evoked biphasic changes in NKCC, the short-lasting enhancement (at 15 min in all genotypes, most pronounced in nn pigs-increase of 73%) was followed by subsequent suppression at 240 min (decrease of 71%) in stress resistant NN pigs.
These data suggest that time dependent differences in IL-2 and IL-12 responses to stress could contribute to stress-induced stimulatory effects on blood NKCC. Moreover, the intensification of reactivity of different compartments of the immune system during stress could depend on stress susceptibility status of an animal, which probably involves hitherto unknown mechanisms underlying gene dependent modulation of individual stress response. Neuromyelitis optica (NMO), a severe demyelinating disease, is the subject of increasing interest since the discovery of the highly specific serum biomarker NMO-IgG. The target antigen aquaporin-4 (AQP4), a water channel protein on astrocytic endfeet, is expressed in the form of full length M1 or shorter M23 AQP4 isoforms. Previous data from our group did not only confirm the presence of AQP4-IgG in the sera of NMO and high risk patients, but also indicate that the conformational epitopes of M23 AQP4 are the primary targets of AQP4-IgG. Based on these results, we intend to investigate the ability of AQP4-IgG to induce cellular and complement mediated cytotoxicity (CDC).
AQP4 M23 and M1 transfected HEK293 cells were exposed to heat inactivated samples of 20 NMO patients and 20 controls. After addition of active complement, complement dependent cytotoxicity (CDC) was analyzed by the presence of the terminal complement complex (TCC) using a live cell staining immunofluorescence (IF) assay and flow cytometry. Our results show cell bound components of the activated complement cascade on cells incubated with high titer AQP4-IgG positive samples, but not in healthy controls or patients with multiple sclerosis. Besides CDC, antibody dependent cellular cytotoxicity (ADCC) is currently evaluated by incubating transfected cells with surface-bound AQP4-IgG from patients and human NK cells. Cell death of AQP4 transfected cells and the activation status of NK cells is examined by flow cytometry.
The analysis of CDC and ADCC mediated by human anti-AQP4 antibodies might provide new insights in the role of these antibodies in NMO. Several studies reported cytotoxic mechanisms in many pathological events. In the accidental pathology like scorpion envenomation, complex immune response reaction with release and activation of inflammatory mediators and cellular infiltration are observed. The pharmacological actions of scorpion venoms from North Africa are relatively scarce and the direct action of the crude venoms has not been assessed using cell culture models.
In this work, the investigation of toxicity of venom in cell culture and in mice is undertaken. The in vivo effect of Androctonus australis hector (Aah) venom on macrophage and spleen cells are examined. In addition, obtained peritoneal macrophages from male BALB/c mice and culture cells were stimulated in vitro with Aah venom.
They showed that Aah venom induced cytotoxicity activity on macrophages and splenocytes. The determination o the cytotoxicity by the test of crystal violet staining after 24 h of incubation was 70% at a concentration of 10 μg/ml of venom in vivo and 25 μg/ml of venom. Biochemical study was also conducted to evaluate the oxidative stress caused by the venom. Mediators of oxidative balance were measured; malondialdehyde, an index of lipid peroxidation, glutathione and H2O2. After 24 h of stimulation, an increase of 10% production of malondialdehyde was observed with splenocytes compared to control cells. Lipid peroxidation can be either a cause or an effect of toxic reactions induced by scorpion venom. Indeed, the increase of lipid peroxidation correlates with increased production of hydrogen peroxide. A significant release of H2O2 was also obtained after activation of peritoneal macrophages with low doses of Aah venom. Furthermore, an increase of intracellular glutathione, marker of antioxidant balance has been highlighted on the spleen cells and macrophages. The activation of these cell lines with increasing doses of venom caused a maximum production of glutathione to low doses of venom.
The mechanism of action led by the venom is complex, the involvement of inflammatory cells and mediators in the process of cytotoxicity remains unclear.
Differences in cellular immunity associated with active disease in patients with MS and NMO Matsui Makoto ⁎ ,1 , Hashiba Naomi 1 , Araya Shin-ichi 1 , Inada Hiroyuki 1 , Nagayama Shigemi 1 , Tanaka Keiko 1 , Konishi Tetsuro 2 1 Kanazawa Medical University, Uchinada, Japan; 2 Utano National Hospital, Kyoto, Japan
There is ongoing discussion regarding whether multiple sclerosis (MS) and neuromyelitis optica (NMO) should be considered as distinct disease entities, or represent the spectrum of a demyelinating disease. To address the differences in pathogenesis between MS and NMO patients, it is important to compare inflammatory events occurring in the systemic circulation as well as cerebrospinal fluid (CSF) during relapse and active disease states. We investigated cellular immunological events in blood and CSF samples obtained from patients with active MS and NMO using a flow cytometry system.
Fifteen patients with MS (7 women, 8 men) and 7 with NMO (all women) were enrolled in the study. All MS patients had typical MS plaque in the brain that fulfilled Barkhof's criteria and no long spinal cord lesions (LCL) encompassing more than 3 vertebral segments, and were negative for anti-AQP4. In contrast, all NMO patients had LCL and were positive for the antibody. None of the subjects were receiving immunomodulatory drugs at the time of the study. Each was examined during a relapse or active stage of disease, when magnetic resonance imaging (MRI) scanning showed gadolinium enhancement in the brain or spinal cord. Blood and CSF samples were obtained on the same day, and measurements using flow cytometry were performed to determine the percentages of CD3−, CD4−, and CD8positive T cells, B cells, functional CD4+ subsets including CD4+CD25 high regulatory T cells, Th1, and Th2 cells, and CD8+ subsets including CD8+CD11a+ cytotoxic T cells and CD8+CD11a-suppressor cells. In addition, levels of CSF protein, albumin, and IgG, as well as IgG index were determined.
In peripheral blood of patients with NMO, the CD8+CD11asuppressor cell population was significantly decreased as compared to the MS group (2.9% vs. 8.8%), while protein levels were significantly increased in the CSF of the NMO group (56.3 vs. 28.9 mg/dl). To assess the relevance of anti-AQP4 in lesion formation in optic nerves and the spinal cord, all data obtained from patients with spinal or optic-spinal MS (n = 4) and the NMO patients (n = 7) were compared, which indicated that antibody positivity contributed to decreases in both Th1 and Th2 cells in the CSF.
Our results suggest that relapse in NMO patients has a background of systemic immunoregulatory disturbance that is more conspicuous than that in MS patients. Furthermore, the anti-AQP4 antibody has an effect on cellular immunity in the central nervous system.
Does anti-inflammatory cytokine IL-10 help to restore female cycle integrity after immunological challenge?
Barad Zsuzsanna ⁎ ,1 , Barabás Klaudia 2 , Kiss Endre 3 , Kövesdi Dorottya 3 , Sármay Gabriella 3 , Ábrahám István 1 1 Ctr. for Neuroendocrinology, Dept. Physiol, Univ. of Otago, Dunedin, New Ze1land; 2 Inst. of Med. Sciences, Univ. of Aberdeen, Aberdeen, United Kingdom; 3 Dept of Immunol., Eotvos Lorand Univ., Budapest, Hungary
It is well known that peripheral immune challenge can interfere with reproduction. In females immune/inflammatory stress can disrupt the estrus cycle by affecting several levels of the hypothalamus-pituitarygonad (HPG) axis. Previous research has shown that peripheral LPS injection is able to alter the pulsatility of gonadotrophin releasing hormone (GnRH) secretion of gonadotrophin releasing hormone neurons (GnRH neurons) of the medial preoptic area (mPOA) in female rats. The aim of our experiments was to test the effect of a T celldependent antigen (fluorescein isothiocyanate conjugated keyhole limpet hemocyanine, FITC-KLH) and a T cell-independent antigen (FITC conjugated dextran) on GnRH neurons upon immunization in female C57/BL6 or IL-10 knockout (IL-10 KO) mice.
We measured the phosphorylation of the extracellular signalregulated kinase 1/2 (ERK1/2) as an index of intracellular changes. pERK1/2 in GnRH neurons was detected by means of double-label immunohistochemistry at different time points after immunization. T cell-dependent antigen-induced immune challenge led to a significant increase (p b 0.05) in pERK1/2 in GnRH neurons on day 3 with a maximum increase on day 6. In contrast, FITC-dextran administration failed to have any effect on ERK1/2 phosphorylation in GnRH neurons. Interestingly, elevation of the level of anti-inflammatory cytokine, IL-10 (pb 0.05) in the hypothalamus of FITC-KLH challenged mice showed strong synchronization with the increase of pERK1/2 in GnRH neurons, while pro-inflammatory cytokines such as IL-1 or TNFa and prostaglandins were not detected in the hypothalamus of immunized mice at the same time. The role of IL-10 was evaluated by using IL-10 KO mice. Immunization of IL-10 KO mice showed that the pERK1/2 increase in GnRH neurons on days 3 and 6 was completely abolished. The effect of immunization on cycle integrity was assessed by analyzing vaginal smears daily for at least 2 weeks prior and after the administration of FITC-KLH. We found that initial disruption in cycle integrity, which might be caused by the immune challenge, was restored by day 6 in wild type C57/BL6 mice, while IL-10 KO animals showed highly irregular cycles which were further perturbed by immunization.
Taken together our results suggest that IL-10, released during the immune response in a time and antigen dependent manner, may regulate GnRH neuron functions by inducing ERK1/2 phosphorylation, thereby contributing to the integrity of the HPG axis. Background: Multiple sclerosis (MS) is a relapsing-remitting demyelinating disease. Despite evidence supported the idea that MS is a T cell-mediated autoimmune disease, the involvement of autoantigenspecific regulatory and effector T lymphocytes is not fully understood and remain to be established. Objectives: To assess the dynamics of autorreactive effector (Teff) and regulatory T (Treg) lymphocytes during the progression of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, and also made a screening about the balance between both populations. Methods: We induced EAE in C57BL/6 (IA b) and performed quantitative analysis using MOG35-55/IAb (MHC class II) tetramers. Tetramers were used to track autoantigen-specific regulatory (CD4+ CD25high) and effector T cells (CD4+CD45RO+CD69+) during the disease. Detection and characterization of antigen-specific T cells was realized in spleen, lymphatic nodes, peripheral blood and CNS tissue during the progression of EAE and analyzed by flow cytometry. Results: We observed the increase of both populations Teff and Treg specific for MOG after the induction of the disease. We are currently performing the analysis of both populations in the brain tissue. In addition we observed a subpopulation of CD4-cells crossreacting with the MOG tetramer that is going to be further characterized.
Conclusions: Better understanding of effector and regulatory T cell dynamics will allows improving the knowledge of the pathogenesis of autoimmune diseases. Further studies are required in order to find new therapy targets. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a signature cytokine of autoimmune pathogenic T cells and is known to be absolutely required for the generation of an encephalitogenic T cells response. The mechanisms of action are beginning to become unravelled and include both the priming and the effector phase of the response.
We found that intracerebral production of GM-CSF in healthy mice suffices to recruit identical dendritic cell (DC) populations as in EAE into the CNS. These populations consisted of a minority of lymphoid DCs and a majority of myeloid and 'inflammatory' DCs. Both in EAE and after intracerebral GM-CSF treatment, DCs found within the CNS prove to have the same origin and functional properties. Lymphoid and myeloid DCs are almost exclusively blood-derived whereas 'inflammatory' DCs are constituted of both blood-and CNS-derived cells. Furthermore, lymphoid and myeloid DCs were found to stimulate T cell proliferation and cytokine secretion strongly while peripheral 'inflammatory' DCs were inferior in these functions. In contrast, CNS-derived 'inflammatory' DCs as well as microglial cells treated in vitro with GM-CSF were found to be able to inhibit T cell proliferation. Despite the capacity of GM-CSF to induce CNS-derived inhibitory inflDCs, the administration of GM-CSF into mice with EAE resulted in exacerbated disease.
GM-CSF has a dual role in the CNS: it directs both CNS-derived DCs towards an inhibitory phenotype and recruits peripheral DCs exhibiting pro-inflammatory functions. Additional CNS-specific effector functions are under investigation and will be discussed. Vascular cell adhesion molecule 1 (VCAM-1) has been shown to be a key factor for the initial steps of leukocyte extravasation in MS lesions and serves as ligand for VLA-4 expressed on leukocytes. VCAM-1, and consecutively, its soluble form (sVCAM)is up-regulated during endothelial activation in inflammatory diseases. Natalizumab, a recombinant monoclonal antibody directed against VLA-4 downregulates cellular trafficking across the blood brain barrier. The effect of VLA-4 blockage on its counterpart VCAM-1 has not been investigated in vivo.
We wanted to investigate how sVCAM-1 serum levels are affected by Natalizumab treatment compared to various control groups and if sVCAM-1 levels may serve as a marker of Natalizumab bioavailability.
We investigated the effect of Natalizumab on serum sVCAM-1 levels, measured by ELISA, in MS patients (95 samples) compared to various control groups including ten healthy persons, and untreated as well as interferon-beta treated MS patients (103 samples). In a subset of patients and controls VLA-4 expression was measured by fluorescence activated cell sorting on lymphocytes and monocytes. Neutralizing antibodies (NAB) against Natalizumab and interferonbeta have been determined by standard methods.
Median serum sVCAM-1 concentrations were significantly lower in samples from Natalizumab treated patients as compared to all control samples (256 vs 597 ng/ml). There was no significant difference between control samples and NAB positive Natalizumab samples (597 vs 602 ng/ml) and a significant difference between NAB positive versus NAB negative samples (602 vs 235 ng/ml). A sVCAM-1 concentration of less than 382 ng/ml indicated Natalizumab treatment with an 87% sensitivity and 90% specificity. sVCAM-1 concentrations in follow-up samples of Natalizumab treated patients did not change over time. VLA-4 expression was present on nearly all lymphocytes and monocytes in controls and almost entirely blocked in Natalizumab treated patients.
We believe that the study results indicate a dual role of Natalizumab by blocking VLA-4 and down-regulation of VCAM-1. Both mechanisms may be involved in the reduced cell trafficking into the CNS leading to clinical effect of Natalizumab but also to the rare albeit evident adverse event of progressive multifocal leukoencephalopathy observed in MS patients during this treatment. sVCAM-1 appears to be a good biomarker of Natalizumab bioactivity which might be used for therapy monitoring. MS is a chronic inflammatory demyelinating disease of the central nervous system and it is also regarded as T cells-mediated autoimmune disease. Natural killer (NK) cells are a major component of innate immune system and are capable of killing target cells without prior immunization. Several reports suggested a protective role for NK cells in MS patients. But how NK cells kill autologous T cells has not been defined so far. The goal of the current study is to identify the mechanism utilized by CD56bright NK cells for killing autologous T cells.
We used polyclonally activated T cells, isolated from peripheral blood mononuclear cells (PBMC) of daclizumab-treated MS patients by negative selection, as target cells. Target cells were effectively killed by autologous NK cells (0.1:1 E:T ratio) in modified flowcytometry based killing assay, which demonstrated predominant degranulation of CD56bright, as opposed to CD56dim NK cells. In contrast, resting T cells were not killed. In order to identify mechanism of killing, several blocking reagents were applied. EGTA, which inhibits the degranulation of NK cells, had more than 80% blocking effects, while Z-VAD-FMK, which is pan-caspase inhibitor, had only 20% blocking effects on the killing. The neutralizing anti-Fas Ab or TRAIL R2-Fc had no effect. By confocal microscopy, we observed transfer of granzymes (Gzm A, -B and -K) from effector cells to activated T cells within 3 h of co-culture. Quantitatively, Gzm K, which was almost completely absent in activated CD4+ T cells had highest fold increase. Because granzymes including GzmA, GzmB and GzmK kill target cells by inducing mitochondrial dysfunction and reactive oxygen species (ROS), we measured mitochondrial transmembrane potential and intracellular ROS formation and found both significantly impaired in the activated T cells upon co-culture with NK cells.
Granule exocytosis pathway contributes to NK cells-mediated killing of autologous T cells in Multiple Sclerosis. Chemokines and their receptors were originally described as chemotactic cytokines involved in leucocyte trafficking. However, further research has shown that chemokine receptors are not restricted to leucocytes. One of those receptors, CCR5, regulates both trafficking and effector functions of Th1 cells, macrophages, NK cells, and immature dendritic cells. A common 32-basepair deletion (CCR5delta32) in the coding region of CCR5 gene originates a truncated non-functional receptor with reduced expression on the cell surface. Multiple Sclerosis (MS) patients have elevated percentages of blood T cells expressing CCR5 compared with healthy controls, suggesting that this genetic polymorphism could modulate disease susceptibility or progression.
To investigate whether the CCR5delta32 deletion is associated with susceptibility and/or severity in Portuguese MS patients.
A total of 424 MS patients and 230 ethnically-matched controls were studied. A subset of 136 patients with disease duration of at least 10 years, was divided into 2 groups according to severity: 96 patients were considered to have benign MS (EDSS b= 3) and 40 aggressive MS (EDSS N= 6). Kaplan-Meier survival analysis of the distribution of time to reach mild (EDSS = 3) and severe disability (EDSS = 6) was performed. Differences between distributions were tested using Log Rank test.
No significant difference was observed in the allelic frequency of CCR5delta32 between patients and controls (5.7% in MS vs. 8.3% in controls, OR = 0.666; p = 0.069). Concerning disease severity, CCR5delta32 frequency was significantly higher in the aggressive group (5.2% in benign MS vs. 13.7% in aggressive MS, p = 0.016). For EDSS = 3 as the end point, the median progression time was 6 years for delta32 positive patients and 11 years for delta32 negative (p = 0.011). To reach an EDSS = 6 the median progression time was 18 years for delta32 positive group and 23 years for the other group (p = 0.039).
In this study MS patients carrying the CCR5delta32 deletion appear to have a worse prognosis, in agreement with a previous study from Gade-Andavolu and colleagues [Genet Med 2004; 6(3) :126-131] that reported a strong association of this deletion with early death. The 32 deletion may protect against the development of MS, although we could not confirm it in this study. However, once the disease is diagnosed, carriers may progress more rapidly to an advanced EDSS, suggesting a possible dual effect of this deletion. Objectives: (i) To confirm the HSP70i differential gene expression between MS patients and HD by using real-time PCR, and (ii) to study the HSP70i putative role in MS pathogenesis.
HSPA1A (HSP70i) gene expression was determined by real time-PCR in both fresh and frozen peripheral blood mononuclear cells (PBMC) from relapsing-remitting (RR) MS patients and HD. In contrast to previous results, HSP70i expression in fresh PBMC from RRMS patients was significantly increased compared with HD (n = 30 per group; fold change = 2.5; p = 0.036). As HSP70i expression is induced by stress conditions and previous studies had used fresh and/ or frozen PBMC, we analyzed whether freezing could modify HSP70i expression and explain the controversial observed data. However, HSP70i expression did not significantly change when comparing fresh and frozen PBMC from either HD (n=10; fold change=1.39; p = 0.106) or RRMS patients (n = 9; fold change = −1.79; p = 0.91). However, HSP70i gene expression increase in RRMS patients was not associated with changes in the protein expression in CD4+ and CD8+ T cells and monocytes.
To study the putative role of HSP70i in MS, we induced experimental autoimmune encephalomyelitis (EAE) in HSP70i KO mice. In two independent studies, no clinical or histopathological differences in HSP70i KO mice were found compared with wild-type mice. Even though differences were not statistically significant, EAE incidence reduction (78.6% vs 65.4%) and increased disease severity (maximum score: 4.0 ± 0.0 vs 4.5 ± 0.9) were observed in HSP70i KO mice.
In the present study, previous data derived from microarray studies regarding HSP70i expression in MS patients could not be confirmed by using an RT-PCR approach. Conversely, HSP70i gene expression was found to be increased in RRMS patients compared with HD. These data suggest a relation between HSP70i and an enhancement of immune response. Additional studies are needed to gain more insight into the biological relevance of HSP70i in the MS pathogenesis. Virus-primed memory CD4 T cells protect immunodeficient SCID recipients following CNS infection by neurotropic coronavirus (JHMV), whereas IFN-g deficient (GKO) donor CD4 T cells result in rapid mortality. Although virus replication remains uncontrolled without demyelination in control infected mice, both recipient groups controlled virus replication and exhibited similar demyelination. However, distinct from wt CD4 T cell recipients, mortality of GKO recipients was associated with massive CNS neutrophil infiltration and detection of IL-17 mRNA.
To determine the interplay between IFN-g, IL-17 and neutrophils in disease severity, equal populations of wt and GKO CD4 T cells were co-transferred to SCID mice. Despite the sparse accumulation of wt versus GKO CD4 T cells in the CNS (20 vs 80%), this minor population abrogated the detrimental effects of GKO CD4 T cells. CNS neutrophil infiltration was decreased coincident with reduced neutrophil-attracting chemokines, and disease severity was ameliorated promoting survival. A protective role for IFN-g in limiting CNS neutrophil recruitment was supported by rapid lethality associated with massive CNS neutrophil infiltration in wt/GKO CD4 T cell recipients treated with anti-IFN-g. Surprisingly however, WT recipients treated with anti-IFN-g were spared from accelerated mortality despite substantial CNS neutrophil infiltration. Analysis of IL-17 expression in the CNS suggested that rapid fatal disease outcome was only observed in IFN-g deficient recipients that expressed IL-17 mRNA. Inhibition of IL-17 in GKO recipients indeed reverted the lethal phenotype despite retention of elevated neutrophils, demonstrating that IFN-g afforded protection by inhibiting IL-17 mediated proinflammatory responses.
These data demonstrate that IFN-g downregulates neutrophil accumulation independent of IL-17. However, neutrophil dysregulation in the absence of IFN-g only has overt clinical consequences in an environment expressing IL-17. Importantly, under conditions expressing both IFN-g and IL-17, IFN-g can override an otherwise detrimental IL-17 effector response.
Impact of IL-17, and IL-23p19 on the neutrophil and T-cell response in murine brain abscess Held Josephin ⁎ , Richter Lydia, Meisen Michael, Heppner Frank L., Stenzel Werner
Brain abscess, a severe inflammatory condition of the CNS is characterized by different phases with an orchestrated immune response, which is elicited by cells of the peripheral immune system as well as CNS-derived cells. Recently, we have analyzed various aspects of the neutrophil response, the T-cell response as well as the role of macrophages and microglial cells, in this context.
Since the recruitment and activation of these cells is regulated mainly by IL-12p35, IL-23p19, and IL-17, as constitutes of the Th1 and the Th17 response, we have used gene-targeted mice deficient for the mentioned cytokines to understand their sequential impact and kinetics in adaptive and innate immune processes during the course of abscess development and resolution.
Especially IL-17 is known to partially play a role in neutrophil recruitment, but is also produced by a recently described subset of T helper cells (Th17 cells), thus having an impact on the early phase characterized by a dominant neutrophil influx, as well as on the later phases of brain abscess, where the adaptive immune response is active.
In this work, we provide evidence that IL-17 is an important mediator of brain abscess development, we discuss the sequential roles of cytokines of the IL-12 family, and compare their role in the initial vs. the late phase of the disease.
138 Impaired responses to gut microbes and gliadins in splenocytes from mice with altered stress-related behavioral and premature immune senescence Stress is associated with an impaired bi-directional communication between the nervous and the immune systems leading to loss of homeostasis, which might play a relevant role in disease risk. Celiac disease (CD) is a chronic inflammatory disorder of the small intestine caused by an abnormal immune response to gluten proteins (gliadins) in genetically predisposed individuals. The ingestion of gluten is the main trigger of the disease, whose typical cases often present in early life after gluten introduction into the diet. However, the prevalence of CD in adulthood is increasing, suggesting that factors other than gluten intake can be involved in the presentation of this disorder. Moreover, CD has been recently associated with alterations in gut microbiota composition, which may also favour the development of the disease and the ongoing inflammation in the small intestinal mucosa.
The aim of this study was to assess whether splenocytes from prematurely aging mice (PAM), a model based on altered stressrelated behavior response and immune senescence, show different responses to gliadins and intestinal bacterial strains, including potential probiotics and pathogens, than those cells from nonprematurely aging mice (NPAM).
Two groups of female adult outbred ICR-CD1 mice, 10 PAM and 10 NPAM obtained according their different behavior in a T-maze test, were used. Splenocytes from these animals were incubated for 24 h with the following stimuli: LPS (1 μg/ml), bovine serum albumin (BSA) (100 μg/ml), gliadin fragments (100 μg/ml) and 4 different bacterial strains (10^6 CFU/ml), Bifidobacterium longum subsp. infantis I1, B. longum F1, E. coli CBL2 and Lactobacillus casei Y1. The levels of cytokine (TNF-alpha and IL-10) secretion were determined in supernatants by ELISA.
Splenocytes from PAM released significantly higher levels of TNFalpha than those from NPAM after stimulation with LPS and all bifidobacteria (P = 0.001-0.007), as well as of IL-10 with one of the bifidobacteria (B. longum F1; P = 0.001). In addition, after stimulation with gliadins, PAM splenocytes secreted significantly higher levels of TNF-alpha and smaller of IL-10 than these cells from NPAM (P = 0.009).
Immune cells from adult PAM mice, with altered stress-related behavioral, show an inflammatory stress response to gliadins, which could lead to a higher risk of developing CD.
Nuyts Amber ⁎ ,1 , Cools Nathalie 1 , Van Camp Kirsten 1 , Lenders Kevin 1 , Stein Barbara 1 , Nagels Guy 2 , D'hooghe Marie Beatrice 2 , Willekens Barbara 3 , Berneman Zwi N. 1
Dendritic cells (DC) are a specialized population of white blood cells that play an important role in the modulation of the immune system. In the present study, we hypothesize that an alteration in the function of DC results in a disturbance of the balance between immunity and tolerance in multiple sclerosis (MS).
Monocyte-derived DC from MS-patients (12 males, 10 females) were generated and compared with age-and gender-matched healthy controls (n= 22). Next, we evaluated, the phenotype and cytokine expression profile of immature DC (iDC) and cytokine cocktail-matured DC (mDC), the capacity of DC to induce CD4+CD25+FOXP3+ regulatory T-cells (nTreg), and their potency to activate autologous myelin-specific T-cells in vitro.
We show that, upon maturation, mDC of MS-patients display significant lower expression levels of MHC class II, costimulatory and migratory molecules as compared to mDC of healthy controls. In addition mDC of MS-patients secrete lower amounts of IL-6, TNFalpha and IL-10 compared to mDC of healthy controls. Moreover, they induce a significantly lower number of nTreg as compared to mDC of healthy controls. In contrast, no difference in the induction of myelinspecific IFN-gamma-producing T-cells was found.
In conclusion, our results show that the phenotype and function of mDC is impaired in MS-patients, which influences the induction of nTreg. These observations have important implications with regard to development of DC-based vaccine strategies against MS. Immunologists have been looking in the past at T cell differentiation as a one-way process leading to terminal and irreversible commitment to one or the other T cell phenotype. We have now to face the concept of plasticity, implying that cells, including T cells, may change their fate according to the needs imposed by the environment and the consequent epigenetic stimuli.
We induced relapsing-remitting EAE in SJL/j FoxP3gfp.KI mice, in which Foxp3+ T cells express a fluorescent, functional fusion protein, allowing for identification and sorting of living FoxP3(gfp)-expressing Treg cells. To investigate if polarized TH cell differentiation stimuli allow for simultaneous expression of different lineage markers, we sorted from these mice CD4+CD62LhiFoxP3gfp-naïve T cells. At the peak of disease, cells from draining lymph nodes were cultured in the presence of a polyclonal stimulus, cytokines, and blocking antibodies to induce differentiation towards TH1, TH2, TH17, and iTreg cells. Surprisingly, we found that FoxP3 mRNA was expressed above background for up to 48 h after stimulation in untreated cultures and in cultures treated to become TH1, TH17, but not TH2 cells. Further, we sorted CD4+CD62LhiFoxP3gfp-T cells from naïve SJL/j FoxP3gfp.KI mice and measured, during the same culture conditions as above, the expression of FoxP3, Tbx21, Gata3, Rorc, IFN-g, and IL17. We confirmed in naïve T cells that FoxP3 is expressed during early time points in untreated, TH1, TH17, and, differently from RR-EAE, also in TH2 cultures. At later time points (48 h), however, FoxP3 was up-regulated only in TH17 and, as expected, in iTreg cultures. Other unexpected findings were the up-regulation of Rorc in iTreg cells. Finally, while IFN-g was especially expressed in untreated and TH1 cultures, IL17 mRNA appeared to be transcribed in all conditions, remaining expressed, although at apparently low levels, long-term only in TH17 culture conditions.
Thus, it appears that early after activation through a polyclonal stimulus, independently from culture conditions, it is possible to detect, in the same cells, markers and effector cytokines from supposedly different lineages. Indeed we found that T cells from autoimmune mice express different lineage-specific nuclear transcription factors in the first hours upon activation suggesting that data produced in the past should be revisited according to this concept of mixed identity. Celiac disease is an enteropathy triggered by the ingestion of cereal gluten proteins (gliadins) in genetically predisposed individuals. This disorder has also been associated with alterations in the composition of the gut microbiota, which in vitro seem to contribute to inducing a Th1-type cytokine profile characteristic of the disease.
The aim of this work was to study the possible role of intestinal bacteria (bifidobacteria and Gram-negative bacteria) in the phenotypic and functional maturation of dendritic cells (DC) in the presence of gliadins.
Monocyte-derived DC were stimulated with potentially probiotic bacterial strains (Bifidobacterium longum ES1 and Bifidobacterium bifidum ES2) and Gram-negative bacterial strains isolated from the gut microbiota of CD patients (E. coli CBL2 and Shigella CBD8) alone or in co-culture with intestinal epithelial Caco-2 cells. Surface protein expression and cytokine production by DCs were evaluated under the effects of the bacterial strains alone and in combination with gliadins by ELISA and flow cytometry.
The Gram-negative bacteria induced higher secretion of Th-1 type cytokines, TNF-alpha, IL-12 and IFN-gamma than the two bifidobacteial strains tested. The induction of the expression of CD40 and CD86 in DC was also higher in the presence of both Gram-negative bacteria than in the presence of the two bifidobacterial strains. Co-cultivation of monocytes derived DCs with Caco-2 cells resulted in reduced cytokine secretion by DCs, and only the two Gram-negative bacteria exerted a pro-inflammatory effect.
Cytokine production and co-stimulatory protein expression in DCs could be influenced by specific components of the gut microbiota, which together with gliadins may also influence their interaction with effector T-cells and the polarization of the immune response. In this study we evaluated the percentage of Perforin+CD8+ cells, Perforin+CD8+CD56− T cells, Perforin+CD8+CD56+ cells, Perforin+CD8+CD56+ dim and Perforin+CD8+CD56+ bright NK cells in peripheral blood from 86 untreated relapsing remitting (RR; 35 in relapse and 51 in remission), 12 untreated secondary progressive (SP), 12 untreated primary progressive (PP) multiple sclerosis patients and 40 age and sex matched healthy subjects.
The percentage of circulating Perforin+CD8+ cells and Perforin Mean Fluorescence Intensity (MFI) in CD8+ cells was higher in RRMS patients, both in relapse and in remission, in SPMS and in PPMS patients than in controls. The percentage of circulating Perforin+CD8+CD56− T cells was significantly higher in SPMS, PPMS patients than in controls while it was increased in RRMS patients without reaching significant value. PPMS patients showed higher percentage of Perforin+CD8+CD56+ dim NK cells than RRMS patients both in relapse and in remission and controls whereas there was no difference in the percentages of Perforin +CD8+CD56+ bright NK cells among the different groups of patients and controls.
The percentages of circulating Perforin+CD8+CD56− T cells and Perforin+CD8+CD56+ dim NK cells positively correlates with the 6 months confirmed EDSS score in all MS patients. MS patients with confirmed EDSS N3 showed higher percentages of Perforin+CD8+ CD56− T cells and Perforin+CD8+CD56+ dim NK cells than patients with EDSS = 3.
Our study demonstrate increased percentages of Perforin+ CD8+CD56− T cells and Perforin+CD8+CD56+ dim NK cells in peripheral blood of MS patients that correlate with confirmed disability. Perforin+CD8+CD56− T cells and Perforin+ CD8+CD56+ dim NK cells may play a role in MS disability progression.
Partially Traditionally, the temporal order of Multiple Sclerosis (MS) lesion development starts with an active lesion, followed by a chronic active lesion in which macrophages accumulate at the rim as myelin in the centre is progressively removed. Finally, the lesion dies leaving a hypocellular scar-like inactive lesion. Still, the most intriguing issue remains the question how MS lesions really start. The earliest pathological sign of damage in MS are clusters of activated microglia in normalappearing white matter that we have termed preactive lesions. Given the frequency of preactive lesions we suggest that most probably spontaneously resolve, without progressing to an active demyelinating stage. Microglia activation at this stage may therefore reflect immune-regulatory or immune-suppressive rather than pro-inflammatory activity. The goal is to examine how preactive lesions arise.
Small heat shock proteins (sHSP) may play a key role in preactive lesions since we have shown that alpha B crystallin (CRYAB) accumulate in oligodendrocytes in preactive MS lesions and that CRYAB directly contacts the microglial surface. In vitro CRYAB activates human microglia inducing an immune-regulatory phenotype typified by production of IL-10, TGF-beta and TNF-alpha, and suppression of IL-12, consistent with the previously demonstrated anti-inflammatory effects of sHSPs in animal models. In vitro CRYAB is unregulated in stressed oligodendrocytes following treatment with monoclonal antibodies (mAb) directed to myelin oligodendrocyte glycoprotein (MOG). These mAbs augment clinical disease and demyelination in mice with EAE. Moreover antibodies to MOG, but not mAb to other myelin proteins induce stress in oligodendrocytes inducing repartitioning of MOG changes in intracellular signalling and cell morphology.
In this paper we will present our hypothesis that in MS antibodies directed to MOG could be derived from EBV infected B cells in MS. These antibodies initiate stress in oligodendrocyte and upregulate CRYAB as a protective mechanism. Such expression of CRYAB however triggers the activation of microglia which in MS leads to the formation of preactive lesions.
We therefore hypothesize that pre-active lesions reflect a mild form of reversible neuroinflammation, governed by predominantly immune-regulatory and neuroprotective processes. We believe preactive lesions thus hold the key to understanding natural mechanisms of immune regulation and repair in the human CNS.
Mannose binding lectin in Guillain-Barré syndrome Uchibori Ayumi ⁎ , Chiba Atsuro Kyorin University, Tokyo, Japan Background: In Guillain-Barré syndrome (GBS), it is suggested that complement activation is an important mechanism for the postinfectious immune-mediated peripheral nerve damage. Mannose-binding lectin (MBL), which recognizes sugar structures such as mannose and N-acetyl-glucosamine on the surface of pathogens and activates the complement system, has been reported to be related with severity of GBS. Objective: To elucidate the roles of MBL in pathogenesis and exacerbation of GBS, we investigated chronological change of serum MBL concentration especially in early period of neurological onset. Methods: Peripheral blood samples were serially obtained from 12 patients with the IgG anti-GQ1b antibody-positive variant of GBS: 10 with Miller-Fisher syndrome and 2 with GBS with ophthalmoplegia. Serum MBL concentration was determined by ELISA using mannan as the ligand. Results: Patients had wide-ranging serum MBL concentration. In the study on serial samples, the MBL concentration reached its peak around the same time as the bottom of clinical symptoms, and then decreased in four patients. One patient had the peak of the MBL concentration at neurological onset. In two patients who were measured for only a symptomatic worsening phase, the MBL concentration increased. The MBL concentration increased gradually and then decreased over the 15 disease days in a case taking the most severe clinical course, in whom the point of nadir was unclear. In the other two cases, the MBL concentrations were unchanged throughout the clinical course. Conclusions: In the serial study, the chronological change of the MBL concentration showed a similar tendency. The MBL concentration level increased during clinical exacerbation and reached a maximum level just around the bottom of clinical symptoms, and then decreased gradually. These results suggest that complement activation mediated by MBL could be associated with pathological condition of GBS, especially extent of nerve damage. MBL might be another target point for therapeutic approach to prevent the progression of the disease.
Mapping of the corticotropin-releasing factor (CRF) family of peptides and receptors in fibroblasts-like synoviocytes from osteoarthrosis and rheumatoid arthritis patients It is well known the presence of hormones and neurotransmitters (including neuropeptides) in the joints of patients with rheumatic diseases including osteoarthrosis (OA) and rheumatoid arthritis (RA). The origin could be from the central and peripheral nervous systems or produced at local level by the cells of the joint. In this sense, we recently have demonstrated a differential expression of VIP and its receptors, VPAC1 and VPAC2, in OA-FLS and RA-FLS. CRF-UCNs system includes four peptides, corticotropin releasing factor (CRF), urocortin 1 (UCN1), urocortin 2 (UCN2) and urocortin 3 (UCN3), and two receptors the CRFR1 and the CRFR2, which are expressed in both, nervous and peripheral tissues, including immune system. The CRF system is crucial for the regulation of mammalian stress and inflammatory responses, and they are also involved in disorders such as anxiety, depression and drug addiction.
Hypothalamic CRF produces immunosuppressive and anti-inflammatory effects through endogenous glucocorticoid released by activation of the hypothalamus-pituitary-adrenal (HPA) axis. Moreover, at peripheral sites the CRF system promotes direct both, inflammatory and immunostimulatory actions. Local production of peripheral CRF and UCN1 has been demonstrated in experimental models of RA, including streptococcal cell wall-and adjuvant-induced arthritic joints of rats and in the joints of patients with OA and RA. Thus, CRH system could contribute to the pathophysiology of rheumatic diseases. However, to date there is no data about UCN2, UCN3 and the CRF receptors in RA. To establish the role this system plays in this pathophysiology, it is crucial to identify the cellular type involved in its expression.
In the present study we describe the presence of CRF, UCN1, 2 and 3 in FLS from OA and RA patients, expressed at mRNA and protein levels, detected by real time PCR, enzyme immunoassay (EIA) and immunocytochemistry. Moreover, CRFR2 receptor is expressed in OA-and RA-FLS, showing no expression of CRFR1 receptor. CRF and UCN1 are more abundant in RA than in OA, being UCN2 and 3 more expressed in OA. Regarding CRFR2 receptor expression is similar in FLS from both pathologies.
In summary, our study shows that the RA pattern of the CRF peptides in human FLS is: an increased expression of the proinflammatory peptides, CRF and UCN1 and a decreased expression of the predominantly anti-inflammatory peptides UCN2 and 3.
Midkine exacerbates experimental autoimmune encephalomyelitis through suppressing expansion of regulatory dendritic cells Yoshifumi Sonobe ⁎ , Hua Li, Hideyuki Takeuchi, Tetsuya Mizuno, Akio Suzumura Nagoya University, Nagoya, Japan
We have shown previously that RNA aptamer against midkine, a heparin-binding growth factor, attenuates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, through the expansion of CD4+CD25+Foxp3+ regulatory T cells (Treg). However, the precise mechanisms remain unclear. Here, we show that inhibition of midkine expands regulatory dendritic cells (DCreg), which have capacity to induce differentiation of naïve T cells to Treg, which results in attenuating EAE.
DCreg stimulated with midkine decreased allogenic CD4+CD25+ Foxp3+ Treg expansion. Midkine impaired STAT3 phosphorylation (pSTAT3) in DCreg and induced IL-12 production through the induction of src homology phosphatase-2 (SHP-2). Inhibition of IL-12 or SHP-2 cancelled the suppressive effect of midkine on Treg differentiation, suggesting that midkine induced IL-12 production via dephosphorylation of pSTAT3 by SHP-2 in DCreg and attenuated DCreg-induced Treg differentiation.
Administration of midkine-stimulated DCreg to EAE worsened the symptoms of the disease accompanying the decrease of CD4+ CD25+Foxp3+ Treg, as compared to the administration of DCreg. Anti-midkine RNA aptamer attenuated EAE doe to the CD4+CD25+ Foxp3+ Treg and the CD11c+CD45RB+DCreg expansion. IL-12p35 and IL-12p40 mRNA expression levels in DC were downregulated in the lymph nodes of the treated mice.
These results indicate that midkine inhibits DCreg expansion and increases production of IL-12 via dephosphorylation of pSTAT3 by SHP-2, which results in suppression of the CD4+CD25+Foxp3+ Treg differentiation.
Black Jennifer ⁎ ,1 , Freedman Mark S. 2 1 University of Ottawa, Ottawa, Canada; 2 The Ottawa Hospital, Ottawa, Canada Neurofascin (NF) is an integral nodal protein serving a structural role as well as involved in neuro-transmission. Antibodies (Abs) specific for NF are detectable in the sera of multiple sclerosis (MS) patients. MS patient-derived FcR-expressing gamma delta (gd) Tcells have been shown to exert antibody-dependent cellular cytotoxicity (ADCC), and the frequency of this subset of gd T-cells increases with MS progression. These findings led us to propose a possible role for anti-NF Abs and gd T-cells in the pathogenesis of MS.
Using enzyme-linked immunosorbent assays anti-NF Abs were detected in both the cerebrospinal fluid (CSF) and sera of MS patients. The CSF titres were greatest in RRMS patients (p b 0.0001 vs. controls) whereas the reverse was observed in the sera, where the highest titres were detected in patients with progressive forms of disease (p b 0.001 and p b 0.05 for SPMS and PPMS respectively, vs. controls). To initiate the investigation of a possible role for anti-NF in the pathogenesis of MS, we first screened a series of neuronal cell lines using Western blots, flow cytometry and immunohistochemistry to identify those expressing NF. The screen revealed that human NT2/D1 teratocarcinoma cells express NF, and retinoic acidinduced differentiation of NT2/D1 cells under various conditions results in the derivation of a heterogeneous population of classical neurons and astrocytes, in addition to neurite-expressing neurospheres that continue to express NF. Commercial and MS-patientderived anti-NF antibodies (mixtures of IgG1, IgG2 and IgG3) recognized and bound to the NT2/D1 cells, both axonally and on the cell body. Currently we are establishing a system whereby we can examine the cytotoxicity of gd T-cells targeted towards cells of neuronal lineage, in the context of ADCC.
These studies will help to determine if gd T-cells injure neuronal cells in MS through direct and indirect cytotoxic mechanisms.
Opposing and disease stage specific activity of interferon gamma in EAE modulated by type I interferon signals
Raman Chander ⁎ ,1 , Naves Rodrigo 1 , Singh Simer Preet 1 , Cashman Kevin 1 , Axtell Robert 2 , Steinman Lawrence 2 , De Sarno Patrizia 1 1 University of Alabama at Birmingham, Birmingham, United States; 2 Stanford University, Palo Alto, United States
The physiological activity of interferon gamma (IFN-g) in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) remains controversial. Additionally, we recently reported that interferon gamma (IFN-g) signals are necessary for the immunosuppressive activity of IFN-beta (IFN-b) in experimental autoimmune encephalomyelitis (EAE). This study resolves controversies on the role of IFN-g in EAE and integrates it with the biological activities of type I IFN (IFN-a/b) signals.
We performed a comprehensive analysis to determine the extent of cooperation between type I interferons (IFN-a or IFN-b) and type II interferon (IFN-g) dependent signals in the development and progression of experimental autoimmune encephalomyelitis (EAE). The onset and severity of myelin peptide-induced EAE in IFN-a/b receptor null mice and wild-type (WT) mice were similar. In contrast, EAE in mice lacking the IFN-g receptor (IFN-gR −/− ) was delayed in onset but significantly more severe. Remarkably, mice lacking the receptors to both IFN-a/b and IFN-g (IFN-a/b/gR −/− ) developed a milder form of EAE. From these results we inferred (1) that IFN-g is disease promoting during induction phase but protective during the chronic phase and, (2) type I IFN-activity in the absence of IFN-g signaling exacerbates disease. In order to directly test for the opposing activity of IFN-g in EAE, WT mice were injected with recombinant IFNg (rIFN-g) daily for 10 days starting after induction of disease or after onset of disease symptoms. IFN-g treatment during the induction phase worsened disease, and remarkably, treatment after onset of clinical symptoms completely suppressed disease progression. The suppressive effect of rIFN-g treatment was dependent on STAT-1, a transcription factor that integrates both type I and type II IFN-receptor signaling, and functional IFN-a/b receptor. Naïve WT mice adoptively transferred with encephalitogenic T-cells from type I, type II or double IFN-receptor deficient animals revealed that both type I and type II IFNs were required for the persistence of TH1 disease but not TH17 disease. Importantly, functional loss of either IFN-a/b signaling or IFNg signaling led to a more severe TH17 disease.
The data reveal that IFN-g has an opposing disease stage specific activity in the pathogenesis of EAE. We further show that systemic administration of rIFN-g during active disease is therapeutic, but this activity is dependent on type I IFN-signals.
Perry Justin, Han SungPil ⁎ , Kennedy Lucy Boyce, Bibiana Bielekova
Objective of this study was to use unbiased approach to determine full mechanism of action (MOA) of daclizumab (DAC) in multiple sclerosis (MS).
Combining clinical trial of selective immunomodulator (DAC) with in-vivo immunization provides powerful tool to study human immunity. We studied changes in the immune subpopulations induced by influenza haemagglutinin (Flu-HA) immunization in MS patients on DAC therapy versus age/sex matched controls by multicolor flow cytometry. To further corroborate the findings, we analyzed cryopreserved peripheral blood mononuclear cells (PBMC) from NIH DAC trials. Finally, we performed extensive functional studies on purified immune subpopulations to determine the mechanisms underlying observed changes.
We identified a significantly lower proportion of adult LTi cells in DAC-treated patients vs. controls before Flu-HA immunization, which was further exaggerated post-immunization. We confirmed that DAC therapy diminished LTi numbers as early as 3 months post-treatment and that decline in LTi cells correlated with previously-reported expansion of CD56bright NK cells. Furthermore, by analyzing lymph node (LN) biopsy specimens we demonstrated that observed decline in circulating LTi cells in the DAC-treated patients is not due to their increased homing to LN. We hypothesized that DAC induces enhanced differentiation of CD56bright NK cells
Adult LTi cells promote development of chronic inflammation through induction of tertiary lymphoid follicles in target tissue. In contrast, CD56bright NK cells inhibit immune responses. We demonstrated that CD25 and CD127, two molecules genetically linked to MS, regulate differentiation of HPC toward LTi versus CD56bright NK cell lineage. Thus, small differences in IL-2/IL-7 signaling lead to profound changes in the outcome of the immune response from proinflammatory to tolerance-promoting phenotype, and may underlie development of autoimmunity in MS.
Sonia Adi-Bessalem ⁎ , Sassia Sami-Merah, Amina Mendi, Djelila Hammoudi-Triki, Fatima Laraba-Djebari USTHB University, Algiers, Algeria Voltage-gated Na+ (Nav) channel neurotoxins are the most important components of the scorpion venom and the main agents responsible for the toxic effects of scorpion envenoming. They induce a massive release of neurotransmitters and inflammatory modulators. These mediators lead to a cascade of pathological events in central nervous and cardio-respiratory systems but the involved mechanisms in immunological and inflammatory responses have not been extensively evaluated. The aim is to investigate the different types of mediators involved during experimental scorpion envenomation (Androctonus australis hector) using selected anti-inflammatory drugs (Indomethacin and Hydrocortisone) or adrenergic antagonist (Metoprolol).
Our findings showed that the inflammatory process induced by scorpion venom is characterized by hyperleucocytosis (neutrophil, eosinophil and lymphocytes) with elevated production of inflammatory cytokines (IL-6, IL-1β, TNF-a, IL-4, IL-5 and IL-10) in peripheral blood and in tissue of the animals. The migration of neutrophils and eosinophils in lungs was confirmed by the release of reactive oxygen species such as myeloperoxydase (MPO) and pulmonary eosinophil peroxidase (EPO). The hypoalbuminemia, hyperglobulinemia and hypergammaglobulinemia accompanied by a significant activation of complement system are also the predominant abnormalities found in envenomated animals. These inflammatory disturbances have resulted in the lungs and heart by an exudation of plasma proteins and fluid into the tissue forming a pulmonary and myocardic edema. Treated animals with drugs which interfere with arachidonic acid metabolism pathways prior to envenomation, reduced significantly the hyperleucocytosis and the cellular peroxidase activities (EPO and MPO). Reduction of fluid accumulation in lungs and heart, and a normalization of seric protein level compared with animals envenomed are also observed.
Metoprolol, beta-adrenergic blockers, is effective to reduce edema in heart and lungs as confirmed by structural analysis tissue. However, this antagonist did not prevent the increase of rate inflammatory mediators and serum proteins level.
These results suggest that edema induced by Androctonus australis hector venom could be mediated mainly by cellular mediators (neutrophils, eosinophils…) and also by multiple molecular mediators including eicosanoids, and partially by beta-adrenoceptors. We explore the role of serotonin in the animal model of MSexperimental autoimmune encephalomyelitis (EAE)-using fluoxetine to increase serotonergic activity. We showed previously that fluoxetine ameliorated paralysis significantly in EAE. Exploring the mechanism of this effect, we find that in vitro fluoxetine decreases cytokine production by spleen cells in response to inflammatory stimuli, with a major effect on T cells. In pure cultures, in vitro fluoxetine decreased T cell proliferation and increased Fas-dependent activation-induced cell death of T cells. T cells do not express SERT, the well-known site of action of fluoxetine. However, proliferation of T cells is primarily dependent upon the subtype of 5HT receptor, 5HTR1B, which is also known to bind fluoxetine. We therefore, hypothesized that fluoxetine may function by blocking 5HTR1B signaling. However, adding exogenous serotonin to flow cytometrically purified CD4 and CD8 T cells did not rescue this effect of fluoxetine at a high dose. We further explore this mechanism using another SSRI, citalopram, and SERT knock-out animals.
Fluoxetine ameliorates EAE and this is in part due to immunomodulation specifically affecting T cell proliferation and apoptosis. The significance is this effect in vivo is unknown and warrants further exploration. Multiple Sclerosis (MS) is a demyelinating disease with a complex and unknown aetiology. The association between MS and the HLA-DRB1*1501 haplotype has been proven to be strong, but the molecular basis of this link remains unclear. Vitamin D receptor (VDR) gene variants have been proposed to modulate this association, although the link between them and the illness remains controversial. Facing these problems, the following objectives were set: 1) test whether HLA II gene expression could be affected by the *1501 haplotype; 2) test the association between VDR variants and MS and check whether those variants modulate the risk conferred by *1501; and 3) study whether VDR variants affect HLA II gene expression.
Peripheral blood from 364 MS patients and 513 healthy controls was obtained and DNA (all samples) and total RNA (170 patients and 140 controls) were extracted from peripheral blood mononuclear cells. HLA-DRB1, DRB5 and DQA1 gene expression measurements and *1501 genotyping were performed by qPCR using specific taqman probes. VDR variants were genotyped by PCR-RFLP. *1501 positive samples showed a significant overexpression of DRB1 (p b 0.0005), DRB5 (p b 0.0005) and DQA1 (p = 0.009) in patients. DRB1 (p = 0.002) and DRB5 (p b 0.0005) were also overexpressed in *1501 controls. Our data confirm that the *1501 haplotype confers a higher risk of suffering from MS (odds ratio = 1364; 95%C.I. = 1107-1681). No association was found between VDR variants and MS, but ApaI variants were shown to modulate the risk conferred by *1501. Anyway, no HLA II gene expression differences were found among VDR variants.
Our results suggest that HLA II gene expression differences could be part of the molecular mechanism upon which the MS-*1501 association relies. Moreover, ApaI variants in the VDR gene seem to modulate the risk conferred by *1501, although this effect does not seem to happen via gene expression regulation.
The presence of myeloid suppressor cells is related to lymphocyte apoptosis in Multiple Sclerosis Multiple Sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system and the Relapsing-Remitting (MS-RR) is the most frequent variant, characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, in which the patients partially recover. However, the immunosuppressor cell types involved in this process are not well characterized.
Myeloid Derived Suppressor Cells (MDSCs) form a heterogenic population of immature cells with the capacity of suppressing the inflammatory response. This cells act, among other mechanisms, through Arginase-I (Arg-I) on T-cell activity. Previous studies showed an increment in the Arg-I activity in spleen MDSCs and their immunosupressor role on T cells in cancer, infections and the relapsing phase of EAE. However, the nature of the Arg-I+ cells within the CNS has not been identified.
Our study aims to detect and identify Arg-I+ cells in the spinal cord during EAE time course: onset (10 dpi), peak (15 day postimmunization-dpi-), remitting period (25 dpi) and chronic phase (35 and 63 dpi). Our results indicate the presence and density of Arg-I+ cells in EAE mice is parallel to the different disease periods. In fact, their number is highest at 15 dpi, significantly decrease at 25 dpi and completely disappear from 35 dpi onwards. The Arg-I+ cells are detected in both the demyelinated plaque and in the periplaque, and always show the characteristic MDSC phenotype: Arg-I+/CD11b+/ Gr-1+/CD115+. In addition, apoptotic T cells are exclusively detected at the same stages in which MDSCs are present and frequently appear in contact or in the proximity of an Arg-I+ cell. Indeed, apoptotic CD4+ (15 dpi) and CD8+ (25 dpi) T cells present significant direct and opposite correlations, respectively, with the number of MDSCs observed within and around the demyelinated plaques.
Our data show the important role played by MDSCs in the transition from the relapsing to the remitting phase of MS, by immunosuppression of T cells, which favours the anti-inflammatory response and, subsequently, the relative recovery within the disease. The modulation of the MDSC population could be a therapeutic alternative in MS-RR to favour immunosuppression, and therefore, to accelerate the return to the remitting phase. Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease, thought to be of autoimmune origin. Elevated levels of antibodies are present in the cerebrospinal fluid and brain of patients with MS. However, the target antigen of these antibodies has not yet been fully characterised. Post-translational modification of myelin basic protein (MBP) and other CNS-specific proteins, including citrullination of arginine residues, results in conformational changes in the protein. This leads to increased degradation by proteases and exposure of new epitopes, which may then cause production of autoantibodies targeting the myelin sheath. Citrullination is carried out by a family of enzymes called peptidylarginine deiminases (PADs), with PAD2 and PAD4 reported to be present in the brain. Excess citrullination occurs in MS, with MBP from MS white matter being more highly citrullinated than in control white matter. In this context we investigated PAD2 and PAD4 expression by primary human astrocytes and a human brain endothelial cell line, under inflammatory conditions, and their expression and colocalisation with citrullinated proteins in MS and control tissue. This will provide a better understanding of the process of citrullination in CNS inflammation and its possible role in MS pathogenesis.
Cells were treated in vitro with pro-inflammatory cytokines for 24 h, followed by quantitative real-time PCR to determine PAD2 and PAD4 mRNA expression. PAD2 mRNA was decreased by IL-1-b, TNF, and IFN-g in primary human astrocytes. PAD2 mRNA was increased by IL-1-b in the human hCMEC/D3 brain endothelial cell line. PAD4 mRNA expression was undetectable in both cell types. PAD2 and PAD4 protein expression was assessed by immunocytochemistry and immunohistochemistry (IHC), with preliminary data indicating their presence in primary astrocytes in vitro, and in MS and control tissue. IHC showed increased citrullinated proteins in MS lesions and normal appearing white matter (NAWM) compared to control tissue.
PAD2 mRNA is downregulated in primary human astrocytes and upregulated in a human brain endothelial cell line, under inflammatory conditions. PAD2 and PAD4 proteins were detected in primary human astrocytes, and MS and control tissue. Staining of MS lesions and NAWM showed increased citrullination, compared to control white matter. These data indicate that PAD2 and PAD4 may have a role in the pathogenesis of MS through increased citrullination of CNS proteins. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) mainly affecting young adults. Uptake of apoptotic cells by dendritic cells (DC) has been involved in tolerogenesis but also immune activation in different autoimmune disease. Treatment of cells with the chemical crosslinker ECDI has been shown to promote tolerance in several animal models of autoimmune disease. Here we analyzed whether uptake of apoptotic autologous blood mononuclear cells (PBMC) affect the maturation and activation of human monocyte-derived DC and describe their immunostimulatory capacity and compared different methods to induce apoptosis. DC were generated from untouched CD14 positive cells in the presence of IL-4 and GM-CSF. On day 6, cells were stimulated with either ECDI-treated, UV-, gamma-irradiated or without autologous PBMC. The next day, cells were further stimulated with LPS or a defined maturation cocktail, consisting of various pro-inflammatory cytokines. Cell activation and differentiation was analyzed by the expression of cell surface markers (e.g. CCR7, CD1a, CD83, CD86, HLA-DR and PDL-1), and the concentrations of various pro-and antiinflammatory cytokines and chemokines (e.g. TGF-beta1) in the corresponding cell culture supernatants. Furthermore, the immunostimulatory capacity of apoptotic-cell loaded DC was assessed in allogeneic mixed leukocyte reactions (MLR).
We found that the different strategies to induce apoptosis had profound influence on the maturation and activation of DC. In more detail, DC stimulated with ECDI-treated PBMC showed an impaired maturation revealed by expression of surface markers and a reduced capacity to promote allogeneic T cell proliferation.
Our results indicate an immunomodulatory effect of ECDI-treated PBMC on DC like altered maturation and a reduced immunostimulatory capacity in allogeneic MLR. These results provide further evidence for the importance of DC in antigen specific tolerization with ECDI-treated antigen-coupled cells, a novel therapeutic approach in MS. Human cytomegalovirus (HCMV) is the most common viral infection transmitted from mother to unborn child and is a significant infectious cause of developmental CNS disorders in industrialized nations. Neurological deficits resulting from CMV infection are well documented; yet, the mechanisms responsible remain unknown. We have developed a newborn mouse model of CMV infection that mimics many of the characteristics of human infection, including cerebellar deficits. CMV infection of newborn mice results in a focal encephalitis; however, CNS damage is global and symmetric. Therefore, we hypothesize that CNS deficits following CMV infection result from host inflammatory responses to viral replication. Furthermore, we hypothesize that damage to the developing CNS can be limited by treating infected mice with the anti-inflammatory, Dexamethasone.
MCMV infection of newborn mice results in altered cerebellar development.
MCMV infection was analyzed by in situ hybridization as well as staining for IE-1. Histological staining was used to analyze cerebellar morphology, area and volume.
Treatment with Dexamethasone attenuates MCMV induced CNS inflammation.
Groups were treated with 2 doses of Dexamethasone on PND 5 and 6 or 3 doses from PND 4 to 6. Expression of IFN stimulated genes was analyzed by quantitative real-time PCR (Q-PCR). Viral titers were determined by reverse-transcription PCR.
Dexamethasone treatment decreases activation of CNS macrophages in MCMV infected mice.
Activation of microglia was analyzed by immunofluorescence (IF) for Iba-1. Mononuclear cells were isolated from brain and stained with F4/80 and CD45, then analyzed by flow cytometry. MHCII expression was analyzed by IF and flow cytometry.
Dexamethasone normalizes altered developmental and proliferative markers in MCMV infected mice.
Q-PCR was used to assay differences in developmental genes. Phospho-histone 3 was analyzed by IF. Cyclin B1 activation was analyzed by Western Blot analysis.
Treatment of MCMV infected newborn mice with Dexamethasone attenuates inflammation and normalizes the expression of developmentally regulated genes within the CNS. These findings support our hypothesis that cerebellar deficits in MCMV infected mice are secondary to host inflammatory responses to viral infection. These results suggest that modulating inflammation following MCMV infection can limit cerebellar damage; however a better understanding of the inflammatory mechanisms leading to these abnormalities is necessary.
Dendritic cells control tolerance in EAE via the induction of Tregs by the PD-1 signalling pathway Dendritic cells (DCs) are considered as the most efficient antigenpresenting cells in the initiation of immune responses. Under naïve conditions in the absence of danger signals such as toll like receptor (TLR) ligands, DCs are in a steady state and are shown to be important for tolerance and maintenance of the natural T regulatory cell (nTreg) pool. During experimental autoimmune encephalomyelitis (EAE), DCs are thought to play first a role in the priming phase and later during the central nervous system (CNS) infiltration phase. Several DC-depleting models have previously been developed. We wanted to analyze which role do DCs play in the different phases of EAE progression. Our major goals were to find whether EAE induction is possible in the absence of DCs and which is the primary role of DCs in EAE, immunity or tolerance.
We used three distinct genetic means to deplete DCs from mice. First we used the CD11c-DTR model, second, we used a new model, CD11cCre/iDTR mice. In both models depletion of DCs is induced by injections of diphtheria toxin (DT). As third model we used CD11c-Cre/DTA mice. In these mice the DT is expressed in DCs from birth onwards leading to mice lacking DCs constitutively. As further model we used mice in which the MOG35-55 peptide can be inducible expressed and presented by DCs (CD11cCreER/IiMOG).
To our surpise EAE could be induced in all DC-depleting model systems. Even more, EAE development was in all systems enhanced compared to their littermate controls. To test whether the tolerogenic role of DCs was more prominent than the priming role we used CD11cCreER/IiMOG mice and found that EAE was nearly fully prevented when MOG35-55 expression was induced before but also after the induction of EAE. These results hint to a critical role for DCs in the induction of Tregs. We found that DCs induced upregulation of PD-1 on antigen specific T cells and iTreg differentiation. We further showed that DCs lead to iTreg differentiation in a PD-1 dependent manner and that recombinant PD-ligands can complement for the lack of DCs in iTreg differentiation.
First, our data show that during active immunization, either extreme low numbers of DCs or cells other than DCs are able to prime encephalitogenic T cells and that DCs also fulfill a regulatory role during the priming phase probably via the induction of regulatory T cells. Hereby the PD-1 pathway seems to play a predominant role. Interferon beta (IFN-beta) is an anti-inflammatory drug of the first choice in treatment of relapse-remitted multiple sclerosis (MS). Though the mechanism of action of IFN-beta in MS is unclear it is supposed to have antiviral effect, inhibit the immune cells trafficking across blood-brain barrier, regulate T-cells activation, etc. Only half of MS patients show a significant positive response to IFN treatment, so the discovering of predictive biomarkers of the response to IFN-beta treatment is extremely important. The main objective of the research is to perform a mathematical model of the IFN-beta signaling pathway and to predict the behavior of the key molecules of the pathway upon the IFN-beta treatment.
The systems biology methodology is used to perform a kinetic model of IFN-beta signaling pathway using ordinary differential equations (ODE). The pathway includes main molecules of JAK-STAT signaling pathway: IFN-beta, isomers of STAT1 and STAT2 proteins and phosphorylated STAT1 and STAT2, and the inhibitor SOCS1 at mRNA and protein level (measured by PCR, ELISA, Western blot and Flow Cytometry).
In vitro experiments in the Raw mouse macrophage cell line stimulated with LPS and IFN-beta for different times identified the oscillations in concentrations of IFN-beta, phospho-Stat1 proteins and SOCS1 mRNA involved in the IFN-beta signaling pathway. The mathematical model was able to reproduce the oscillatory behavior identifying the negative feedback provided by SOCS1 on the critical element.
Type I IFN pathway is tightly controlled by the negative feedback provided by SOCS1. Kinetic changes in the IFN pathway might indicate response to IFN-beta therapy in MS. Alzheimer's Disease (AD) is a severe neurodegenerative disorder characterized by progressive loss of memory and cognitive functions. According to the "amyloid hypothesis", formation and accumulation of amyloid-β peptide (Aβ is the initiating cause of pathogenic lesions. Vaccines targeting Aβ represent promising therapeutic options, and active immunization against Aβ provided encouraging results in experimental mouse models and, to a lesser extent, in a subsequent clinical trial (AN1792). However, the AN1792 trial had to be interrupted due to the occurrence of meningoencephalitis in 6% of the treated patients. While these severe side effects were attributed to pro-inflammatory T cell responses, preclinical murine models did not show evidence of T cellrelated side effects. In addition, several reports suggest that Aβ-specific CD4+ T cells may be implicated in the natural course of AD and could have a strong therapeutic potential as well. Hence, better understanding adaptive T cell responses to Aβ is crucial for the development of innovative vaccine strategies in AD. As a prerequisite for deciphering the immunobiology of Aβ-specific CD4+ T cell responses in the course of AD, we characterized the parameters controlling CD4+ T cell responses to Aβ in normal mice, independently of the pathological setting. Our data indicate that CD4+ T cell responsiveness to Aβ is highly heterogeneous between mouse strains of different H-2 haplotypes, with SJL (H-2 s) mice displaying a strong response to Aβ10-24 and C57BL/6 (H-2b) mice showing only a weak responsiveness to Aβ16-30, as previously reported. Surprisingly, C57BL/6 mice congenic for the H-2 s haplotype (B6.H-2 S), which display a "permissive" MHC-II allele for the presentation of the immunodominant Aβ10-24 epitope, show a very weak CD4+ T cell response to Aβ similar to C57BL/6 mice, suggesting that MHCindependent factors down-modulate Aβ-specific CD4+ T cell responses in C57BL/6 background. Transient depletion of regulatory T cells (Treg) indicated that CD4+ T cell responses to Aβ are significantly enhanced in both C57BL/6 and B6.H-2S mice upon Treg depletion, while Treg-depleted SJL mice display unaltered Aβ-specific T cell responses. Altogether these results suggest that the magnitude/strength of CD4+ T cell responses to Aβ is controlled by both MHC-dependent and -independent factors in mice, the latter including the overall ability to develop Aβ-specific Treg cell responses.
Kondo Takayuki ⁎ ,1 , Matsuya Nemu 2 , Komori Mika 3 , Nomura Kyouichi 4 , Matsuo Hidenori 2 1 Kitano Hospital, the Tazuke Kofukai Foundation, Medical Research Institute, Osaka, Japan; 2 National Hospital organization Nagasaki Kawatana Medical Center, Nagasaki, Japan; 3 Kyoto University; kyoto; Japan; 4 Saitama Medical Center, Kawagoe, Japan Neuromyelitis optica (NMO) is an inflammatory disease of the central nerve system (CNS) with autoimmune pathogenesis. It was evidenced that anti-aquaporine (AQP) 4 plays a crucial role in the pathogenesis of NMO. In contrast, roles of T cells in NMO have remains to be established yet. In this study we succeeded in revealing participation of T cells in NMO pathogenesis.
11 NMO patients seropositive for anti-AQP-4 antibody10 relapsing-remitting MS patients and 10 healthy subjects (HS) were enrolled. Three with NMO were monitored for T cell activation in vivo between relapses and remissions. With flow cytometry using antibodies, CD3-fluorescein isothiocyanate (FITC), CD69-phycoerythrin (PE), and CD4-phycoerythrin-cyanin (PE-Cy5). Frequencies of CD69+ activated T cells were shown to increase at relapses.
Next, we analyzed antigen-specific T cell response. For this purpose 32 overlapping peptides of AQP-4 and 6 myelin peptides, MOG13-28, MOG145-160, PLP95-116, PLP139-154, PLP185-209 and MBP83-99 were prepared. AQP4(41-60), AQP4(121-140), AQP4 (161-180), and AQP4(191-210) were insoluble and excluded for the study. The remaining peptides were used as stimulating antigens at final concentration of 10 μg/ml. Mixture of AQP4 peptides (AQP4-M) was adjusted at final concentration of 2 μg/ml. 5 × 105 PBMC freshly isolated were seeded and cultured in a well of 96-well atbottom culture plates. Antigen was added to wells after 2 days culture for resting, and the samples were incubated with antigens for 4 hours thereafter. Then the CD69+ CD4 T cells were analyzed by flow cytometry. In this assay we determined three times increase of CD69 + cells as positive. While eight of 10 NMO showed positive to AQP4-M, only two of MS and none of HS responded significantly. Nine of 10 NMO responded to AQP4(11-30) and AQP4(91-110). While AQP4 (91-110) induced positive response in 8 out of 11 MS patients and 5 of 9 HS, only two of 10 MS and 2 of 9 HS reacted to AQP4(11-30). The observation indicates that AQP4(11-30) is NMO-specific T cell epitope. Unexpectedly, seven of 10 with NMO showed response to PLP(95-116). On the other hand, only one of 10 MS and one of 10 HS showed positive.
We demonstrated that T cell immunity against AQP4 and PLP was increased in NMO. Taken together with demonstrated evidence of anti-AQP-4 antibody, we speculated that both of T cells and B cells are essential in development of NMO. Autoaggressive T cells and auto-antibodies are thought to attack the central nervous system in the disease Multiple Sclerosis (MS). This autoimmune condition is characterized by inflammation and demyelination around the axons of the brain and spinal cord. Despite intensive work, the immunopathogenesis of MS is still unknown. In particular, the molecular targets recognized by these autoaggressive T cells are still unidentified.
Our group has recently succeeded in 'reviving' autoaggressive T cells from frozen biopsy samples of MS patients. First, we investigate the T cell repertoire in autoimmune tissue lesions and identify clonal expansions by CDR3 spectratyping. Second, we identify these autoaggressive T cells from human biopsy tissue by T cell receptor (TCR) specific antibodies. Third, we characterize the antigen specific TCR alpha-and beta-chains by a multiplex PCR. Fourth, we functionally express these TCR molecules in mouse hybridoma cells, and lastly, investigate their antigen(s).
So far we have identified four T cell Receptors from the biopsy tissue of one MS patient. Sequence analysis of the primary structure of these TCRs points to a converging antigen recognition motif shared amongst them. We have reconstructed and functionally expressed these autoaggressive TCRs and are currently investigating their antigen-recognition properties. Using autologous EBV-transformed B cells and mononuclear cells HLAmatched donors as presenting cells, we found that they do not recognize peptides from candidate antigens such as MBP, MOG, or EBV. Currently we are screening cDNA libraries from several sources. Age and sex matched 11 healthy subjects, 22 MS patients (RR type) and 6 non-inflammatory neurological diseases (NIND) patients were enrolled in this study. Simultaneous staining of 4 chemokine receptors (CCR2, CCR4, CCR5, and CCR6) on memory CD4+ T cells of PB and CSF were performed and frequency of each T cell subset (CCR4+CCR6+, CCR2+CCR5−, etc.) were measured by flow cytometry. In the PB, no particular memory T cell subset was increased from MS as compared with HS or NIND irrespective of the disease state. Next, we compared the frequency of each T cell subset between CSF and PB from each relapsing MS patient and from each NIND patient. Interestingly, in both groups of patients, the frequencies of CCR2+CCR5− and CCR4+ CCR6+ cells were lower in CSF than in PB. Notably, only in MS but not in NIND patients, the frequency of CCR2+CCR5+ cells was higher in CSF than in PB, suggesting that it is preferentially recruited to the inflamed CNS. By polyclonal activation, it produced both IL-17 and IFN-g and the frequency of both IFN-g and IL-17-producing cells as measured by intracellular cytokine staining was also higher in this subset than CCR2−CCR5+ Th1 subset. Matrix metalloproteinase 9 (MMP-9) can degrade collagens of extracellular matrix and is assumed to play a role in the breakdown of Blood Brain Barrier. We found that the expression level of MMP-9 as well as its activity as measured by zymography were significantly higher in this subset than that in any other T cell subset. Moreover, the expression level of osteopontin, whose transcript is abundantly expressed in active MS lesions, was also significantly higher in this subset.
We have identified a unique T cell subset enriched in CSF in the relapse of MS. This subset, CCR2+CCR5+ cells, not only produce both IFN-g and IL-17 but also equipped with tissue invasive machinery. Targeting this subset can be a novel therapeutic approach where immune surveillance of uninflamed brain is not interfered. The importance of different T cell subsets in the pathogenesis of Multiple Sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) is controversial. The induction of chronic autoimmune CNS inflammation in EAE has been shown to depend on IL-6 and IL-23, which are both involved in the generation of CD4+ Th17 cells, whereas the role of CD8+ T cells in EAE is still unclear. Here, we investigated the requirements for the generation of highly encephalitogenic myelin oligodendrocyte glycoprotein (MOG)-specific CD4+ Th17 cells for EAE induction and the effect of the presence of CD8+ T cells on the EAE course.
We reconstituted Rag1 −/− mice with CD4+ and/or CD8+ T cells and induced active EAE with MOG. Restimulation protocols for the induction of passive EAE with in vitro-generated MOG-specific CD4+ Th17 cells derived from T cell receptor transgenic (2d2) mice were established. We correlated the disease course with flow cytometric profiles of ex vivo isolated T cells. Actively induced EAE in Rag1 −/− mice, which had been reconstituted with CD4+ with or without CD8+ T cells, was comparably severe in the presence and absence of CD8+ T cells. Reconstitution with CD8+ T cells alone however induced only lowlevel clinical disease with infiltrates mainly consisting of IFN-g producing CD8+ T cells. In contrast, severely disabled animals of CD4+ T cell reconstituted mice showed brain infiltrates of IL-17producing CD4+ T cells, highlighting the correlation of the presence of Th17 cells with the clinical severity. In order to induce a stable and highly encephalitogenic Th17 population in vitro, repetitive stimulation of 2d2 CD4+ T cells was essential. When transferred adoptively into Rag1 −/− mice, even low cell numbers induced fulminant and severely progressive EAE. Disease induction correlated with enrichment of transferred 2d2 Th17 cells in the CNS of affected animals.
Taken together, we found that CD8+ T effector cells have only a minor pathogenic potential in MOG-induced EAE even in lymphopenic hosts, which is shown by low-level clinical disease even in the presence of profuse CD8+ T cell infiltrates in the CNS. In contrast, already very low numbers of CD4+ Th17 cells were sufficient to induce severe, often atypic and non-remitting neurologic deficit which led to rapid death by CNS damage. These findings underline the relevance of CD4+ T cell differentiation for the quantity and quality of neurologic deficit in autoimmune neuroinflammation.
Gonzalez Hugo ⁎ ,2 , Prado Carolina 1 , Barrientos Magaly 2 , Pacheco Rodrigo 1 1 Fundacion Ciencia para la Vida, Santiago, Chile; 2 Fundacion Ciencia para la Vida, Santiago, Chile
Despite the role of dopamine (DA) in the central nervous system has been well characterized, recent studies have revealed that some immune cells, such as T-cells, express dopamine receptors (DARs). However, little is known about the impact of regulation mediated by DA on the function of T-cells. In this study, we aimed to further characterize immuno-biological effects of DARs stimulation on murine T-cells.
Accordingly, we first analyzed DARs expression by flow cytometry and RT-PCR. Results show that D1, D2, D3 and D5 were expressed on unstimulated CD4+ and CD8+ T-cells. Importantly, some DARs were differentially expressed upon T-cell activation. Specifically D2 and D3 receptors were up-regulated and D5 receptor was down-modulated after CD8+ T-cell activation, whereas expression of D2 receptor was up-regulated upon activation of CD4+ T-cells. As a functional analysis of T-cell response we evaluated the effect of DA in T-cell activation as secretion of IL-2 after anti-CD3 + anti-CD28 stimulation. Our results show that DA inhibits CD8+ and CD4+ T-cell activation in a dosedependent way. Moreover, the signaling pathway analysis shows that stimulation of dopamine D1-like receptors by the agonist SKF-38393 elicited an increase on intracellular calcium levels of CD4+ T-cells, as measured by fluo-3AM. On the other hand, stimulation of D3 receptor by the selective agonist PD128907 promoted a decreased production of cAMP in forskolin-treated CD4+ T-cells.
In conclusion, our data shows that T-cells express functional dopamine receptors in an activation-dependent manner. Whereas D1-like stimulation elicits intracellular calcium mobilization, D3 couples with inhibition of adenilate cyclase. Moreover, DARs stimulation regulates activation of CD4+ T-cells. In summary, our results suggest that DA may exert a complex regulation of T-cell physiology. Understanding of the role of stimulation of each DAR on Tcell could be helpful to the design of therapies for the treatment of autoimmunity, immunodeficiency and cancer. PBMC. Also, several studies suggest the involvement of the CD8 compartment in MS. On this line we hypothesized that the possible link between MS and EBV could be ascertained from the study of the frequency of EBV-specific CD8+ T cells in MS patients in different stages and time points of the disease compared to healthy donors.
To this aim, we stained PBMC with MHC pentamers coupled with known antigens of the latent (EBNA-3A and LMP-2) and of the lytic EBV infection (BMFL-1 and BZLF-1) and with multiple surface markers to carefully define EBV-specific cell subsets. Our data show that the frequency of CD8+ T cells specific for antigens expressed during the latent phase of the virus is significantly higher in CIS patients and in MS patients with shorter disease duration compared to other MS patients and to healthy donors, while the frequency of CD8+ T cells specific for antigens expressed during the lytic phase of the virus is significantly higher in patients undergoing clinical relapse compared to patients in the stable phase of the disease and healthy donors. We then performed a detailed characterization of EBV specific CD8 T cells by using polychromatic flow cytometry: multiple surface markers and functional assays were thus carried out in order to monitor the quality of the immune response directed against EBV and CMV. Specifically, the relative proportions of cytotoxic or cytokine-producing T cell subsets, within CD8+ EBV specific T cells for antigens of the lytic phase and of the latent phase of virus infection, were analyzed. We found that the subset of cytotoxic perforin+ T cells was confined only to CD8+ T cells specific for antigens of the latent phase and in CMVspecific CD8+ T cells, whereas CD8+ T cells specific for antigens of the lytic phase contained granzyme B and produced large amounts of cytokines.
These results advance our understanding of the relationship between distinct immune responses at different stages of infection and at different stage of MS disease. Premature immunosenescence has been linked to many autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). In particular, CD4+ T cells gain aberrant, possibly cytotoxic functions after repeated antigenic stimulation or homeostatic proliferation. Until now, the absence of CD28 has been used as a marker for these senescent CD4+ T cells. However, a marker which is present on the surface of these cells can greatly benefit the isolation and further characterization of this subset of T cells. Therefore, a phenotypic characterization of these cells was performed using flow cytometry.
Several markers (CD11a, CD49d, CD54, CD56, and NKG2D) were significantly upregulated (p b 0.05) on CD4+CD28null T cells of either healthy controls (HC; n = 9) and patients (MS: n = 8; RA: n = 23) in comparison to CD4+CD28+ T cells. Interestingly, CX3CR1 (fractalkine receptor) was present on the vast majority of CD4+CD28null T cells (HC: 88.2 ± 3.9%; MS: 82.4 ± 4.7%; RA: 90.4 ± 1.5%) and mostly absent on CD4+CD28+ T cells (HC: 2.6 ± 0.9%; MS: 1.5 ± 0.6%; RA: 2.4 ± 0.5%). We further demonstrated that only CD4+CD28null T cells migrate towards fractalkine in a transwell system, thereby proving that CX3CR1 is functional on these cells. Moreover, immunofluorescence stainings of MS brain lesions demonstrate the presence of CD4+CX3CR1+ T cells in the brain of 6 out of 17 donors tested. Once in the target tissue, these cells might exert their cytotoxic properties. Preliminary results show that CD4+CX3CR1+ T cells degranulate after TCR and NKG2D stimulation, as well as after stimulation with the MS related antigens MBP and MOG.
These results indicate that CD4+CD28null T cells contribute to the pathogenesis of autoimmune diseases such as MS and RA.
In particular, we found that the number of CD25high Treg cells expressing the ectonucleotidase CD39 is significantly reduced in peripheral blood mononuclear cells (PBMC) of MS patients compared to healthy donors. Since CD39 expression is confined to cells endowed with the most powerful suppressive abilities, the reduction in CD39+ T cells in MS patient's points to a deregulation of the balance between pathogenic effectors and protective regulators. We analysed the role of plasmacytoid (pDC) and myeloid (mDC) dendritic cells in the establishment of Tregulatory/Teffector cell balance.
We stimulated purified pDC and mDC from healthy donors or MS patients with TLR agonists and we cocultured them with naive or memory CD4 T cells. We studied the frequency of both effector and regulatory T cells after 6 days of coculture. We found that stimulated pDCs are potent inducers of T cells with regulatory properties, while mDCs induce high levels of T cells producing effector cytokines.
These results indicate that pDCs and mDC have a differential role in maintaining the balance between regulatory and effector T cells in MS. Such knowledge will be important to design new therapeutic approaches aiming at shifting the balance towards an anti-inflammatory immune response in MS patients. Multiple sclerosis (MS) is a central nervous system (CNS)-directed inflammatory disease. Leukocytes found in lesions are CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes and antigenpresenting cells. The role of CD4 cells in MS and in its animal model, experimental autoimmune encephalomyelitis (EAE), has been extensively studied. Nevertheless, recent studies have shown that CD8 cells have the capacity to access the CNS as these cells have been found in parenchyma of active lesions of MS patients and EAE animals. Also, clonally expanded CD8+ T lymphocytes have been found in MS lesions and in the cerebrospinal fluid (CSF) of MS patients. However the phenotype, origin and route of entry of CD8+ T lymphocytes found in MS or EAE lesions have yet to be identified. Our objective is to evaluate the phenotype of migrating CD8+ lymphocytes and the mechanism by which such cells cross the blood-brain barrier (BBB).
Using CSF from 17 relapsing-remitting MS patients, from spinal cord of MOG(35-55)-induced EAE and from coronavirus-induced encephalitis, we demonstrate by flow cytometry and by immunostainings that CD8+ T lymphocytes are mostly of the effector memory (EM) phenotype (CD62Lneg CCR7neg GranzymeBhi). We further show that purified human CD8+ TEM lymphocytes transmigrate more readily across BBB endothelial cells than non-EM CD8+ lymphocytes and that BBB endothelium promotes the selective recruitment of CD8+ EM lymphocytes. Furthermore, we provide evidence for a selective recruitment of IFN-g-and IL-17-secreting CD8+ lymphocytes by human and mouse BBB endothelium, in vitro and in vivo. Finally we show that in vitro and in vivo migration of CD8+ lymphocytes to the CNS is dependent on alpha-4 integrin.
Our study provides evidence for an active role of the BBB in the recruitment of CD8+ TEM lymphocytes to the CNS and defines alpha-4 integrin as a major contributor of CD8+ lymphocyte entry into the brain.
Effect of glucocorticoid and Adrenomedullin on AM1 and AM2 expression by T cells Adrenomedullin (AM) is a novel vasodilatatory peptide which acts primarily through the calcitonin receptor-like receptor (CLR) in combination with either receptor-activity-modifying-protein (RAMP) 2 or 3 (receptors, AM1 and AM2 respectively). AM has been shown to play an important yet unclear role during inflammation: increasing following cytokine treatment and promoting macrophage action in situ (pro-inflammatory), but also by down-regulating TNFalpha and increasing IL-6 expression by macrophages (anti-inflammatory). Indeed, and specifically, we have previously demonstrated AM elevation during the development of neuroinflammatory lesions in an animal model of multiple sclerosis. Furthermore AM, constitutively expressed by T cells, is highly expressed during hypoxic conditions, exerting an important role in T cell adaptation and performance at low oxygen concentrations. Finally, the immunosuppressant glucocorticoids (GC) have been shown to regulate AM, AM1 and AM2 in a variety of cells. RAMP2 and CLR mRNA production have been previously detected in Jurkat leukaemia cells under normoxic conditions, but RAMP3 and all AM receptor components at a protein levels have never been studied. The study presented, examines the expression of component parts of the AM receptors in primary human T cells and the effect of AM and GC on that expression. RAMP2, 3 and CLR expression was examined in primary human T cells (PCR and flow cytometry). All AM receptor components were found to be physiologically expressed in unstimulated T cells, both intracellularly and on the cell surface. PHA-stimulation appeared to decrease all receptor proteins, significantly so for CLR and RAMP3 (p b 0.05). Following AM treatment (10-6 M; 24 h) RAMP3 expression was significantly decreased in unstimulated cells (p b 0.05). On the contrary, GC treatment (10-6 M; 24 h) affected only stimulated cells increasing intracellular RAMP 3 levels (p b 0.05).
Our findings indicate a close relationship between RAMP3 expression and the activation state of the T cell. A potential novel mechanism for 'sensing' the T cell environment through the AM2 receptor will be discussed.
Gendron Steve ⁎ , Prat Alexandre
Multiple sclerosis (MS) is characterized by immune cells infiltration in the central nervous system (CNS), particularly T cells of the Th1 and Th17 lineage. One important event of this infiltration is the transmigration of these cells through the blood-brain barrier (BBB) which is mainly constituted of endothelial cells and extracellular matrix (ECM) proteins. Th cells transmigration is largely attributable to integrins of the beta1 family expressed on the surface of these cells. Integrins alpha4beta1 (VLA-4) is known to be the most important beta1 integrin for this process but its blockade do not completely abrogate MS and EAE symptoms. Data from our lab suggest that effector T cells express the integrin alpha8beta1, an integrin which has never been described in T cell before. The goal of this study is to characterize the expression of integrin alpha8beta1 on CD4 Thelper cells and whether this integrin is involved in the transmigration of lymphocytes through the BBB during experimental autoimmune encephalomyelitis (EAE) and MS.
Western blot and RT-PCR analysis demonstrate that integrin alpha8beta1 is found on human CD4CD45RO Th0, Th1, Th2 cells and is strikingly up-regulated with a higher expression on Th17 lymphocytes. As well, we found that nephronectin (NPNT), the main ligand of this integrin, is produced by primary cultures of human brain endothelial cells (HBECs) and is expressed on vascular structures in human and rodent CNS. Our preliminary data on the specific role of integrin alpha8beta1 in the migration of Thelper lymphocytes across brain vsacular structures is being addressed using a known RGD-based blocking peptide of alpha8beta1-NPNT interaction, both invitro and invivo in EAE.
This work aims to identify a potential new interaction between pathological T cells expressing integrin alpha8beta1 and NPNT expressed by brain endothelial cells. This interaction might be lead to the identification of novel therapeutical targets for the treatment of MS.
Erasmus MC, University Medical Center, Rotterdam, The Netherlands Introduction: One of the first validated single nucleotide polymorphisms associated with an increased risk for multiple sclerosis was the IL-7Ra. However, limited information is available on the expression of this receptor in different lymphocyte subsets and a possible differential signalling capacity in MS compared to healthy controls (HC). Aim of the study: To determine whether frequencies and expression levels of the IL-7Ra on functional T-cell subsets differs between MS patients and healthy controls. Moreover, functional differences in STAT5 signaling upon IL-7 stimulation were tested in MS patients.
Methods: Flowcytometric study on PBMC of 77 MS patients in remission and 53 healthy controls. A. naïve (CD45RA+CD27+), B. memory (CD45RA-CD27+) and C. effector (CD45RA-CD27−) CD4 and CD8 populations were stained for the expression CD127 (IL-7Ra). Moreover, IL-7 stimulation on sorted T-cells was performed to study functional parameters. Results: As expected, the percentages of both CD4 and CD8 memory Tcells were significantly increased in MS patients. Interestingly, a significant increased frequency of CD127+CD8 effector memory (CD8EM) was found in MS (MS 65.4%, HC 48.8%, p = 0.0005). Moreover, CD127 expression levels were significantly increased in all CD8 T-cell subsets except the CD8 memory T-cells and in CD4 memory T-cells of MS patients (all p b 0.05).
MS patients showed a significantly stronger increase of phophorylated STAT5 in CD8EM T-cells compared to healthy controls (p b 0.05) when stimulated with IL-7 independently of CD127 membrane expression. In both MS and HC, IL-7 stimulation of Tcells lead to an increased expression of genes involved in cytotoxicity (granzyme B and perforin-1, all significant). Specifically in MS patients, an upregulation of granzyme A expression was observed (p = 0.001). Discussion: The IL-7Ra/IL-7 axis is more active in MS patients, possibly related for the maintenance of the memory T-cell pool. Recently, it was shown that MS lesions produce IL-7. The lower threshold for IL-7 induced cytotoxicity in MS patients may further enhance pathogenicity of CD8+ T-cells. This appears of special interest in light of the demyelinating and cytotoxic effects by granzyme A. Neuromyelitis optica (NMO) is associated with aquaporin-4 (AQP4)-specific IgG1 antibodies. Human IgG1 is a T cell-dependent antibody subclass, suggesting that AQP4-specific T cells may participate in NMO pathogenesis, a possibility supported by observations that AQP4-specific antibodies alone are not pathogenic in the absence of CNS inflammation. In order to understand the potential role of T cells in NMO pathogenesis and to develop a model of NMO, we have been studying T cell recognition of AQP4 in NMO patients and in mice. The goal of this investigation was to identify AQP4-specific T cell determinants in C57BL/6 and SJL/J mice.
AQP4 was scanned for T cell reactivity using intact AQP4 and overlapping 20mer peptides encompassing the entire sequence of mouse and human AQP4. Mice were immunized with AQP4 peptides or intact AQP4 in complete Freund's adjuvant. Proliferation was measured by 3H-thymidine incorporation. By direct immunization of AQP4 peptides into C57BL/6 mice, T cell determinants were identified within peptides (p) 21-40, p91-110, p166-180, and p261-280. In SJL/ J mice, T cell determinants are situated in p11-30, p21-40, p101-130, and p261-280. These T cell determinants, identified in both strains, are located within putative cytoplasmic domains of AQP4. MHC IIrestricted CD4^+ T cell lines specific for these individual AQP4 determinants proliferate in response to intact AQP4. However, after immunization with intact AQP4, recall to p21-40 was strongest in both strains, indicating that this region contains the naturally processed immunodominant T cell epitopes. AQP4-specific T cells secreted both IFN-g and IL-17. Using truncated AQP4 peptides within 21-40, these T cell determinants were characterized further. AQP4specific T cells from C57BL/6 and SJL/J mice recognize p25-38 and p23-38, respectively. Preliminary data from proliferation and tetramer analysis indicate that T cells from NMO patients also recognize some of these AQP4 determinants.
AQP4 residues 11-30, 21-40, 91-110, 101-120, 166-180, and 261-280 contain immunogenic MHC II-restricted CD4^+ T cell determinants in C57BL/6 and SJL/J mice. p21-40 contains the immunodominant determinant within intact AQP4. Unlike AQP4 recognition by B cells and antibodies, which frequently recognize extracellular domains of proteins, T cell determinants of AQP4 are located within cytoplasmic domains. These data may be useful for investigators developing AQP4-targeted NMO models.
In vivo visualization of antigen presentation in the CNS Kawakami Naoto ⁎ ,1 , Bartholomäus Ingo 1 , Pesic Marija 1 , Odoardi Francesca 2 , Schläger Christian 2 , Flügel Alexander 2 , Wekerle Hartmut 1 1 Max Planck Institute of Neurobiology, Martinsried, Germany; 2 University of Göttingen, Göttingen, Germany
In the Experimental Autoimmune Encephalomyelitis (EAE) as well as multiple sclerosis, T cells seem to have central roles for clinical outcome. During the acute phase of adoptive transfer EAE, autoreactive T cells infiltrate into the CNS massively. There T cells are highly re-activated, produce inflammatory cytokines and recruit other immune cells, e.g. macrophages and B cells. We asked the questions when, where and how T cell infiltration into the CNS starts.
Using intravital two photon microscopy, we visualized genetically GFP labeled CD4 positive MBP specific T cells (TMBP-GFP cells) infiltration procedures during entire EAE period. Before onset, TMBP-GFP cells appeared in leptomeningeal vessels of the spinal cord. TMBP-GFP cells crawled on intra luminal surface before extravasations. Interestingly, T cells did not penetrate into the CNS parenchyma after extravasations, but continued to scan abluminal surface of vessels. We hypothesized antigen presentation happens at this perivascular location since perivascular/meningeal macrophages locate there. We marked perivascular/meningeal macrophages, potential antigen presenting cells (APCs), by intrathecal injection of fluorochrome-dextran conjugates. Alternatively, APCs were marked by bone marrow transplantation that bone marrow cells derived from GFP transgenic rats were grafted into irradiated recipients. We observed both short-and long-lasting contacts between T cells and APCs, that seemed to represent antigen recognition. Gene profiling analysis supported this observation because TMBP-GFP cells were activated in the meninges compared those in the spleen or blood. Interestingly, meningeal APCs are not saturated with endogenous antigens because exogenous MBP hyper-activated TMBP-GFP cells in the CNS and exaggerated EAE. Furthermore, we showed importance of antigen presentation at CNS meninges using OVA specific T cells. Small numbers of TOVA-GFP cell appeared in the CNS meninges but they did not penetrate into the CNS parenchyma. Intrathecal injection of OVA-pulsed meningeal APCs changed situation dramatically. Higher number of TOVA-GFP cells were recruited into the CNS and showed stationary behavior that seemed to represent antigen recognition.
We showed that antigen presentation in the CNS meninges is a critical step to T cell penetration into the CNS. Currently we are studying relationship between duration of contacts and T cell activation. Furthermore we are characterizing meningeal APC in depth.
Schneider Raphael ⁎ , Yaneva Teodora, Beauseigle Diane, Prat Alexandre, Arbour Nathalie
Interleukin-27 (IL-27), a cytokine of the IL-12 family, has been shown to exhibit both pro-and anti-inflammatory properties. Whereas IL-27 favors the development of Th1 responses it also inhibits Th17 responses. Several studies have documented the IL-27 capacity to suppress experimental autoimmune encephalomyelitis (EAE). Murine astrocytes and microglia have been reported to produce IL-27. Moreover, IL-27 promotes proliferation and cytotoxic functions of mouse CD8 T lymphocytes, but whether it has similar effects on human cells has not been elucidated. Finally, whether human CNS cells could locally provide IL-27 has not been explored.
OUR GOAL is to investigate the impact of IL-27 on human CD8 T cell functions and to identify cellular sources of IL-27 in human central nervous system (CNS).
We use peripheral blood mononuclear cells (PBMC) and isolated CD8 T cells from healthy donors to perform phenotypic and functional assays. We first determined that a subset of human CD8 T cells expresses the complete IL-27 receptor and that such expression was increased upon polyclonal activation. IL-27 induced a rapid phosphorylation of signal transducers and activators of transcription (STAT) STAT1 and STAT3 and enhanced STAT1 protein expression in CD8 T cells. Furthermore, upon IL-27 treatment, human CD8 T cells expressed suppressor of cytokine signaling (SOCS) SOCS-1 and SOCS-3 mRNA. Importantly, these effects were attenuated after inhibition of STAT1 signaling. Addition of IL-27 to anti-CD3 activated naïve CD8 T cells significantly increased the transcription factor Tbet expression levels, cell proliferation, and IFN-gamma and granzyme B production by these cells leading to increased CD8 T cellmediated cytotoxicity. IL-27 detection by immunohistochemistry on human brain tissue sections indicated that microglia and brain endothelial cells are potential local sources of IL-27. In addition, IL-27 was detected in supernatants obtained from primary cultures of human brain endothelial cells treated with pro-inflammatory stimuli supporting the notion that IL-27 can be secreted by these cells in an inflamed environment.
Our results demonstrate a pro-inflammatory impact of IL-27 on human CD8 T cells mediated via STAT1 leading to boosted cytotoxic effector cells. Furthermore, human microglia and brain endothelial cells could locally provide IL-27 and hence impact infiltrating human T cells. Our goals were to study the presence of regulatory CD4+ (Treg) (CD4+CD25+FoxP3+,CD4+CD25medium, CD4+CD25high), and regulatory/suppressor CD8+ (Ts) (CD8+CD25+, CD8+CD28−, CD8+ CD25+CD28−, CD8+CD25+FoxP3+ and CD8+CD25+CD127lo) T cell subsets and of effector CD4+ (CD45Ra+CCR7−) and CD8+ (CD45Ra+CCR7−) T cells in the CSF and peripheral blood (PB) of MS patients at first relapse.
Twenty-five patients with first clinical relapse that were later on diagnosed of MS were consecutively studied. CSF and PB samples from MS patients were analyzed in parallel by multiparametric flowcytometry.
The proportions of Ts CD8+CD28−, CD8+CD25+CD28−, CD8+ CD25+CD127− and CD8+CD25+FoxP3+ were significantly higher in CSF than in PB (p= 0.05; p = 0.06; p = 0.004 and NS, respectively). On the contrary, CD8+CD25+ T cells counts were more elevated in the periphery (p= 0.03). Treg frequencies were also higher in CSF than in PB (CD4+CD25high p = 0.01, CD4+CD25+FoxP3+ p = 0.08, CD4+ CD25high+CD28+ p = 0.06) indicating a specific recruitment of these cells towards the CSF at MS relapse. Interestingly, we found that surface CD28 expression was significantly reduced on CD4+ Treg cells in the CSF compared to the periphery (mean fluorescence intensity, MFI CD4+ CD25med: p b 0.0001; MFI CD4+CD25high p b 0.0001). As expected, CD4 and CD8 naïve T cells percentages were lower in the CSF than in PB (pb 0.0001 and p = 0.004, respectively), while memory (p= 0.002), effector-memory (p= 0.004), and effector (p= 0.07) T CD4+ cells were higher in CSF. Effector-memory (p= 0.004) and effector CD8 T cells were higher in CSF, while memory CD8+ T cells were higher in PB (p= 0.04).
We observed a specific intrathecal enrichment of regulatory CD4 and CD8 T cells, together with effector-memory and effector CD4 and CD8 cells within the CSF in our cohort of MS patients at first MS relapse. Interestingly, down-modulation of the co-stimulatory signal CD28 on T cells and induction of forkhead transcription factor 3 (FoxP3) expressions on Treg and Ts suggest an attempt to inhibit the self-antigen effector CD4 and CD8 specific response and thus control the inflammatory activity of disease.
Smith Kristen ⁎ , Guerau-de-Arellano Mireia, Bottoni Arianna, Racke Michael, Lovett-Racke Amy, Whitacre Caroline
Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that affects more than 3 million worldwide. Autoreactive CD4+ T-Helper (TH) cells of a TH1 phenotype have been implicated in MS pathogenesis. MicroRNAs (miRs) are a class of small non-coding RNAs that negatively regulate post-transcriptional gene expression. Dicer-deficient CD4+ T cells, which lack expression of miRs, produce more inflammatory cytokine upon stimulation. However, the miR(s) responsible for modulating T cell programming have not been defined. This study implicates a specific miR in regulating TH1 responses, making this miR a potential biomarker in MS.
Using target prediction algorithms, we found that miR-29 is predicted to target two key components of TH1 differentiation: Tbet and IFN-g. Importantly, T-bet has been identified as a factor necessary for T cells to infiltrate the CNS. Through targeting both Tbet and IFN-g, miR-29 is positioned to control TH1 programming, function, and encephalitogenicity. We have validated miR-29 targeting of T-bet and IFN-g using gain-and loss-of-function experiments in human and murine TH1-polarized cells. Consistent with a role for miR-29 in TH1 development, miR-29 expression increases more than 7-fold following TH1 polarization, and induction correlates with IFN-g production. Additionally, purified naïve T cells treated with IFN-g up-regulate miR-29. These results indicate that mir-29 is induced through IFN-g signaling and acts in a negative feedback loop to control the amplitude of TH1 responses. We therefore hypothesized that MS patients would have altered levels of miR-29. Purified CD4+ T cells from MS patients have lower basal levels of miR-29 relative to healthy controls, suggesting that a fundamental dysregulation of miR-29 in MS patients may contribute to disease pathogenesis.
These data identify an IFNg-miR29 axis that is initiated in TH1 cells to control inflammation. This axis in disrupted in MS patients, which predisposes individuals to the development of a robust TH1 response. In order to return the immune system to a homeostatic state, MS treatments may work in part by normalizing miR-29 expression, a hypothesis that is being actively investigated. Ultimately, the results of these studies could lead to the development of miR-29 as a biomarker in T cell-mediated diseases such as MS.
Peripheral opioid-induced analgesia is associated with adaptive T cell response Boue jerome ⁎ , Blanpied Catherine, Brousset Pierre, Vergnolle Nathalie, Dietrich Gilles
Painful sensation is a hallmark of the inflammatory response induced by pathogens or tissue damage. A large spectrum of molecules released within the inflamed tissue including neuropeptides, prostaglandins or proteases induces pain by stimulating primary afferent neurons in situ. This activity of primary sensitive fibers can be counteracted by peripheral endogenous regulatory mechanisms involving local opioid release by leukocytes infiltrating the inflammatory site.
In this study, we show that inflammatory pain relief which occurs spontaneously a few days after antigen challenge is caused by opioid release by antigen-primed CD4+ T cells at the site of inflammation. Within draining lymph nodes, specific antigen priming up-regulates opioid synthesis in CD4+ T lymphocytes.
Taken together, our data demonstrate that CD4+ T lymphocytes acquire anti-nociceptive effector properties when specifically primed by antigen during adaptive immune response.
Haile Yohannes ⁎ , Turner Diane, Bleackley Chris, Giuliani Fabrizio
Regulatory T cells (Tregs) play an unequivocal role in modulating a diverse array of immunological responses and maintaining peripheral tolerance. Nevertheless, there are contradictory reports regarding to the role of Treg impairment in multiple sclerosis (MS). Activated cytotoxic T cells release a serine protease granzyme B (GrB) and induce neurotoxicity by degrading the myelin and/or axon in neurodegenerative diseases such as MS. However, the separate effect of T cell subsets in terms of GrB expression and subsequent neurotoxicity has not been explored. This study aims to characterize the level of GrB expression and assess the cytotoxic effect of T cell subsets on human neurons, with special emphasis on Tregs.
Human lymphocytes were activated by anti-CD3 antibody before or after negative/positive selection into CD4+, CD8+ or CD4+CD25+ T cell subsets using magnetic separation. Foxp3, IL-17 and GrB expressions were measured by real-time PCR and flow cytometry. Human cortical fetal neurons were cultured alone or co-cultured with unactivated/activated T cell subsets. Neuronal viability was assessed by immunocytochemistry for microtubule associated protein-2. IFNgamma was measured by cytokine-ELISA. Suppression assay was performed by quantification of 3H-thymidine incorporated T cells.
RT-PCR revealed that activated CD4+CD25+Foxp3+ T cells express 100 fold more GrB compared to unactivated cells and confirmed by FACS that Foxp3+ T cells induced significant amount of GrB and IFN-gamma but not IL-17. Tregs induced severe neurotoxicity (89.5%) which was comparable to that of CD8+ T cells (94.7%). Separate activation of CD8+ T cells significantly enhanced GrB expression (12.4 fold compared to CD4+). CD4+CD25+Foxp3+ T cells failed to suppress the proliferation of effector cells.
The current paradigm considers Tregs beneficial for autoimmune responses and potent suppressive of immune-mediated tissue damage. On the contrary, our findings show that CD4+CD25+ Foxp3+ T cells are neurotoxic after activation. Loss of suppressive function and IFN-gamma expression indicate a shift towards a TH-1 phenotype. GrB induces target cell apoptosis, thus, the neuronal death might be mediated by the presence of high levels of GrB expressed by CD4+CD25+Foxp3+ T cells. These data provide important insights about the limitations of Treg cells in therapeutic applications and the requirement of serious caution in the use of Tregs for disease intervention strategies. While Tcells and macrophages play a crucial role in the primary immune attack, inflammation has also been associated with remyelination in acute MS plaques and in animal models. Since inflammation occurs concurrent with remyelination it is, however, difficult to isolate the beneficial effects of inflammation on remyelination. We have previously shown that adoptively transferred T-cells infiltrate zones of axonal lesion but devoid of autoimmune demyelination. Here, we investigate to which extent infiltrating T cells or factors released from these cells or from activated microglia/macrophages affect oligodendrocyte precursor cell (OPC) proliferation and oligodendrocyte formation. Methods: Groups of mice were subjected to adoptive transfer of 1) proteolipid protein-specific T-cells (TPLP) and axonal lesion, 2) TPLP, 3) ovalbumin-specific T-cells (TOVA) and axonal lesion, 4) TOVA or 4) axonal lesion alone. Mice were injected with the proliferation marker BrdU and the tissues processed for cell counting of CD3+ T cells, NG2+ OPC and CNPase + oligodendrocytes along with double stainings for BrdU. The sprouting of calretinergic axons was examined through areal estimations, and the expression of cytokines was evaluated by quantitative PCR and in situ hybridization techniques. Results: We found that TPLP mice with axonal lesion showed a significant increase in the proliferation of NG2+ OPC 2 days post lesion and in the formation of new CNPase + oligodendrocytes 7 days post lesion compared to lesioned TOVA or naïve mice. The presence of TPLP cells also enhanced the clearance of myelin debris, increased the sprouting response of intact calretinergic fibres within the dentate gyrus, and markedly raised expression of the key inflammatory cytokines IFN and TNFa along with its p75 receptor. Conclusion: These data show that the presence of myelin-reactive T cells may have a stimulatory effect on OPC proliferation and differentiation by affecting several parameters that alone or in combination can facilitate or induce myelination. These results suggest, that in spite of the pathological role of myelin-reactive Tcell in MS the cells may also be associated with beneficial effects on regenerative processes in the brain. Mucin P-selectin glycoprotein ligand-1 (PSGL-1) expressed by all leukocytes is the main ligand for endothelial selectins, and is involved in leukocyte trafficking under physiological and pathological conditions. Whereas PSGL-1 role is emerging in T cell homeostasis, controversial results were obtained in animal models of autoimmune diseases. Our goal was to clarify the role of the mucin PSGL-1 in T cell responses during experimental autoimmune encephalomyelitis (EAE). EAE induced by active immunization was more severe in PSGL-1 −/− mice, whereas Myelin Oligodendrocyte Glycoprotein (MOG)35-55specific T cells obtained from PSGL-1 −/− mice transferred a more severe disease than WT cells, suggesting a role for PSGL-1 in regulatory mechanisms during EAE. As CD4+ T cells isolated form WT and PSGL-1 −/− mice displayed similar proliferative response and pro-inflammatory cytokine production, we studied the role of PSGL-1 in the suppressor activity exerted by CD4+CD25+FOXP3+ regulatory T (Treg) cells in EAE, by using two-photon laser microscopy (TPLM). To this aim we visualized the behavior of encephalitogenic CD4+ T cells in the presence or absence of Tregs in intact draining lymph nodes during early (day 1 post-immunization) and late (day 7 post-immunization) preclinical phase of EAE. TPLM experiments showed that MOG35-55-primed T cells moved slower, had a lower motility coefficient and an enhanced arrest coefficient, displaying a less random movement, when compared to naïve T cells. MOG35-55 but not ovalbumin-primed T cells showed a significant increase in their speed and a decrease of the arrest coefficient in the presence of WT Tregs in draining lymph nodes of immunized mice. Interestingly, PSGL-1 −/− Tregs failed to modulate the motility behavior and proliferation of MOG35-55 CD4+ T cells during late but not early preclinical phase of EAE. In addition, naïve Tregs from PSGL-1 −/− mice presented reduced migration capacity to CNS in the pre-clinical phase of EAE, when compared to WT Tregs, and display a dramatic decrease of adhesive interactions in inflamed brain microcirculation. Finally, PSGL-1 deficient Tregs had a reduced capacity to suppress EAE when compared to WT cells.
Our data suggest that PSGL-1 has a key role in CD4+CD25+FOXP3+ Tregs functions in EAE by mediating cell-cell contacts required for efficient suppression in vivo and by controlling Treg migration in inflamed brain.
Organ-specific autoimmune diseases are triggered often by tissuespecific auto-reactive T lymphocytes. Experimental autoimmune encephalomyelitis (EAE) serves as animal model for Multiple Sclerosis. EAE can be induced by passive transfer of CNS antigen specific T cells which have been stimulated in vitro. However, susceptibility and clinical outcome of EAE are strongly strain and antigen dependent. In Lewis rats, myelin basic protein (MBP) specific T cells (T-MBP cells) triggers a severe neurological disease while T cells specific for myelin oligodendrocyte glycoprotein (MOG) (T-MOG cells) induce only very mild disease despite T cell infiltration in the CNS. Interestingly, in the context of a different genotype, in the Dark Agouti (DA) rats, T-MOG cells induce a severe MBP-like EAE.
Using retrovirally GFP labeled CNS antigen specific T cells and intravital two photon microscopy, we compared the behavior of encephalitogenic T cells in relation to their pathogenicity. We observed that low, as well as highly encephalitogenic T cells infiltrated leptomeningeal area of the spinal cord through the cascade of rolling, crawling and extravasation through the blood vessel. However, while highly encephalitogenic T-MBP cells have crawling phenotype as dominant, in low encephalitogenic T-MOG cells, rolling and crawling were equally observed. Nevertheless, when these T-MOG cells attached to the intraluminal surface of the blood vessel they crawled as long as highly encephalitogenic T-MBP cells. Once extravasated, low encephalitogenic T-MOG cells, made relatively short contact with local APCs comparing to the highly encephalitogenic T-MBP cells. TMOG cells were not activated in the CNS and did not penetrate deep into the CNS parenchyma. Intrathecal injection of APCs that were in vitro pulsed with MOG protein, changed situation dramatically. Those APCs recruited T-MOG cells within the CNS parenchyma and induced significantly severer clinical symptoms.
Our results suggest that low encephalitogenic T-MOG cells are not able to get activation signal from the local APCs and consequently fail to further infiltrate into the CNS parenchyma. We are currently exploring the highly encephalitogenic T-MOG cell behavior in the DA rats. It leads us better understanding for the structures involved in T cell homing and antigen presentation, that potentially qualify as new and selective therapeutic targets in brain autoimmune disease.
The paradoxical role of TH1 effector CD4 T cells during autoimmune chronic inflammation Harrington Laurie ⁎
Many autoimmune chronic inflammatory diseases, including multiple sclerosis, have been previously reported as Th1-mediated disorders; however recent data suggests that Th17 cells are responsible for disease. Paradoxically, the principle Th1 cytokine does not appear necessary for disease, but the key Th1-associated transcription factor Tbet is required. This conundrum propelled us to investigate the regulation of this transcription factor and effector CD4 T cells functions, as well as the role of Th1 cells during autoimmunity.
To address these questions, we have employed the well described mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Following the onset of EAE, we observed a preferential upregulation of Tbet by effector CD4 T cells within the CNS, but not the secondary lymphoid organs. These Tbet+CD4 T cells were capable of producing IFNgamma, and a fraction were able to make both IFNgamma and IL-17. In contrast, the CD4 T cells producing only IL-17 were primarily low for Tbet expression, in keeping with the nonessential role for Tbet during Th17 differentiation. We next examined the requirement of the Th1 promoting cytokines IFNgamma and IL-12 for the induction of Tbet+CD4 T cells during EAE. In the absence of these cytokines, a significant population of CD4 T cells still upregulated Tbet. Consistent with these data, we found that CD4 T cells from IFNgamma reporter/IFNgamma deficient mice with EAE expressed the Thy1.1 reporter molecule, indicating the presence of Th1-like, Tbet+CD4 T cells even in the absence of the cardinal Th1 cytokine, IFNgamma.
In conclusion, these data demonstrate that Tbet+CD4 T cells are highly associated with the onset of EAE. Moreover, these data indicate that Tbet+ effector CD4 T cells arise under non-typical Th1 polarizing conditions, suggesting that alternative mechanisms regulate Tbet expression during chronic inflammation.
The role of glycolipids in the pathogenesis of Multiple Sclerosis Biggs Nancy J., Pon Robert A. ⁎
The search for etiological antigens in multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS), has focused on host-derived protein autoantigens and related microorganism-derived proteins and/or genetic motifs-all with little success. Carbohydrates, in the form of glycolipids, represent an additional class of antigen richly expressed within the CNS and poorly studied as to their role in MS pathogenesis.
In this study, we describe methods to assess this relationship using materials derived from human peripheral blood (PB) and cerebrospinal fluid (CSF). This is accomplished by expanding and characterizing T cells sensitized to glycolipids in the presence of autologous CD1 expressing dendritic cells (DCs) along with the examination of antibody binding profiles against a panel of defined glycolipid antigens.
As the major presenting element for glycolipid antigens, we found that CD1 isoform expression levels on monocyte-derived DCs were sensitive to medium components. CD1a, b, and c expressions were poor in medium containing human as opposed to bovine serum while CD1d expression was relatively unaffected. Since CD1 expression has a direct bearing on our strategic expansion of patient derived glycolipid reactive T cells and more significantly, for the presentation of brain derived glycolipids within the CNS, this matter was further investigated. Interestingly, human plasma as opposed to serum did not negatively affect CD1 expression nor did cultures containing CSF (up to 40%), with enhanced levels of CD1 often observed under these conditions. Primary expansion protocols were established using mixed pools of bovine glycolipid leading to the establishment and immortalization of several MS patient T cell lines. Characterization of these T cell lines revealed glycolipid reactivities through cytokine release and [3H]-thymidine uptake assays. Ongoing screening of glycolipid antibody present within patient CSF and PB detected relatively strong levels of anti-GM2, GD1b, and GQ1b antibodies which varied between compartment assayed and patient samples.
In summary, methods have now been established for a more comprehensive examination of the role brain glycolipids play in the immunopathogenesis of MS. Further studies will address the frequency of glycolipid reactive T cells in blood and CSF of MS and other neurological disease controls with possible correlations to antiglycolipid antibody levels. The two pore domain potassium channel K2P5.1 (TASK2) is constitutively expressed on human T cells. Its pathophysiological relevance during neuroinflammation is shown by an up-regulation of K2P5.1 in CD4+ and especially CD8+ T cells in relapsing-remitting multiple sclerosis patients. A better understanding of K2P5.1 channel function could provide the basis for a selective targeting of pathophysiologically relevant T cells.
K2P5.1 is assumed to contribute to the compensating K+ efflux upon T cell stimulation necessary for enduring T cell activation. We here demonstrate that overexpression of K2P5.1 leads to 2.5 fold increased T cell proliferation and cytokine production. By contrast, pharmacological inhibition of K2P5.1 results in about 50% decrease of proliferation and cytokine production. Proliferation is a basic cell function and relies on a progression of the cell cycle, which is tightly regulated by several promoters and inhibitors of cell cycle. Inhibition of K2P5.1 is followed by a G0/G1 arrest of T cells. It could be shown that Cycline D3 (a cell cycle promoter) expression is inhibited about 65%, whereas p27kip (an inhibitor of cell cycle G1/S transition) is upregulated about 68% after blockade of K2P5.1 channel function. To understand the role of K2P5.1 for T cell functions it is also important to point out the regulation of this channel. The blockade of different intracellular signaling pathways all result in a comparable inhibition of about 50% K2P5.1 up-regulation on mRNA level upon T cell stimulation. K2P5.1 seems also to be regulated on protein level as western blotting data shows a pool of intracellular located K2P5.1 protein as well as a plasma-membrane localized K2P5.1 protein fraction.
Our results point to an essential function of K2P5.1 as a key integrator of extracellular signals on a molecular level. A dynamic interplay between the K2P5.1 pools in plasma membrane and intracellular opens up the possibility of a rapid expression of K2P5.1 in the plasmamembrane for an immediate adaption to extracellular signals. γδT-cells are enigmatic lymphocytes displaying effector as well as immunoregulatory functions and involved in pathogeneses of autoimmune diseases. to estimate pathogenetic role of γδT-lymphocytes in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). peripheral blood mononuclear cells (PBMC), lymph nodes, intraepithelial lymphocytes of intestine, splenocytes and thymocytes of 25 Wistar rats with induced EAE and 12 control animals as well as PBMC from 26 patients with relapsing-remitting MS and 17 healthy donors were used as material. The quantity of γδTcells, myelin-induced activation and proliferation of CFSE-labeled αβT-lymphocytes subsets in the presence or absence of γδTlymphocytes or CD14 + cells (removed from cell culture by magnetic separation) were monitored by flow cytometry analysis.
The investigation of γδTCR+cells in lymphoid organs showed γδTlymphocytes' redistribution from peripheral blood to lymph nodes in EAE rats which correlates with clinical finding of disease. Similarly, in MS patients the number of γδT-cells was reduced among PBMC. The estimation of γδT-cells influence on homing and myelin-induced activation of αβT-lymphocytes in vitro revealed that γδT-cells hold in check CD62L shedding from αβT-lymphocytes surface (pb 0.01) and control the CD25 expression (p b 0.05) on T-cells subsets cultured with autoantigen in both MS patients and healthy donors. Moreover, the presence of γδT-cells suppresses spontaneous and autoantigen-induced proliferation of αβT-lymphocytes (p b 0.01) in healthy donors performing thereby immunoregulatory role. On the contrary, in MS patients γδT-cells promote the increase of CD4 + T-cells myelin-specific proliferation (p b 0.05) assuming their participation in autoantigen presentation to Thelpers. Obtained data were confirmed by αβT-cells myelin-specific proliferation in the absence of potential antigen-presenting CD14 +cells, but not γδT-cells, in PBMC culture only in MS patients.
γδT-cells are involved in pathogenesis of MS having been confirmed on EAE model. γδT-lymphocyte regulate homing and activation of myelin-specific αβT-cells, but in different ways affect αβT-cells subsets proliferation in response to specific autoantigen in MS patients and healthy donors making γδT-cells perspective target for pathogenetic therapy. Experimental autoimmune encephalomyelitis (EAE) is a central nervous system targeting autoimmune disease and a primary animal model of multiple sclerosis (MS). We have used EAE in a small-sized Neotropical primate, the common marmoset (Callitrix jacchus), to unravel cellular mechanisms of demyelination. This animal model is relevant due the clinical and pathological similarity to MS with a high genetic and immunological proximity to humans.
The aim of this research was to assess the phenotype and functionality of a newly discovered subset of auto-reactive cytotoxic T-cells, which mediate demyelination in the absence of autoantibody involvement. Beside this we also identified the MHC-peptide complex targeted by these T-cells.
EAE was induced by immunizing unrelated marmosets with a synthetic peptide representing peptide 34 to 56 of human myelin/ oligodendrocyte glycoprotein (MOG34-56) emulsified in incomplete Freund's adjuvant (IFA). All animals developed neurological deficit and demyelinated lesions in the brain and spinal cord. By FACS analysis cells with CFSE dilution upon stimulation with MOG34-56 are CD3+CD4+, CD3+CD8+ or CD3+CD4+CD8+, co-expressing CD27, CD45RO and CD56, but no CD16, CD28 and CCR7. Moreover these cells displayed specific cytolytic activity to allogeneic and autologous B-cells, with a minimal 9-mer T-cell epitope within the MOG34-46 sequence. B-cell exchange experiments show that cytotoxicity is peptide specific but target cell independent. This indicates that the peptide may be presented by an invariant MHC class I allele, being Caja-E, which is homologue to human HLA-E.
The normal repertoire of marmoset monkeys contains memorytype T-cells that can be in vivo activated with MOG34-56 in adjuvant (IFA) without innate antigen receptor ligands. The striking capacity of T-cells to induce demyelination in CNS white and grey matter is associated with a 9-mer epitope within the MOG34-56 sequence and Caja-E restricted cytotoxicity. We hypothesize that both features are causally related.
Ortlieb Guerreiro-Cacais Andre ⁎ , Beyeen Amennai, Jagodic Maja, Olsson Thomas Department of Clinical Neuroscience, Neuroimmunology Unit, Karolinska Institutet, Stockholm, Sweden VAV1, a member of the family of guanine nucleotide exchange factors (GEF) for the Rho family of GTP binding proteins, is important in hematopoiesis, also playing a role in T-cell and B-cell development, activation, tissue homing and effector function. Vav1 was found within Eae4, a rat quantitative trait locus (QTL) that regulates experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), and overlaps with several QTLs for immunemediated diseases. Analysis of an extensive human cohort also demonstrated association of certain haplotypes within the first intron of the gene with MS, and increased levels of VAV1 mRNA were found in peripheral blood and cerebrospinal fluid of MS patients as compared to matched controls (published results*). In an attempt to further characterize the function of VAV1 in the context of development of autoimmunity we used interval-specific congenic rat lines carrying the disease predisposing or protective Vav1 variants. We show that when immunized with recombinant rat myelin oligodendrocyte glycoprotein (MOG) in incomplete Freund's adjuvant, the disease predisposing variant promotes the development of a high proinflammatory state, with higher numbers of IFNγ + γδT cells and higher numbers of both CD4 + CD8+ (double positive) T cells and CD4+ CD8dim T cells that show increased expression of the activation markers CD28, CD44, CD49d and increased Brdu incorporation, indicative of cells in cycle. Upon antigen-specific in vitro restimulation, cells from the disease predisposing variant exhibit a phenotype with increased production of IFNγ and TNF, while cells from the strain with the protective variant show increased levels of IL10, IL4 and MCP1, as well as higher overall levels of CD3+ CD25+ Foxp3+ T cells, especially within the CD8+ subset. We conclude that VAV1 plays a central role in controlling the inflammatory immune response in a rat model of EAE. Our studies are now aimed at pinpointing which stage of the immune response is specifically controlled by this nucleotide exchange factor. EAE can also be prevented by oral tolerance to MBP. However, although oral tolerance to myelin antigens has been achieved in multiple sclerosis (MS) patients, there was no beneficial effect to the patients. It has recently been found that oral tolerance to egg ovalbumin also occurred in the liver, mediated specifically by plasmacytoid dendritic cells. This systemic tolerance involved delayed type hypersensitivity responses. Plasmacytoid dendritic cells have not been demonstrated in EAE lesions. Plasma cells, identified using the specific marker CD 138, are confined to the perivascular spaces in MS patients and are not present in the brain parenchyma. Cells in the parenchyma with extensive rough endoplasmic reticulum and previously described as plasma cells are probably plasmacytoid dendritic cells, but this needs confirmation using the specific marked CD123.
In order to produce an animal model of non-lepromatous leprosy, Dutch Bantam and English/Lop rabbits were injected with a homogenate of human sensory peripheral nerve plus adjuvant and then skin tested with dilute concentrations of sensory nerve and various human non-myelin antigens. Some of these rabbits have developed a state of granulomatous hypersensitivity, i.e., plasma-like cells with extensive rough endoplasmic reticulum accumulate at skin test sites, both in the dermis and also in the endoneurium with axonal damage and thinning of the myelin sheaths. These plasma-like cells may be plasmacytoid dendritic cells. The most active component of human sensory nerve inducing the granulomatous response was a deoxycholate-extracted membrane fraction from the 'nuclear pellet' of sensory nerve after the myelin has been removed.
It may be possible to induce oral tolerance using this specific antigen. In order to confirm whether tolerance has occurred, there should be an absence of plasma-like cells in the endoneurium of dermal nerves with no axonal damage at skin-test sites following challenge with this membrane fraction in sensitized rabbits. Although the antigen is effective in micrograms of protein, further purification will be necessary to localise a similar antigen in the central nervous system.
It may then be possible to induce tolerance to this antigen, in which case a vaccine could be developed to prevent disability in MS.
Activation of the SphK1/S1P receptor axis in response to anoxia in primary rat astrocytes Fischer Iris ⁎ , Nichols Anthony, Alliod Chantal, Frossard Marie, Pouly Sandrine
Ischemic-type of lesions in the CNS, is a characteristic hallmark of multiple sclerosis (MS) and likely participates in the neurodegenerative processes of the disease. Hypoxia was shown to lead to increased permeability and disruption of the BBB. During hypoxia, astrocytes can retract their end feet processes from the microvessels, resulting in increased permeability and proliferation that may lead to formation of a glial scar. The activation of astrocytes in response to hypoxic stress was also shown to induce the pro-angiogenic factor vascular endothelial growth factor (VEGF), a major target gene activated during hypoxia.
Others have shown that the enzyme sphingosine kinase I is induced under low oxygen levels in a cell line derived from human glioblastoma, which led to increased sphingosine-1-phosphate (S1P) levels. Finally, crosstalk between S1P and VEGF was demonstrated to up-regulate expression of both S1P3 and S1P1 receptors and was associated with an enhanced intracellular signaling in response to S1P.
The goal of the present study was thus to investigate if combined oxygen-glucose deprivation (OGD) and subsequent re-oxygenation activates the SphK/S1P receptor axis in primary rat astrocytes, and if this leads to a transactivation of VEGF.
Primary cultures of rat astrocytes were exposed to OGD for 4 h, followed by reoxygenation for up to 24 h. The regulation of the mRNA levels of the SphK1/2 and the S1P receptors were measured as well as the influence on ERK-and Akt-signaling. S1P receptor specific signaling was investigated using the CellKey technology which allows the measurement of bioimpedance modulation induced, upon receptor activation, by changes in cell shape. Furthermore the production of VEGF and CXCL1 was measured.
OGD followed by reoxygenation induced an upregulation of the SphK1 and S1P3 mRNA levels but not of SphK2, S1P1 or S1P2. In addition, ERKsignaling was elevated after 4 h of OGD and during reoxygenation, whereas Akt-signaling was elevated only during reoxygenation. Finally, specific signaling via S1P1 and 3 was increased during reoxygenation and correlated with an increase in VEGF and CXCL1 levels.
These preliminary data demonstrate that ischemic stress followed by reoxygenation activates the SphK1/S1P receptor axis in cultures of primary rat astrocytes. Further experiments will attempt to elucidate the possible involvement of this pathway on the production of VEGF and CXCL1.
NMO-IgG autoantibody is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggest that NMO-IgG may play a central role in NMO physiopathology. The aim of this in vitro-based study was to characterize molecular and functional consequences of interaction between NMO-IgG and astrocytes primary cultures.
Three NMO-IgG positive NMO patients from Neuroimmunology of the Ramos Mejia Hospital, Argentina were selected. As control, we included serum from 3 NMO-IgG negative healthy controls. Primary cultures of rat astrocytes were incubated with heat-inactivated control or NMO patients' sera (dilution 1/50), for 1 h at 4°C or 12 h at 37°C and then immunofluorescence studies as well as water permeability measurements by fluorescence videomicroscopy were performed. Fluorescence was quantified by densitometric analysis using Image-J software.
Immunofluorescence studies showed that cells exposition to NMO patients' sera for 1 h at 4°C (to restrict membrane fluidity) resulted in IgG binding to the plasma membrane in a linear pattern that colocalized with AQP4, while serum IgG from controls did not bind significantly. In contrast, after exposure for 12 h at 37°C, NMO patients' sera containing AQP4 specific IgG resulted in disappearance of AQP4 fluorescence from the plasma membrane while control sera had no effect on AQP4 expression. Even more, 1 h exposition of astrocytes to NMO-IgG does not change the water permeability, indicating that the water pore is not affected by the binding of IgG, but 12 h of exposition to NMO-IgG clearly induced a significant reduction of the water permeability (time constant of water transport, . (s), control vs. NMO-IgG sera 18.04 ± 1.04 vs. 34.96 ± 4.45, p b 0.01).
NMO patients'sera IgG has a selective pathogenic effect on cell membranes expressing AQP4. We demonstrated that NMO-IgG binding to astrocytes alters AQP4 expression and decreases permeability.
Vartak Neha ⁎ , Borgmann Kathleen, Tang Lin, Ghorpade Anuja
Human immunodeficiency virus (HIV)-1-associated dementia (HAD) is a highly debilitating neurocognitive disorder occurring in 20-40% of HIV-1-infected untreated individuals. Astrocytes, the most abundant cells in the brain, play a crucial role in HAD progression and outcome. Recently, a rapid subtraction hybridization approach (RaSH) to determine differentially expressed genes in HIV-1 infected astrocytes, identified a novel gene, astrocyte elevated gene-1 (AEG-1), as a HIV-1 and tumor necrosis factor (TNF)-a inducible transcript. Subsequently, AEG-1 was also shown to be elevated in subsets of malignant gliomas, hepatocellular and breast carcinomas, and was identified as an oncogene responsible for metastasis, anchorageindependent growth and resistance to chemotherapeutic drugs. Although AEG-1 was primarily identified as a HIV-1 inducible gene in astrocytes, its role in HIV-1 infection has not been studied so far. Astrocytes as crucial regulators of brain homeostasis, have been the focus of HAD research for a long time. Therefore, it would be very beneficial to study the role of AEG-1 in the astrocyte responses orchestrated during HIV-1 infection of the brain. Here, we propose that AEG-1 can be a crucial contributing factor to HAD progression as it can modulate the astrocyte responses to inflammation, injury and stress generated during the pathogenesis of HAD by activating the downstream nuclear factor (NF)-kB pathway.
Our preliminary data show that HAD-relevant stimuli, HIV-1, interleukin-1b (IL-1b) and TNF-a, upregulate the expression of AEG-1 both at the mRNA and protein levels in primary human astrocytes. A corresponding decrease in excitatory amino acid transporter (EAAT)-2 mRNA levels and an increase in CXCL8 chemokine production, a characteristic phenomenon of HAD, was also observed. This suggests a probable role of AEG-1 in HAD by modulating the expression of these proteins at the mRNA level. Further, our immunocytochemical studies show that AEG-1 localizes in the cytoplasm and nucleus of primary human astrocytes in response to HAD-relevant inflammatory stimuli but in the nucleolus in response to injury and oxidative stress.
This distinction in the intracellular localization of AEG-1 in response to different HAD-relevant stimuli suggests the involvement of AEG-1 in regulating astrocyte behavior in HAD development and progression. Rheumatoid arthritis, a systemic autoimmune disease, brings about except pain often impairment of logic memory and attention deficit. So far there are no experimental correlates that could sufficiently explain or possibly prevent these problems. The aim of the present study was to analyze neuroendcrine and inflammatory changes in the circulation, and the mRNA expressions of parameters of oxidative stress in the hippocampus before clinical manifestation of the inflammatory disease, on days 2 and 4 after cFA administration and in control male Lewis rats.
The cFA was administered intradermally at the base of the tail. ACTH, corticosterone, and C-reactive protein were assayed in plasma by specific RIA or ELISA, and albumin was measured spectrophotometrically on the base of formation of bromcresol green complex. The mRNAs for NADPH1 oxidase (NOX1), NADPH2 oxidase (NOX2), interleukin-1beta (IL-1beta), and inducible NO. syntase ( iNOS) in the hippocampus were assayed by quantitative real-time PCR using TaqMan probes and primers and are expressed in arbitrary units related to beta-actin expression. C-fos was measured immunohistochemically in the lumbar spinal cord slices. We did not find enhanced c-fos in fibers of the lumbar spinal cord which indicates no activation of pain related neurons from the hind paws. In this preclinical stage of the disease we found enhanced circulating levels of ACTH, corticosterone, C-reactive protein, and reduced levels of albumin. In the hippocampus we found over-expression of mRNA for NOX1, while NOX2 was unchanged. Furthermore the expressions of mRNAs IL-1beta, as well as for iNOS were upregulated.
Microglias upon immune activation are known to enhance NOX1 activity which consequently induces IL-1β, as well as NO production. These products give rise to highly neurotoxic proooxidant peroxynitrite ONOO-and may lead to synapse loss. Based on our results we assume that the changes we found in the early phase of inflammatory disease may progressively induce oxidative stress to the neurons. Furthermore, our results show that the oxidative-inflammatory changes precede any clinical manifestation, and the treatment with antioxidant may possibly help to slower its process.
Supported by: GACR 303/10/1368 and APVV 51-017905.
Changes in neuromuscular junction and peripheral nerve associated to acute and chronic exposition to anti-GM2 and GalNAc-GD1a antibodies Antiganglioside antibodies are one of the most frequently autoantibodies associated to immune mediated neuropathies. Almost 30% of GBS patients demonstrated serum reactivity against those glucolipids. Chronic polineuropathies showed a lower association frequency. Although antibodies recognizing some gangliosides like GM1, GQ1b, and GD1a, are frequently found, the mechanisms implicated in the nerve damage or a possible interference in gangliosides functions in axons or in the neuromuscular junctions are little studied. We have studied the effect of a human IgM monoclonal antibody that recognize specifically the carbohydrate sequence -GalNAc1-4Gal (3-2NeuAc)1], sheared by GM2, GD1a-GalNAc and GD1b-GalNAc on nerve and neuromuscular junction, and in the presence of complement or without.
Neuromuscular junction (NMJ) studies have been performed in mouse levator auris longus muscle. Nerve conduction studies were performed in sciatic rat nerve. We performed: a) immunohistochemical studies to localize the antibodies fixation, the complement activation and structural changes in NMJ and nerves, b) methylene blue studies to analyze structural changes in nerves and c) electron microscopy to evaluate ultrastructural changes. Intracellular recording electrofisiological studies were performed to evaluate NMJ and conventional motor neurographies and electromyography to evaluate rat sciatic nerve.
In the NMJ, we have found an acute and chronic neurotransmitter release blockage without complement implication. Voltage-dependent calcium channels (VDCC) were involved in this effect. Immunoglobulin chronically applied in vivo provoked terminal axon retraction, spouting and expression of VDCC type L, again without complement activation. Passive intra nerve immunoglobulin transfer induced an acute complement dependent partial conduction block. Chronic treatment results in complement dependent mixed axonaldemielinating electrophysiological and morphological changes.
Anti-GM2 antibodies can affect neuromuscular transmission without complement activation, however nerve conduction alterations need membrane structural damage. These gangliosides are involved in the reciprocal Schwann cell-nerve terminal interactions, including structural stability and neurotransmission. Introduction: Inflammatory mechanisms play a key role in the pathophysiology of postischemic inflammation following stroke. Resident microglial cells and invading immune cells are major contributors to the postischemic inflammation. The different immune cell subpopulations are integrated in a complex cytokine network, which is composed of pro-and antiinflammatory cytokines. The goal of our study is the elucidation of immunological mechanisms underlying the activation of the immune system in the ischemic brain. Methods: Intracellular cytokine production was analyzed by fluorescence activated cell sorting (FACS) analysis in immune cell subpopulations following temporary occlusion of the middle cerebral artery (MCAO) and subsequent purification of immune cells out of the brain homogenate. Results: At day 1, the cytokine production in T cells is dominated by Interferon gamma (INFg) and TNF alpha in CD4 and CD8 positive T cells. On day 3, gamma delta T cells (gd T cells) emerge in the ischemic hemisphere, which are the major producers of IL17A, followed by CD4 positive T cells and NKT cells producing smaller amounts of IL17A. Regarding antigen-presenting cells (APCs), macrophages and microglial cells are the major producers of TNF alpha, whereas neutrophils contribute substantial amounts of IL6. Next, we utilized lymphocyte deficient Rag1 mice to analyze the interplay between T cells and APCs. According with published data Rag mice exhibited smaller infarct volumes 3 days post stroke. Additionally, the deficiency in lymphocytes resulted in substantial decrease in TNF production in macrophages whereas the microglia-derived TNF production was unaffected. Conclusion: Initially, the cytokine milieu in the ischemic hemisphere is dominated by INFg and TNF alpha produced by CD4 and CD8 T cells. Then atypical T cells producing IL17A invade the ischemic hemisphere. In parallel macrophages and microglial cells produce large amounts of TNF. Remarkably, T cells seem to play a central role already in the orchestration of the initial immune response, since the activation of pro-inflammatory macrophages is decreased in Rag mice. The further characterization of the immunological mechanisms of the postischemic inflammation will provide valuable insights into the role of different immune cell population and their activation and modulation by cytokine pathways.
Cumulative autoimmunity: Myelin-specific CD4 T cells co-recognise neurofilament-medium Despite extensive efforts, the antigen-specificity of the inflammatory response driving Multiple Sclerosis (MS) remains poorly defined. Its determination is hindered by the presence of CNS-specific T cells in healthy individuals, but above all by the fact that the immune response in MS is composite and varies over time.
Myelin Oligodendrocyte Glycoprotein (MOG) is a candidate auto-antigen for MS. Its positioning on the outermost lamellae renders MOG directly accessible to pathogenic immunoglobulins that are found within the CSF of MS patients. We aimed to further explore the pathogenic traits of the MOG-specific CD4+ T cell response by making use of TcR-transgenic (2D2) mice in which N95% of CD4+ T cells are specific for MOG35-55:I-A.
Using 2D2 mice we made the paradoxical observation that the spontaneous EAE persists even in the absence of the target antigen MOG. 2D2 mice crossed on a MOG-deficient background preserved the same incidence of spontaneous EAE as 2D2 MOG sufficient mice. Using a proteomics approach we could identify an alternative target for the 2D2 T cell response. Neurofilament medium (NF-M) is a cytoskeletal protein exclusively expressed in neurons. NF-M15-35 was shown to share the same TCR binding residues as MOG35-55 when presented in the context of I-Ab, and could stimulate CD4+ T cells from 2D2xRAG −/− mice in vitro. NF-M is the alternative target for the pathogenic MOG35-55 specific CD4+ T cell response as the adoptive transfer of pathogenic MOG-specific CD4+ T cells into syngeneic recipients lacking both MOG and NF-M (MOG -/-NF-M -/-) double-deficient mice failed to induce disease. To assess the individual contribution of either MOG or NF-M to disease severity we used MOG or NF-M single deficient mice as recipients of pathogenic 2D2 T cells. In the absence of MOG expression we observed a disease characterized by a delayed onset, but with a similar maximum severity. On the other hand, mice lacking NF-M developed a disease with similar onset, but with a reduced disease severity. Furthermore, we recently observed that 2D2xNF-M-deficient mice preserved the same 18% disease-incidence of spontaneous EAE as 2D2 wild-type mice or 2D2xMOG-deficient mice.
These data reveal that molecular mimicry between the neural antigens MOG35-55 and NF-M15-35 can aggravate an autoimmune disease by permitting a clonal CD4+ T cell response to target more than one cognate auto-antigen simultaneously. We coined this observation cumulative autoimmunity. Macrophages play an important role in the pathogenesis of multiple sclerosis (MS), having both beneficial and detrimental effects. Several studies found correlations between macrophages and axonal damage: i) elimination of infiltrating macrophages reduced axonal damage and clinical signs in EAE; ii) a correlation was found between the number of infiltrating macrophages and axonal damage. Links between macrophages and axonal regeneration and repair are reported: i) macrophages phagocytose myelin debris, which is growth inhibiting; ii) macrophage infiltration corresponded with an upregulation in expression level of growthassociated protein-43, a marker for axonal growth. Our hypothesis is that these different effects of macrophages on axonal damage and repair are due to different subtypes of macrophages. The extreme subtypes of macrophages are: classically activated (CA) macrophages, which are cytotoxic, and alternatively activated (AA) macrophages, which are growth promoting. Here we investigate the effects of differentially activated macrophages on neuronal integrity.
Differently activated macrophages were generated by 2 day exposure to LPS and IFN-g to induce CA macrophages, or IL-4 to induce AA macrophages. Mouse primary neuron cultures were exposed to either differently activated macrophages or the conditioned medium. The effect on neuronal vitality and morphology was determined.
We determined three aspects of macrophage functioning: migration, induction of damage and phagocytosis. First attraction of CA and AA macrophages by different CNS cell types was determined. Neuronally conditioned medium attracted AA macrophages in significantly higher numbers compared to CA macrophages. The CA macrophages were more attracted towards astrocyte conditioned medium. Next, we investigated the induction of neuronal damage. When neurons were exposed to either CA macrophages or CA conditioned medium a reduction in neuronal vitality was found. Both exposure to AA macrophages and AA conditioned medium did not reduce neuronal vitality. Finally, we determined the capacity of phagocytosis of differently activated macrophages. The CA macrophages were more efficient in phagocytosing neurons and neuronal fragments compared to AA macrophages. CR3 did not influence phagocytosis of neuronal fragments, while myelin phagocytosis was significantly reduced.
In summary, CA macrophages damage neurons and are able to clear the debris more efficiently compared to AA macrophages. Members of the TNF receptor family elicit a wide range of biological responses, including cell death, cell proliferation, inflammation and differentiation. The cytotoxic effects of these receptors are mediated by the activation of an apoptotic cell death program in which the Fas associated death domain protein (FADD), a key adaptor protein that facilitates the recruitment of caspase-8 to the receptor, was shown to be crucial. Targeted inactivation of FADD in mice results in early embryonic lethality with signs of cardiac failure and hemorrhage. Fibroblasts from FADD knockout mice are completely resistant to death receptor (DR)-mediated apoptosis, and thymocytes from FADD knockout chimeric mice display reduced proliferation in response to mitogens, indicating that the FADD-signaling pathway also plays a role in T cell development and activation. Since oligodendrocytes are thought do die by apoptosis during the course of multiple sclerosis (MS) and its main animal model experimental autoimmune encephalomyelitis (EAE), we sought to further elucidate the role of FADD in EAE.
As FADD deficient mice die in utero, we generated a conditional FADD allele using the Cre/LoxP recombination system allowing the study of the role of FADD in adult mice. To investigate the role of FADD in the pathology of MS, we generated mice lacking FADD in oligodendroctes, by crossing FADDFL/FL mice with a knockin line that expresses Cre under control of the endogenous oligodendrocytespecific myelin oligodendrocyte glycoprotein promoter (MOGi-Cre), resulting in mice lacking FADD specifically in oligodendrocytes (FADDolg-ko). EAE was induced in these FADDolg-ko mice in order to evaluate the role of FADD-dependent death signaling in oligodendrocytes in the development and progression of EAE. These mice are protected against EAE, reflected in both a milder clinical disease course as well as a reduced inflammatory infiltration in the spinal cord, otherwise a typical hallmark of EAE. Furthermore, this reduced inflammatory phenotype was not the result of an aberrant peripheral immune response in FADDolg-ko mice.
Previous reports have pointed out a role for oligodendrocyte apoptosis in EAE. The results presented here indicate that DR signaling through FADD in oligodendrocytes is crucial in the development and progression of EAE.
Ström Mikael, Al Nimer Faiez, Lindblom Rickard, Piehl Fredrik ⁎
Neurodegeneration is an important feature of neurologic disorders such as Multiple Sclerosis, Alzheimer's, ALS and Parkinson's diseases. The common forms of these diseases are caused by a complex interaction between genetic and environmental factors. We have applied an unbiased genetic dissection using a mechanical nerve injury model (ventral root avulsion; VRA) to identify factors responsible for differences in neurodegeneration among different rat strains. VRA1, a QTL regulating nerve cell death was previously found in a F2 DA × PVG intercross and the effect has been reproduced and fine mapped in congenic strains and advanced intercross lines. We now combined this with a F2 DA × PVG intercross where global gene expression profiling using Affymetrix Rat Exon 1.0 chips was performed in 144 animals subjected to VRA at 5 days after injury. Linkage analysis in the VRA1 region displayed four cis-regulated eQTLs, of which only one, GSTA4, with certainty is included in the smallest congenic fragment. Effect plots and independent verification with RT-PCR demonstrate lower expression of GSTA4 in the DA strain displaying lower motorneuron survival.
Neuroinflammation with activation of microglia leads to production of reactive oxygen species (ROS) and subsequently to lipid peroxidation. 4-Hydroxynonenal (4-HNE) is a highly reactive endproduct of lipid peroxidation and GSTA4 has an extremely high catalytic efficiency in reducing it by conjugation to glutathione. 4-HNE reacts with proteins and form stable adducts that alters the protein function has been found in elevated levels in several neurodegenerative diseases and is also present in MS lesions.
We have studied the effect of VRA1 in traumatic brain injury (TBI) and experimental autoimmune encephalomyelitis (EAE) in congenic rat strains. In TBI, VRA1 leads to higher expression of GSTA4 in the brain and lower levels of histidine-conjugated 4-HNE in cerebrospinal fluid (CSF) measured with ELISA and in a pilot MOG-induced EAE experiment VRA1 regulates disease severity. Levels of 4-HNE was also found to be elevated in CSF of a sub-population of patients with MS.
Together, these data suggest that naturally occurring genetic variability in Gsta4 may regulate susceptibility to neurodegeneration in trauma and autoimmune neuroinflammation. Multiple sclerosis (MS) is associated with cognitive deficits, developing independently from motor disorders. These deficits are a manifestation of neurons damage. In this study, using the experimental model for MS-autoimmune encephalomyelitis (EAE), we investigated whether EAE will result in the damage of hippocampal neurons and selective deficits in learning and memory, as investigated by the Morris water maze test.
Lewis female rats 3 months old were injected with 4 millions of anti-MBP CD4+ T cells to evoke EAE. Animals suffered from tail and hind limb paresis and recovered completely after about 10 days post inoculation (dpi). T cells infiltrated spinal cord and many brain regions including hippocampus. We demonstrated the decrease of pyramidal neurons in CA1 and CA4 region by about 20%, as evaluated by stereological measurements, at 21 and 90 dpi. This was preceded by prolonged glial activation as well as by a rise of the pro-inflammatory cytokine mRNA expression (IL-1beta, IL-6 and TNF alfa). Increased expression of some cytokines mRNA was observed also at 90 dpi. However, contrary to our hypothesis, no differences in the water maze test were detected between the EAE and control groups, neither on 21 dpi nor on 90 dpi.
Anti-MBP CD4+ T cells are capable of injuring hippocampal pyramidal neurons during EAE, probably, through the secretion of pro-inflammatory cytokines, also by activated glial cells. However, in the studied conditions, hippocampal neurodegeneration caused by T cells did not result in memory disturbances.
Supported by the grant no. N401-1293-33 of the Ministry of Scientific Research and Information Technology in Poland.
IL-13 stimulates neurite outgrowth in primary neurons and organotypic brain slices Vidal Vera Pía ⁎ , Lemmens Evi, Boato Francesco, Nelissen Sofie, Vangansewinkel Tim, Hendrix Sven
There is increasing evidence that inflammatory processes after spinal cord injury (SCI) may exert detrimental as well as beneficial effects. Previous studies indicate that T helper type 2 cells may influence axon regeneration via secreted anti-inflammatory cytokines such as IL-4. Inside this family the biological functions of IL-13 resemble mostly those of IL-4; they share the IL-4 receptor a-chain which is thought to be responsible for most of the functional characteristics of these cytokines. Here we investigated the effect of recombinant IL-13 on neurite outgrowth in outgrowth assays based on neuronal cell cultures and organotypic brain slices.
In cortical primary neuron cultures IL-13 significantly increased the average axonal length. To get closer to the in vivo situation we also analyzed IL-13 effects on entorhinal cortex explants embedded in a three dimensional collagen matrix from two-old wildtype mice as well as on entorhinal cortices derived from enhanced green fluorescent protein-positive mice co-cultured with hippocampus from wildtype littermates. In both models, IL-13 was found as a potent stimulator of neurite outgrowth in a dose-dependent manner. Analyses using MTT assays on primary neuron cultures indicated no change of metabolic activity, suggesting no neuroprotective or toxic effects to be responsible for increased neurite outgrowth.
In conclusion, these results suggest that IL-13 is a potent promoter of axonal regeneration in vitro, which occurs independent of neuronal cell survival. Pro-inflammatory cytokines such as Interleukin-1 beta (IL-1β) are considered to exert detrimental effects in the context of brain trauma e.g. by inducing neuronal cells death. Consistent with this concept it has been demonstrated that IL-1 suppresses neurotrophin-mediated neuronal cell survival. Therefore, we have investigated here whether IL-1 has a similar impact on neurotrophin-induced neurite growth.
We have analyzed neurite density and length of organotypic brain and spinal cord slice cultures under the influence of the neurotrophins Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and NT-4. Only NT-3 significantly promoted neurite growth and increased the number of slices displaying maximal growth. Similarly, IL-1 promoted neurite density and extension as well as the number of slices showing maximal growth. Brain slices from IL-1 knockout mice revealed that endogenous IL-1 is not crucial for spontaneous neurite growth. Coadministration of IL-1 and NT-3 still significantly stimulates neurite density and length as well as the number of slices displaying maximal neurite growth. This effect appears to be brain-specific since spinal cord slices were only stimulated by NGF but not by NT-3, IL-1 or coadministration of both factors.
Thus, in contrasts to its inhibitory effect on neurotrophinmediated neuronal cell survival IL-1 stimulates neurite growth and does not impair NT-3-induced neurite growth. These data suggest that anti-inflammatory treatments may prevent neuronal cell death at the cost of neurite regeneration.
Haile Yohannes ⁎ , Simmen Katia, Touret Nicolas, Simmen Thomas, Bleackley Chris, Giuliani Fabrizio
Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by demyelination and neurodegeneration. Infiltrating T cells and macrophages are constituents of MS lesions. Activated cytotoxic T cells (CTLs) release a serine-protease granzyme B (GrB) which has been shown to induce neurotoxicity. CTLs-mediated neuronal microtubule disruption was reported in animal model of MS, experimental autoimmune encephalomyelitis. Recent studies also showed alpha-tubulin, one of the main constituents of microtubules, as a new target for GrB. Therefore, the objective of this study is to investigate the mechanisms of GrBmediated neuronal injury.
Human cortical foetal neurons (HFNs) were cultured alone, treated with GrB, co-cultured with unactivated or anti CD3activated T cells on poly-ornithine coated culture plates. Viability assay was performed using immunocytochemistry for microtubule associated protein-2 or β-tubulin to assess the survival of neurons. Expression of GrB by T cells was measured by RT-PCR. Confocal microscopy was used to follow the interaction of T cells and neurons, internalization of GrB into neurons and induction of apoptosis. Cleavage of alpha-tubulin, GrB substrate, was evaluated using Western blotting.
The viability of HFNs was significantly low in cultures treated with activated T cells (32 ± 7%) or GrB (36 ± 9%). In mouse system, activated T cells from GrB knock out mice failed to kill mouse neurons. GrB entered into neurons via mannose-6-phosphate receptors (M6PR) and induced perforin-independent neuronal apoptosis. Western blotting showed caspase-dependent cleavage of alphatubulin in presence of GrB or activated T cells.
This study shows that GrB internalization into neurons is independent of perforin and occurs through M6PR mediation. In addition, GrB destabilizes the cytoskeletal proteins and induce caspase-dependent neuronal apoptosis. These data show GrB as new potential target for neuroprotective strategies in MS treatments. The alpha7 nicotinic acetylcholine receptor (nAChR) is a wellknown mediator of inflammation. Activated alpha7 nAChRs expressed on macrophages suppress the synthesis and release of proinflammatory cytokines, including TNFalpha. It remains possible that the immune system itself reciprocally regulates the functions of the alpha7 nAChR on microglia, astrocytes and neurons in the brain. Furthermore, inflammation in the brain is associated with multiple disorders, including autism spectrum disorders (ASDs). Hence, understanding the functional consequences of a potential bi-directional cross-talk between the immune system and the alpha7 nAChR is important. Our laboratory is interested in the relationship between the alpha7 nAChR and ASDs because genome-wide association studies link the alpha7 nAChR to ASDs; post-mortem analyses of specific regions of ASD patient brains show an increase in alpha7 nAChR levels; and we identified a functional association between nAChRs and neurexins, presynaptic cell adhesion molecules. Bi-directional interactions between neurexins and their postsynaptic binding partners, the neuroligins, promote synapse maturation by recruiting specific pre-and post-synaptic molecules, including nicotinic receptors to synapses. Deletion and mutation of both neurexin and neuroligin genes are strongly linked to ASDs.
In this study, we examined the association between the alpha7 nAChR and specific neurexin isoforms. Rat hippocampal neurons were cotransfected with alpha7 nAChR and neurexin isoforms and cocultured with non-neuronal tsA 201 cells transfected with neuroligin isoforms. When alpha7 nAChR is expressed alone in neurons, it does not target to neuroligin-1-expressing tsA 201 cells. However, when alpha7 is coexpressed with neurexin-1beta, it targets to neuroligin-1-expressing tsA 201 cells in a small percentage of the neurons examined in culture.
These data suggest that neurexins influence the targeting of alpha7 nAChRs in a specific subset of neurons, whose identity is under investigation. Thus, dysfunction of either inflammatory processes and/or of synapse maturation, could promote changes in the expression of alpha7 nAChR observed in ASD brains and consequently some of the behavioral deficits associated with this disorder. Parkinson's disease (PD) patients have impaired spatial working memory, problems with organizing and using new materials as well as applying strategies. Striatal dopamine (DA) deficiency is not solely responsible for the genesis of the non-motor symptoms. In the present study 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine MPTP administration and influence of neurodegeneration on memory impairment has been studied.
One year old C57/BL male mice received MPTP 40 mg/kg, and control group received 0.9% NaCl. In order to evaluate learning and memory abilities, the Morris water maze (WM) behavioral test was provided. The brain concentration of: DA, norepinephrine (NE), 5hydroxytryptamine (5-HT) and glutamate (Glu) were estimated by using high-performance liquid chromatography (HPLC). The ratio of metabolites (MHPG, HVA, 5-HIAA) to their parent amines in the selected brain regions were estimated. Neuron-specific nuclear protein (NeuN) immunohistochemistry and unbiased stereological methods were used to determine the total number of neurons in the CA1, CA3 subregions of the hippocampal formation. Statistical significant decrease in the levels of monoamines was noticed in MPTP mice group in striatum (DA, NE) hippocampus (NE) and prefrontal cortex (DA, NE, 5-HT content) 7 days and 3 months after intoxication. Statistical significant differences in NE content in the hippocampus (p b 0.01) and prefrontal cortex (p b 0.01) remained also 6 months after intoxication. MHPG/NE ratio was increased in hippocampus (p b 0.05), in prefrontal cortex (p b 0.001), HVA/DA ratio was increased only 7 days after intoxication in striatum and prefrontal cortex (p b 0.001). We noticed subtle differences in the long-term memory test provided 6 months after MPTP intoxication compared to controls. The MPTP group mice spent less time in the target quadrant (SE) after intoxication than control mice (− 31%). Swim distance to reach the hidden platform was negatively correlated with NA content in the hippocampus (r = − 0.62, p b 0.05).
Our results evidence that acute systemic administration of MPTP can lead to serious alternation in NE and 5-HT neurotransmission. Accelerated turnover of DA and NE in the absence of decline in the number of cell bodies, may indicate the existence of a compensation mechanism. The differences in spatial long-term memory also make the suggestion that noradrenergic function is required for hippocampal, but not cortical dependent memories. Increasing evidence suggest that inflammatory processes after spinal cord injury (SCI) may cause detrimental as well as beneficial effects. Previous studies indicate that T cells may influence axon regeneration via secreted anti-inflammatory cytokines such as interleukin-4 (IL-4) and IL-13. The anti-inflammatory cytokines IL-4 and IL-13 have multiple functions in common and share the IL-4 receptor a-chain (IL-4Ra) which is thought to be responsible for most of the functional characteristics.
In the current study we have investigated the therapeutic potential of recombinant IL-4 and IL-13 administered locally in gelfoam beads above the lesion at selected time points after SCI. Single administration of recombinant IL-4 immediately and 1 day after the injury significantly improved locomotor restoration as assessed by two behavioral tests, the open-field Basso Mouse Scale (BMS) for locomotion and Rotarod treadmill experiments. Histological analysis revealed that recombinant IL-4 significantly stimulates axon regrowth in the lesioned spinal cord in vivo. Surprisingly, immediate local administration of recombinant IL-13 worsens clinical outcome after SCI.
In summary, our results indicate that acute treatment with recombinant IL-4 significantly promotes functional recovery after SCI, while IL-13 exerts opposing effects and thus impairs clinical outcome after SCI in vivo although both cytokines share the IL-4Ra.
The role of fibroblast growth factor-9 in chronically demyelinated Multiple Sclerosis lesions Multiple sclerosis (MS) is the leading cause of non-traumatic neurological disability among young adults in North America and Western Europe. The pathological hallmark of this inflammatory demyelinating disease is the formation of chronically demyelinated plaques of gliotic scar tissue in the central nervous system (CNS). Why these lesions fail to remyelinate is unclear, as endogenous repair mechanisms normally act to ensure rapid and essentially complete remyelination in the CNS. Several reports suggest that failure of remyelination in MS may be due over-or under-expression of factors influencing oligodendrocyte progenitor cell (OPC) migration and/or differentiation. Experimental studies have identified numerous potential candidates that may affect OPC function in MS, but those that are clinically relevant remains unclear.
A low-density microarray study investigating growth factors, cytokines and their receptors in MS lesions (developed and performed at the Max Planck Institute for Neuroimmunology-Mohan and Meinl), revealed an association between the failure of remyelination and an up-regulation of FGF9 expression. To investigate the functional significance of this observation we treated myelinating CNS cultures with exogenous FGF9. In this experimental setting, FGF9 inhibited the ability of mature oligodendrocytes to myelinate axons in a dose dependent manner; an effect associated with a corresponding decrease in transcripts encoding myelin proteins and myelin related transcription factors. However, a genomewide microarray analysis revealed that in addition to this negative effect on myelination, FGF9 also significantly enhanced expression of genes implicated in neuroprotection and neuronal survival. These account for 15% of the hundred genes most highly up-regulated by FGF9. We hypothesize that FGF9 mediated neuroprotective effects contribute to the survival of axons that traverse persistently demyelinated gliotic plaques in MS, albeit at the expense of impairing remyelination. Understanding the molecular basis of these diverse effects may lead to improved neuroprotective therapies for MS. Japanese encephalitis (JE) is a leading form of viral encephalitis in the world and is caused by the Japanese encephalitis virus (JEV) which belongs to the family flaviviridae. Evasion of peripheral immune system facilitates the entry of the virus into the central nervous system (CNS) where is causes neuronal death that results from the inflammatory milieu generated in the CNS due to microglial activation. Peripheral macrophages constitute an important part of the immune surveillance. It has been proposed that after entry into the body, the virus may be carried into the CNS by peripheral immune cells such as the macrophages, which acts as 'Trojan horses'. JEV has been shown to persist within macrophages long enough to be carried into the CNS. We have shown earlier that the blood-brain barrier is critically compromised in JE which results in massive infiltration of peripheral cells in the CNS. However, it is unclear whether these accumulating virus-infected peripheral macrophages in CNS, can also contribute to death of potentially viable neurons.
Using peritoneal macrophages as a model of peripheral macrophage, we have shown that upon infection with JEV, there is significantly increased release of proinflammatory cytokines from them, as well as reactive oxygen and nitrogen species. The expression of transcription factors associated with the proinflammatory cytokine gene expression, are also found to be elevated in macrophages following JEV-infection. On addition of the culture media from the JEV-infected macrophages on primary cortical neurons, significantly reduced survivality was observed, when compared to neurons on which media from mockinfected macrophages were added. Annexin-PI staining showed that the numbers of necrotic and late apoptotic neuronal cells were also increased along with the levels of pro-apoptotic markers. Addition of specific inhibitors (N-acetyl cysteine, celecoxib and aminoguanidine) to the macrophages post JEV infection, led to reduction in the level of the proinflammatory soluble factors from the macrophages. Addition of this conditioned media from macrophages on neurons resulted in significantly higher survivality.
Thus it seems possible that JEV-infected macrophages add to the bystander death of neurons in JE that results from the activation of microglia, the resident macrophages of the CNS. Activated microglia and infiltrating immune cells are prominent pathological features in amyotrophic lateral sclerosis (ALS). In transgenic mSOD1 mice, wild-type microglia are more neuroprotective than mSOD1 microglia, and the absence of CD4+ T-cells, is associated with faster disease progression, shortened survival, and increased spinal cord expression of markers of microglial activation (NOX2 and TNF-a). Transplantation of bone marrow restores CD4+ T cells, attenuates microglial activation, and slows disease progression. These data suggest that CD4+ T-cells may be neuroprotective by suppressing injurious microglia. Our goal was to define the specific subpopulation of CD4+ T-cells involved and the potential mechanisms for attenuating the microglial-mediated toxicity.
Using flow cytometry and RT-PCR, CD4+CD25+FoxP3+ T-cells were increased in blood, lymph nodes, and spinal cords during early stages of disease and significantly decreased during late stages of disease in mSOD1 transgenic mice. Passive transfer of these early stage CD4+CD25+ T-cells effectively slowed disease progression and prolonged survival of mSOD1/ RAG2 −/− mice. Microglia in vivo as well as those isolated from spinal cord during early stages manifested hallmarks of the neuroprotective M2 phenotype using RT-PCR, but the balance shifted to a toxic M1 phenotype in microglia in vivo as well as those isolated during the rapid progression phase of disease. In vitro, early stage mSOD1 CD4+CD25+ Treg-cells, isolated from spleen and lymph nodes of mSOD1 mice and cultured with adult SOD1 microglia, suppressed the expression of the M1 marker NOX2, while mSOD1 CD4+CD25−(Teff)-cells had minimal effects. The in vitro suppressive effect of mSOD1 Treg-cells was mediated by IL-4 and was blocked by IL-4+ AB.
CD4+CD25+ T-cells present during the early stable phase are effective in slowing disease progression, possibly by secreting IL-4, thereby maintaining an M2 neuroprotective microglial phenotype. The marked decrease of Treg cells in late disease, is associated with a more injurious microglial M1 phenotype. These results suggest that mSOD1 Treg cells may sustain an M2 microglial phenotype and thereby provide motoneuron protection. The use of CD4+CD25+ regulatory T-cells to enhance microglial neuroprotection may offer a novel therapeutic option for ALS.
A critical pathogenic aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS), which induce inflammation, demyelination and axonal damage causing neurological deficits. Current therapies for MS reduce cell infiltration or induce immunosuppression but additionally disturb the immune balance, illustrating the need for a locally acting drug to treat MS. Tissue Transglutaminase (TG2) is an enzyme that is ubiquitously expressed and becomes locally active upon inflammation. TG2 is known to act as a co-receptor for βintegrin subunits and is involved in cytoskeletal remodelling. This enzyme could be of interest in immune cell adhesion and migration processes occuring during MS. We therefore performed studies to determine the possible role of TG2 activity in some of the cellular processes contributing to MS.
We demonstrate that during MS and its animal model chronic relapsing experimental autoimmune encephalomyelitis (cr-EAE) TG2 levels are significantly increased. Reduction of TG2 activity by a specific inhibitor, KCC009, in cr-EAE animals dramatically attenuated clinical deficits. Neuropathologically, KCC009 treatment clearly reduced infiltration of MHC II positive cells. These monocytes resided in the blood vessel lumen and perivascular space, but did not migrate into the CNS tissue parenchyma. Additional in vitro experiments supported the role of TG2 in monocyte adhesion and migration. The reduced migration of monocytes into the CNS is due to attenuated monocytic cytoskeletal flexibility and RhoA GTPase activity. As a possible consequence of the reduced number of activated monocytes present in the CNS, a reduction in TNFa and NOS2 expression in CNS but not in spleen of KCC009 treated cr-EAE animals was found. This could explain the attenuated demyelination as observed in KCC009 treated cr-EAE animals. Interestingly, the expression levels of various other inflammatory genes were not affected in either CNS or spleen compared to untreated cr-EAE animals.
We conclude that selectively blocking monocyte migration into the CNS, but not monocyte adhesion to brain endothelium by inhibition of TG2 activity results in less demyelination and opens new avenues for therapeutic intervention in MS, which are seemingly not complicated by disturbance of the peripheral cytokine response.
A new player in neuroinflammation CRMP2 is critical for T lymphocyte motility and emerges as a potential peripheral indicator of inflammation
Lymphocyte motility is crucial for tissue invasion during neuroinflammation and is intimately regulated by chemoattractant including chemokines. We have identified the phosphoprotein CRMP2 in immune cells and demonstrated its role in T-lymphocyte polarization and migration (Vincent et al., JI 2005) . CRMP2 level expression was elevated in blood immune cells of patients suffering from retrovirus-induced neuroinflammation and also in lymph node-and brain-isolated immune cells of mice infected with neurotropic viruses (Vuaillat et al., JNI 2007) . This led us to investigate the mechanisms underlying CRMP2 function in immune cell motility and the potentiality of CRMP2 to be a marker of neuroinflammation.
In contrast to neural cells where CRMP2 responds to semaphorin signal, CRMP2 appeared a novel transducer of chemokine signal in T-Workshops lymphocyte. The chemokine CXCL12/SDF1 induced a dynamic relocalization of CRMP2 at uropod, the flexible structure of motile lymphocyte and increased its binding to the cytoskeleton protein vimentin. As mutation strategy in T lymphocyte showed the crucial role of CRMP2 phosphorylation in protein function, we examined the CRMP2 phosphorylation status under chemokine signal. SDF1 modified the balance between the GSK-3β-targeted site CRMP2-Thr509 and the Yes-targeted site CRMP2-Tyr479 site. Y479F mutation strongly reduced T-lymphocyte motility, evidencing the functional importance of this newly reported CRMP2 phosphosite. Investigation of CRMP2 in blood immune cells of healthy controls and multiple sclerosis patients showed that CRMP2 was diversely expressed in lymphocytes, natural killers and monocyte subtypes. An antibody directed against the pCRMP2-Y479 form showed its upregulation in activated (HLADR+) T lymphocyte and its specific presence in divers5 neuroinflammatory situations including multiple sclerosis.
To conclude, T-lymphocyte migration that requires the integration of several processes, also involves the phosphoprotein CRMP2, downstream the SDF1 chemokine signal. Our data also suggest that CRMP2 modulation by chemokine of brain origin could be crucial in the diver's steps leading to immune cell neuroinvasion during neuroinflammation.
Infiltration of blood-derived monocytes is required for disease progress in experimental autoimmune encephalomyelitis (EAE)
Microglia, the principal immune cells of the Central Nervous System (CNS), are exquisitely sensitive to CNS injury and disease, fueling a reactive-state called microgliosis. We have used parabiosis, as surgical procedure that allows the creations of peripheral blood chimeras without transplantation, to show that unlike most other tissue-resident macrophages that rely on circulating blood-borne precursors for their replacement, microglia are capable of selfrenewal within the CNS. We also showed that blood-born monocytes are excluded from the CNS in murine models of denervation or neurodegeneration (Ajami et al., Nature Neuroscience 2007) .
In contrast, certain inflammatory pathological conditions such as multiple sclerosis and its murine model, experimental autoimmune encephalomyelitis (EAE), are known to lead to the recruitment of inflammatory monocytes to the CNS.
Is the entry of these cells, normally excluded from the CNS, a causal factor in disease progression?
To address this question we developed a new experimental model, based on parabiosis and differential bone marrow irradiation, which allows the precise distinction between peripheral blood-derived monocyte/macrophages and resident microglia, thus enabling us to investigate the kinetics of microglia activation, blood born monocytes entry in the CNS and their differentiation into macrophages during EAE progression.
Our data reveals a dynamic interplay between macrophages and microglia and strongly supports a causal link between myelomonocytic cell invasion and disease progression. Indeed, specifically blocking circulating inflammatory monocytes from entering the CNS prevented progression beyond disease stage 1.5 in our experiments. Finally, we were able to demonstrate that the presence of blood-borne cells in the CNS parenchyma is transient and that even when they successfully invade the CNS, these cells do not contribute to the microglial pool. In summary, our data identified the invasion of circulating monocytes into the CNS parenchyma as a major adverse event in EAE progression, supporting therapeutic strategies specifi-cally aimed at inhibiting the migration of myelomonocytic cells rather than that of leukocytes in general.
Petermann Franziska ⁎ ,3 , Rothhammer Veit 3 , Claussen Malte C. 3 , Haas Jan D. 4 Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated autoimmune disease that serves as an animal model for multiple sclerosis. Classically, EAE has been thought to be mediated by CD4+ MHC class II-restricted T cells that are reactive to central nervous system (CNS)-derived autoantigens. However, recently, there has been much evidence suggesting that non-conventional gd T cells (T-gd) may also contribute to the pathogenesis of EAE because a subset of T-gd constitutively expresses the IL-23 receptor (IL-23R) and responds vigorously to IL-23 by producing inflammatory cytokines.
Here, we used an IL-23R (GFP) reporter mouse approach to investigate the role of IL-23R + T-gd in EAE in vivo. Upon immunization with MOG35-55 emulsified in CFA, IL-23R + T-gd expand in the secondary lymphoid tissue and accumulate in the CNS at the peak of disease. Interestingly, control of myelin antigen specific CD4+ T cells by regulatory T cells (Tregs) in the CNS is only operative after contraction of the IL-23R + T-gd population suggesting that T-gd inhibit Treg responses. Indeed, supernatant from IL-23-activated T-gd abrogates Treg mediated suppression of effector T cells. Moreover, IL-23-activated T-gd are extremely potent in producing factors that inhibit TGF-driven conversion of conventional T cells into Foxp3+ Tregs in vitro and in vivo. As a consequence, T cell receptor delta (Tcrd) KO mice, which lack Tgd cells, exhibit exaggerated Treg responses that attenuate the development of EAE. Depletion of Tregs in Tcrd KO mice restores full susceptiblity to EAE in these animals.
In conclusion, we provide evidence that IL-23 arms T-gd to inhibit Treg responses. Thus, the fast kinetics of T-gd responses to IL-23 creates an environment in which productive adaptive T cell responses can unfold in an uninhibited manner. These results point to IL-23 driven pathogenic properties of T-gd that might be particularly relevant at epithelial surfaces and in neuroectodermal tissue.
We recently identified a gene expression signature in CD4+ T cells of individuals with clinically isolated syndrome (CIS) that highly correlated with a rapid progression to MS. This signature included upregulation of genes that promote T cell activation as well as downregulation of genes that promote quiescence. Among these, the antiproliferative gene TOB1 was 7-fold downregulated. We then hypothesized that Tob1 deficient mice will show an earlier EAE onset, a more severe phenotype, or both.
In this study, the immunological and neurodevelopmental properties of Tob1 −/− mice were characterized. Tob1 −/− T cells proliferated more, and expressed more IL-17 and lL-23 than their WT counterparts. Tob1 −/− mice experienced both an earlier onset and a more severe EAE. Analysis of motoneuron function and apoptosis suggest the earlier onset is associated with neural toxicity in the Tob1 −/− animals. To evaluate a potentially independent role for Tob1 in the CNS, we measured its expression during myelination by in-situ hybridizations and immunostaining for myelin antigens. Interestingly, Tob1 −/− mice showed a significant delay in spinal cord myelination at P5 when compared to WT mice. Finally, we found that the HDAC inhibitor TSA (which increases Tob1 expression by N10-fold in vitro) ameliorated EAE. Strikingly, Tob1 −/− mice with EAE showed no improvement after TSA administration, suggesting that Tob1 expression is required for the beneficial effects of TSA. We confirmed these results with in-vitro experiments.
Our results suggest a dual role of Tob1 in the CNS and the periphery. We found that while Tob1 is required for keeping quiescence in T cells in the periphery, it may also be critical for oligodendrocytes to (re) myelinate axons in a timely manner. Tob1 is required for TSA to exert its beneficial effect in EAE and to suppress T cell proliferation in vitro, thus a potential therapeutic target in EAE and MS.
and thus play an important role in maintaining the balance between immunity and tolerance. In this study, we investigated the phenotype and T-cell stimulatory capacity of standard IL-4 immature DC (IL4-iDC) and IL-10-modulated immature DC (IL10-iDC) with respect to antigen-specific production of cytokines and expression of regulatory T-cell markers.
We observed a diminished expression of CD86 on IL10-iDC compared to IL4-iDC, indicative of a more pronounced immature phenotype. Both IL4-iDC and IL10-iDC suppressed CMV-specific IFN-production of CMV pp65-stimulated lymphocytes compared to stimulated lymphocytes alone. More importantly, antigen-specific IFN-production by autologous lymphocytes to myelin-associated antigens (MBP/MOG) was diminished after coculture with IL4-iDC as well as with IL10-iDC. We could not demonstrate a difference in suppressive potential between IL4-iDC and IL10-iDC; neither did we observe a difference in their capacity to induce Tregs. However when iDC were subjected to an inflammatory cytokinecocktail, IL10-iDC were less prone to upregulate maturation markers, such as CD80, CD86 and HLA-DR as compared to IL4-iDC, indicating a more stable immature DC phenotype.
Currently, the use of immature DC as a therapeutic intervention for down-modulation of exuberant immune responses, e.g. in diabetes and in graft-versus-host disease is being investigated. In this study, we demonstrated that both IL4-iDC and IL10-iDC are effective in suppressing viral immune responses as well as MS-associated pathogenic responses. However, IL10-iDC might be more interesting for clinical use since the tolerogenic phenotype of IL10-iDC is more stable compared to IL4-iDC.
Establish tolerance in multiple sclerosis (ETIMS), an investigatorinitiated cell therapy for early multiple sclerosis: Putative in vitro mechanisms of action Multiple sclerosis (MS) is a devastating autoimmune inflammatory disease of the brain and spinal cord. Current therapies for MS inhibit the autoimmune response in a non-specific manner, are only moderately effective and can have significant side effects. We adopted a very promising tolerization strategy that employs autologous peptide-coupled antigen presenting cells as tolerogen. This therapy has proven excellent efficacy in animal models of MS and different T cell-mediated autoimmune diseases. We have started to apply this tolerization regimen in MS patients and to assess its safety and efficacy in an open label MRI-controlled phase I/IIa clinical trial.
Our aims are to analyse the in-vitro mechanisms of action of a novel tolerization strategy using autologous antigen-coupled, fixed antigen-presenting cells.
In-vitro mechanisms of action of EDC-treated cells (tPBMC) were analysed in PBMC from healthy donors. Inhibitory capacity of tPBMC on T cells was analysed using a T cell clone obtained from an MS patient. Proliferative capacity of T cells was assessed by thymidine incorporation or CFSE dilution assays. We analysed the effect of tPBMC on the phenotype, maturation and cytokine expression of dendritic cells or monocytes.
We have proven the capacity of EDC to couple peptides to the surface of the cells. Our data suggest that the peptide is bound unspecifically to the surface of cells. Besides the coupling reaction, treatment of PBMC with EDC leads to caspase activation and apoptotic cell death. Using a well characterized T cell clone we demonstrated that tPBMC are effective in inhibiting T cell proliferation and that this inhibition is doseand cell-cell contact-dependent. We find that the presence of tPBMC affects the maturation of dendritic cells and monocytes/macrophages, as measured by the expression of CD83, CD80 and CD86. Dendritic cells and monocytes incubated in the presence of tPBMC produce considerable amounts of transforming-growth-factor-β (TGF-beta).
Tolerance by EDC-coupled cells involves several direct and indirect mechanisms of action including caspase-dependent apoptotic cell death and uptake of dead cells by antigen presenting cells. Plasmacytoid dendritic cells (pDC) are emerging as a possible therapeutic tool within the complex immunomodulatory network in autoimmune diseases. In contrast to classical DC, pDC may not be essential for initiating immune responses, but crucial for regulating severity, extent and quality of autoimmune diseases. This study provides strong evidence that preventive and therapeutic adoptive transfer of PDCA-1+ cells has the potential to modulate EAE in C57Bl/6 mice, focusing on the properties of two distinct PDCA-1+ pDC subsets, PDCA-1+CD4− and PDCA-1+ CD4+ pDC.
Both subpopulations are characterized by a differentiated expression pattern of PDCA-1 and an antigen, recognized by the specific interferon type I secreting pDC antibody PDC/IPC (120G8). Additionally, the abundance of typical pDC markers differ: In comparison to PDCA-1+CD4+ pDC, PDCA-1+CD4− pDC express more B220, whereas expression of Ly6c and SiglecH, a lectin exclusively found on pDC or specific interferon type I secreting pDC (IPC), are decreased. Furthermore, the expression of costimulatory molecules like, CD80, CD273, CD86 and MHC class II are reduced indicating a less activated state of PDCA-1+CD4− pDC.
In vivo experiments reveal that preventive or therapeutic intravenous injections of PDCA-1+CD4− pDC ameliorate EAE more effectively than PDCA-1+CD4+ pDC. In vitro mixed cultures demonstrate a lack of proliferation of MOG-specific responder T cells in the presence of PDCA-1+CD4− pDC in contrast to PDCA-1+CD4+ pDC. Adding anti-FasL abrogates this unresponsiveness, whereas adding neither anti-CD40 nor anti-ICOSL displays any effect. However, the addition of the pDCstimulating TLR 9 ligand, CpG, results in an overall inhibition of proliferation, maybe due to the induction of IL-12p40 and IL-10 secretion. Furthermore, since the apoptosis and necrosis rates are comparable in both settings, cell death may not explain the decrease of potentially encephalitogenic T cells.
Thus, this study rather leads to the conclusion that in EAE the immunomodulatory function of PDCA-1+CD4− pDC is associated with regulatory T cells as well as reducing proinflammatory conditions. Investigating subpopulations of pDC may increase the efficacy of the therapeutic potential.
Supported by BMBF 01 GZ 0708.
Weber Clinical data indicate that anti-CD20 B cell depletion may be effective in treatment of multiple sclerosis (MS). Our goals were to investigate the influence of B cells on T cell activation and differentiation in EAE, and the mechanisms of immune modulation induced by CD20 B cell depletion. We examined two murine EAE models, one in which B cells participate in EAE pathogenesis, and one that does not require B cells for EAE induction.
EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), or MOG peptide (p)35-55, a B cell-independent EAE model. Immunization with MOG protein promoted expansion of activated B cells. When serving as antigen presenting cells (APC), these activated B cells efficiently presented MOG protein or MOG peptide to naive MOG p35-55-specific T cells, and promoted Th1 and Th17 differentiation. CD20 B cell depletion prevented or reversed established MOG protein-induced EAE, which was associated with less CNS inflammation, elimination of meningeal B cells, and reduction of MOG-specific Th1 and Th17 cells. In contrast, B cells from mice immunized with MOG p35-55 did not become activated, and similar to naive B cells, they could present MOG peptide, but not MOG protein, to MOG p35-55-specific T cells. Further, although MOG p35-55 binds MHC II directly on unactivated or naive B cells, when used as APC, these B cells did not efficiently promote Th1 or Th17 differentiation. In EAE induced by MOG p35-55, anti-CD20 treatment exacerbated EAE, and did not alter development of Th1 or Th17 cells. Irrespective of the EAE model used, B cell depletion reduced the frequency of CD25 + FoxP3 + regulatory T cells (Treg). After CD20 depletion, remaining myeloid (CD11b + ) APC produced more TNF and less IL-10, and when serving as APC promoted development of encephalitogenic T cells. These findings suggest that B cells may regulate other APC.
Our study highlights distinct roles for B cells in pathogenesis and regulation of CNS autoimmune disease. Activated MOG-specific B cells, but not naive or unactivated B cells, are efficient APC, and are capable of inducing differentiation of proinflammatory myelinspecific T cells. The clinical benefit observed from CD20 depletion in MOG protein-induced EAE, may relate primarily to the elimination of proinflammatory B cell APC function. However, in certain clinical settings, elimination of unactivated B cells, which participate in regulation of T cells and other APC, may be undesirable. Multifocal motor neuropathy (MMN) is an autoimmune disorder of peripheral nerves causing muscle weakness and wasting. Intravenous immunoglobulin (IVIg) is the standard treatment; however, its effect is short-lived and does not halt long term progression. Trials of other immunomodulatory agents have shown no benefit to date. MMN is known to be an antibody-mediated disease; up to 80% of patients are positive for anti-GM1 glycolipid antibody. Animal models of anti-GM1 antibody-mediated neuropathy require complement activation for neural destruction. Complete inhibition of nerve damage is displayed when terminal complement activation is inhibited by Eculizumab, a humanised monoclonal antibody which specifically prevents cleavage of C5 and thus terminal complement activation. Eculizumab is licensed to treat paroxysmal nocturnal haemoglobinuria (PNH). Trials of Eculizumab in other autoimmune conditions are ongoing; however, no trial has yet been conducted in neurological disease, or during an ongoing IVIg treatment.
In this trial, 13 patients with MMN were given Eculizumab for 14 weeks. The primary outcome was the safety of Eculizumab in this group concurrently receiving IVIg. The major secondary outcome was a change in IVIg dosing frequency. At monthly treatment intervals, subjects were assessed for other efficacy measures, including MRC grade, dynamometry and 9-hole peg test, and subjective scorings. Electrophysiology was performed at baseline and at the end of the treatment period.
Treatment emergent signs and symptoms were highest during the first four weeks of treatment; the most common was headache, accounting for 33% of all adverse events. Headache is also a common side effect of IVIg treatment. No patient discontinued treatment due to adverse event. We have shown that Eculizumab can be safely used in conjunction with IVIg.
During the treatment period, 9 out of 10 patients on IVIg maintenance continued to require doses of IVIg. In two patients the intertreatment interval was lengthened by 6-7 days. Half of the group were subjective responders, including 2 of the 3 patients who were not receiving IVIg. There were some individual improvements in clinical and electrophysiological parameters, but as a group, no significant overall trend towards sustained improvement.
The use of complement inhibitors in treating immune mediated neuropathies merits further investigation; however, they may be more efficacious in the hyperacute phase of these disorders. Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). While the majority of currently used MS medications target the inflammatory aspects of the disease, treatments to induce remyelination are lacking. The multiple nature of neuroinflammation is being increasingly recognized, where inflammatory cells and molecules on the one hand can damage neural elements, but where beneficial aspects of neuroinflammation that can promote repair also occur. The challenge of harnessing beneficial aspects of neuroinflammation is to avoid its potential detrimental consequences. Given the increasing evidence that activated monocytoid cells (macrophages and microglia) have roles in CNS repair, we have tested the hypothesis that medications that stimulate monocytoid cells can be used to stimulate remyelination.
We sought first to identify medications that could stimulate monocytoid cells. Using cytokine production by human microglia as the initial screen, we tested a collection of 1040 compounds already in clinical use for various indications and found a single medication, Amphotericin B (AmpB), as a microglia activator. To facilitate the transition to a mouse model in vivo, we tested and found that AmpB similarly increased the activity of macrophages cultured from the bone marrow of mice; activity was determined by expression of toll-like receptors (TLRs), scavenger receptors and cytokines. Moreover, the increase in activity of cultured mouse macrophages by AmpB was enhanced by simultaneous treatment with another agent known to have effects on monocytoid cells: macrophage-colony stimulating factor (M-CSF). We next produced demyelination of the mouse spinal cord by the local injection of the lipid-disrupting agent lysolecithin into the dorsal column of the T3/T4 region. Three days after, significant demyelination in the spinal cord was accompanied by elevation of several markers of monocytoid activity. Importantly, the systemic injections of the combination of AmpB and MCSF further elevated these monocytoid markers. Moreover, when tissue was processed 28 days after the lysolecithin insult for lesion volume and G-ratio analyses, we found that the combination treatment of AmpB and M-CSF improved remyelination in comparison to single or control treatment.
Our results support the concept that there are benefits of neuroinflammation, and they show that the stimulation of monocytoid cells by Amphotericin B and M-CSF enhances remyelination. Inclusion body myositis (IBM) is the most common acquired myopathy beyond age 50. A histological hallmark is non-necrotic myofibers that are attacked by CD8+ T-cells. In contrast to myofibers from healthy individuals, both attacked and non-attacked myofibers of patients with IBM express high levels of human leukocyte antigen HLA class-I (HLA-I) molecules. HLA-I expression is a prerequisite for antigen presentation to CD8+ T cells, but only a subgroup of HLA-I+ myofibers is attacked while others are spared. To identify changes that are specific to attacked myofibers, we applied laser microdissection to separately analyse a) healthy control myofibers, b) non-attacked IBM myofibers, and c) attacked IBM myofibers.
By immunohistochemistry (IHC) we classified myofibers from five patients with IBM as attacked or non-attacked depending on the presence of adjoining CD8+ T cells. We then microdissected the intracellular contents of healthy control myofibers, non-attacked myofibers, and attacked myofibers. Pools of microdissected material from the three different groups were analysed by microarray hybridization and quantitative PCR. HLA-I upregulation was present in attacked and non-attacked IBM myofibers, whereas healthy control fibers were negative for HLA-I. In contrast, the inducible chain of the IFN-g Receptor (IFNGR2), and a number of IFN-g-induced genes were upregulated in attacked myofibers compared to nonattacked fibers and healthy controls. IHC verified IFNGR2 expression on the protein level. Confocal microscopy showed segmental IFNGR2 upregulation on the membranes of attacked myofibers, which positively correlated with the number of adjacent CD8+ T-cells.
Our results suggest an "amplification loop hypothesis" of inflammation in IBM muscle. First, an unknown etiological factor leads to ubiquitous upregulation of HLA-class I in all myofibers. Next, a CD8+ T cell recognizes an unknown antigen displayed on the surface of a HLA-I + myofiber. Initially this is a rare chance event. Subsequently, activated immune cells produce proinflammatory cytokines which induce the local expression of IFNGR2 as well as major downstream effectors of IFN-g signalling pathways by the attacked myofibers. Thus, the attacked fibers are stimulated to release factors attracting more inflammatory cells, which further amplify the focal inflammatory changes. Multiple sclerosis (MS) is a chronic inflammatory response against constituents of the central nervous system. It is known that regulatory T cells (Tregs) play a key role in autoimmune balance and their improper function may facilitate the expansion of autoaggressive T cell clones. Recently, microRNAs (miRNAs) have been involved in autoimmune disorders and their loss-of-function in immune cells was shown to facilitate systemic autoimmune disorders. Here, we analyzed the miRNA expression profile in Tregs from MS-RR.
We assessed miRNA genome-wide expression profile by microarray analysis on CD4+CD25+high T regulatory cells from 12 MS relapsingremitting patients in stable condition and 14 healthy controls. In order to validate these results, we are performing a quantitative RT-PCR on CD4+CD25+high CD127dim/− cells. We found 23 human miRNAs differentially expressed between Treg CD4+CD25+high cells from MS patients versus healthy donors. Among the deregulated miRNAs, members of the miR-106b-25 cluster were found up-regulated in MS patients when compared to healthy donors. miR-106b and miR-25 were previously shown to modulate the TGF-β signaling pathway through their action on CDKN1A/p21 and BCL2L11/Bim. TGF-β is involved in T regulatory cells's differentiation and maturation. Therefore, the up-regulation of this miRNA cluster may alter CD4+CD25+high T regulatory cells activity in course of MS, by blocking TGF-β biological functions. Functional type I interferon (IFN) signaling is critical for the host response to viruses. Cellular responses to type I IFNs depend largely on STAT1, STAT2 and IRF9. Here we studied the effects of IRF9deficiency on the host response to a viral infection in the CNS.
Wild-type (WT) mice and mice lacking IRF9 (IRF9 KO) were infected intracranially with lymphocytic choriomeningitis virus (LCMV). In WT mice, a lethal lymphocytic choriomeningitis (LCM) occurred by day 7 with characteristic cerebral seizures and LCMV being largely confined to the CNS. In contrast, LCMV-infection of IRF9 KO mice caused a transient non-fatal clinical disease and virus spread to peripheral organs. Viral RNA levels decreased slowly over time and became undetectable in some peripheral organs such as liver and spleen but remained detectable in the CNS for more than 150 days. In the CNS, sites of viral infection were associated with foci of activated microglia/macrophages, multi-nucleated giant cells and moderate Tcell infiltrates. The presence of virus and immune pathology in the CNS and peripheral organs was paralleled by significantly increased expression of various cytokine mRNAs, including IFN-gamma and TNF, as well as of the co-inhibitory molecule B7-H1 (PD-L1 or CD274) mRNA.
In conclusion, these findings indicate that the absence of IRF9 prevents lethal LCM but results in persistent infection and chronic inflammation in the CNS. The presence of T-cells and the slow decrease in viral RNA levels in the CNS and peripheral organs argue for a retarded rather than an exhausted or incapacitated anti-viral response.
Tregs with a subsequent alteration of the disease course. Information about human leukocyte E (HLA-E) restricted CD8+ T cells is limited. This study aims at elucidating the involvement of HLA-E restricted CD8+ T cells in immunoregulatory alterations observed in MS.
First, the regulation of HLA-E surface expression on different immune subsets was studied. While HLA-E expression was transiently upregulated on all cell types upon polyclonal activation, B cells exhibited a significantly higher upregulation in MS patients compared to healthy controls (p b 0.01). In addition, pro-inflammatory cytokines (IL-12, IFN-and IL-27) but not anti-inflammatory cytokines (IL-4 and IL-10) induced increased HLA-E expression on the cell surface of CD4 T cells. These findings indicate that activation of immune cells in proinflammatory conditions such as MS upregulates HLA-E. In a second part, an MS association study of HLA-E polymorphisms was performed in HC (n = 1078) and MS patients (n = 832). Plink option analysis identified a significantly higher frequency of the GC haplotype in MS patients compared to HC (p = 0.04). Conditioning for DRB1*1501 revealed that this association merely reflected the well described effect of DRB1*1501 on MS susceptibility. Still, MS patients carrying the GC haplotype showed a significantly lower (p b 0.05) potential to upregulate HLA-E surface expression on T and B cells after activation. In a final part, HLA-E restricted CD8 T-cells were characterized based on the expression of its major ligands namely CD94/NKG2A and CD94/NKG2C. While the number of NKG2A+CD8+ T cells was not different between HC and MS patients, the frequency of NKG2C+CD8+ T cells was significantly lower in MS patients (p b 0.05). Moreover, regulatory markers Foxp3 and CD122 were significantly down regulated (p b 0.05) in NKG2C+CD8+ T cells of MS patients compared to controls. Co-culture experiments with CD4 T cells will point out whether these phenotypical differences correlate to the suppressive capacity of NKG2C+ and 2A+ CD8+ T cells.
This study identified immunological alterations in HLA-E restricted CD8 T cells in MS. The functional consequences of this and the actual role in the disease process needs to be further investigated.
Kawachi Izumi ⁎ , Toyoshima Yasuko, Yanagawa Kaori, Saji Etsuji, Kakita Akiyoshi, Takahashi Hitoshi, Nishizawa Masatoyo Brain Research Institute, Niigata University, Niigata, Japan Neuromyelitis optica (NMO) is a demyelinating syndrome characterized by myelitis and optic neuritis. A crucial role for humoral immunity in the NMO pathogenesis is suggested by the detection of a highly specific serum autoantibody NMO immunoglobulin G that binds to aquaporin-4 (AQP4) water channels, and the pronounced deposition of immunoglobulins colocalizing with products of complement activation in a vasculocentric pattern in NMO lesions. However, levels of several cytokines such as interleukin (IL)-17, IL-8, IL-6, and IL-1β are increased in the cerebrospinal fluid of human NMO patients, and the injection of human AQP4 antibodies in a rodent model with T-cell-mediated brain inflammation induces acute NMO lesions. These findings indicate that the cellular immune response may play a role in induction and effecter phase of NMO lesions in combination with humoral element, but the detailed immunopathologic features of cellular elements in NMO remain elusive. Therefore, we investigated the humoral and cellular immune responses in NMO patients in clinical, immunologic, and pathologic studies.
Radiologic finding from 17 patients with NMO and pathologic findings from 7 autopsied cases of NMO demonstrated the graymatter-predominant lesions as well as subpial white-matter-predominant lesions with longitudinal extension in the spinal cord. Moreover, we confirmed that NMO lesions were accompanied by infiltration of immune cells such as CD45RO+ or CD20+ cells in the leptomeningeal membrane and around the radial vessels in pathologic studies. B-cell follicles were not detected in the meninges. This infiltrating pattern of lymphocytes in NMO cord lesions might be similar to the rodent model of T-cell-mediated experimental autoimmune encephalomyelitis, in which myelin antigen-specific T cells are arrested in the leptomeningeal vessels, immediately monitor the luminal surface, continue to scan the abluminal vascular surface and the underlying leptomeningeal membrane, encounter phagocytes that effectively present antigens, produce inflammatory mediators, and provide the formation of inflammatory infiltrates. These cellular elements in patients with NMO might aid B cells in AQP4 antibody production, and break the blood-brain barrier due to the access of AQP4 antibodies to the extracellular domain of AQP4 at the astrocytic foot process.
We suspect that not only humoral but also cellular immune response might play a critical role in the NMO pathogenesis.
Oestradiol enhances perforin expression on human regulatory T cells. Implications for multiple sclerosis Teijeiro Roseta 1 , Valor Larissa 1 , Faure Florence 2 , De Andrés Clara 3 , Tejera-Alhambra Marta 1 , Alonso Bárbara 1 , Fernández-Cruz Eduardo 1 , Sánchez-Ramón Silvia ⁎ ,1 1 Dept. Immunology, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 2 INSERM U932, Institut Curie, Paris, France; 3 Dept. of Neurology, Hospital General Universitario Gregorio Marañón, Madrid, Spain CD4+CD25+FoxP3+ regulatory T-cells (TReg) suppress immune responses to autoantigens and other diverse antigens, mainly through a cell contact-dependent mechanism not yet fully defined. It has been reported that both human natural and induced TReg exert cytotoxic activity against autologous target cells, which suggests that the perforin/granzyme pathway may be a relevant candidate mechanism for the suppressive function of TReg. During pregnancy, oestradiol (E2) is markedly increased. Previous reports have shown that E2 modulates TReg percentages and function, and has been proposed as a potential modulator of multiple sclerosis (MS).
Our main goal was to quantify the TReg proportions and perforin expression on TReg in MS and healthy women during pregnancy and menstrual cycle; secondly, to determine the effects of E2 on Treg function and on perforin expression in ex vivo cultures.
We studied 30 MS pregnant, 32 healthy pregnant and 12 MS and 45 healthy non-pregnant women at days 1-3 (D1) of menstrual cycle for Treg and perforin ex vivo expression by multiparametric flowcytometry. We performed suppression experiments on autologous responder T cells (CD4+CD25−) after Treg purification to see the effects of E2 on Treg function. Degranulation assays for CD107a/b expression on Treg were also done.
We found statistically higher proportions of circulating TReg in MS and healthy pregnants compared to MS and healthy non-pregnant women, respectively, at D1: CD4+CD25med (p = 0.02 and p = 0.002, respectively), CD4+CD25high (p = 0.05 and p b 0.0001, respectively) and CD4+CD25+FoxP3+ (NS and p = 0.005, respectively). Expression of perforin on Treg was significantly higher at third trimester of MS healthy pregnants than D1 (p = 0.03 and p = 0.01, respectively). Addition of E2 to stimulated-responders induced a significantly higher Treg phenotype compared to non-stimulated responders (p = 0.001) and TCR stimulated responders alone (p = 0.002). TReg effectively expressed both CD107a and b in a similar way as CD8+ (CTL) cells do.
TRegs are expanded and express higher perforin levels during pregnancy in healthy women and in the setting of MS, although at lower levels in the later ones. We show that E2 in vitro expands TReg, enhances TReg function and induces a TReg phenotype in activated responder T-cells, further increasing the expression of perforin on TReg. We found surface LAMP-1 and -2 expression by TReg, which is a sign of cell degranulation and therefore of cytotoxicity by these cells. Inflammatory myopathies are chronic diseases of the skeletal muscle. Typically, autoaggressive CD8+-T-cells attack MHC class I positive muscle fibers, and destroy the target cells. In most cases, these T-cells are oligoclonally expanded and express alpha-beta-Tcell-receptor (TCR) molecules. In a rare form of polymyositis, the myocytotoxic T-cells express a gamma-delta-TCR (gd-TCR). gd-TCRs recognize their antigenslike antibodiesin an MHC-non-restricted way. In one patient, a monoclonal gd-T cell expansion was observed. Therefore the gd-TCR chains could be cloned easily and were then expressed in a T-hybridoma cell line that lacks endogenous TCR chains. In previous experiments, we found that the gd-TCR recognizes an enigmatic antigen that is expressed not only in human muscle fibers but also in other tissues and species. Therefore we surmised that the gd-TCR might recognize not a defined epitope, but rather an evolutionary conserved motif. We here describe the molecular characterisation of the antigenic motif of this pathogenic gd-TCR.
We found that several proteins associated with the RNA translation apparatus of human cells and of bacteria were recognized. Candidate proteins were coated to microtiter wells, incubated with gd-TCR transfectants, and secreted IL-2 was measured. Strikingly, several tRNA-synthetases, including the well-known myositis-antigen Jo-1, were recognized. Using the small soluble translation initiation factor IF1 from E. coli as paradigm, we showed by site-directed mutagenesis that the gd-TCR recognizes a conformational antigenic motif consisting of four amino acids. The motif is located in an alphahelical segment of a surface-exposed loop. Using gd-TCR transfectants with defined amino acid exchanges at different positions of both TCR chains, we showed that the motif is recognized in a specific, CDR3region dependent way.
The antigenic motif described here represents the first motif of a pathogenic human gd-TCR. It is found in human and microbial proteins. Surprisingly, the motif recognized by our gd-TCR is present on several tRNA-synthetases, which are known to elicit B cell responses in myositis patients. This provides an unexpected link between the pathogenic gd T cell response, which is considered to be related to the innate immune system, and myositis-associated anti-AARS auto-antibodies, which usually have undergone extensive affinity maturation.
Koutrolos Michail ⁎ ,1 , Bartholomaeus Ingo 1 , Kawakami Naoto 1 , Sparwasser Tim 2 , Krishnamoorthy Gurumoorthy 1 , Wekerle Hartmut 1 1 Max Planck Institute of Neurobiology, Martinsried, Germany; 2 Technische Universität München, Munich, Germany CD4+Foxp3+ regulatory (Treg) T cells are shown to play a pivotal role in the control of autoimmune responses. The importance of Treg cells has been extensively studied in actively induced Experimental Autoimmune Encephalomyelitis (EAE) models. In this study, we investigated the functional role of Treg cells in a spontaneous opticospinal EAE mouse model, which develops following interactions between myelin oligodendrocyte glycoprotein (MOG) specific T and B cells.
We used a double-transgenic (TCRMOG × IgHMOG) mouse strain, which spontaneously develops opticospinal EAE ("OSE" mouse) at high frequency. We bred "OSE" mice a) with a transgenic strain expressing a diphtheria toxin (DTx) receptor-eGFP fusion protein (DEREG mice) in Treg cells, allowing selective depletion of Treg cells by DTx injection, and b) with a fluorescent Treg reporter mouse (Foxp3-GFP.KI) which expresses eGFP under the control of Foxp3 promoter to track and visualize Treg cells and their interaction with other immune cells in vivo.
We found that chronic depletion of Treg cells in OSE× DEREG mice treated with DTx, starting before the onset of spontaneous EAE, increased disease incidence and burden. Next, we monitored the frequency of GFP+ Treg cells during the spontaneous EAE in OSE × Foxp3-GFP.KI mice by FACS. We found that in the peripheral immune system the fraction of Treg cells was low and remained stable irrespective of the disease status. However, in clinical EAE, numerous Treg cells accumulated in the CNS infiltrates and their frequency was not altered throughout the entire disease episode. These findings were confirmed by immunocytochemistry. Furthermore, while the proportion of MOG-specific tgTCR-expressing Treg cells was elevated significantly in the CNS compared to the periphery, the fraction of MOG-specific tgTCR-expressing Teff cells was decreased. Initial observations using intravital 2-photon spinal cord imaging suggest that Treg cells accumulate in the perivascular milieu and move with low velocity (5 μm/min) in the meninges of mice with chronic spontaneous EAE.
We show that, in a spontaneous EAE model, the frequency of MOGspecific Treg cells remained stable in periphery but increased abundantly in the CNS, indicating a possible role of these cells within the target tissue. Importantly, depletion of Treg cells before the onset of the disease seems to increase the disease incidence, suggesting a protective role of Treg cells in spontaneous EAE. Effector/memory CD4+ T cells specific for myelin antigens perpetuate central nervous system (CNS) inflammation in multiple sclerosis (MS). After becoming activated in the periphery, these CD4+ T cells cross the blood-brain barrier and enter the unique microenvironment of the CNSan environment designed to limit deleterious inflammation to a primarily non-regenerative organ. Once in the CNS, CD4+ T cells are re-activated and create an inflammatory microenvironment that causes damage to the myelin sheath of axons, leading to clinical neurological impairment. Although significant efforts have described the effects of cytokines on naïve CD4+ T cells, much less is known about their ability to influence antigen-experienced effector T cells. TGF-β, a multi-functioning cytokine present in both the healthy and inflamed CNS, has well-characterized suppressive effects on naïve T cell functions. However, the ability of TGF-β to influence Th1 effector cells is not well defined.
Using myelin-specific TCR transgenic mice in the experimental autoimmune encephalomyelitis (EAE) model, we demonstrate that TGF-β elicits differential effects on naïve versus Th1 effector cells. We show that TGF-β exposure during secondary stimulation of Th1 effector cells enhances cellular activation, proliferation, and cytokine productiona response very different from that seen in naïve CD4+ T cells. Interestingly, these cells exhibit a reduction in their encephalitogenicity when adoptively transferred into naïve wildtype mice. We then demonstrate that TGF-β promotes Th1 self-regulation by inducing a population of IFN-g+IL-10+ cells that are responsible for the reduction in encephalitogenicity. Silencing IL-10 with smallinterfering RNA technology restores disease severity.
These data demonstrate a unique mechanism by which TGF-β is able to reduce the pathogenicity of myelin-specific T cells based on the differentiation state of the target cell. Although semaphorins were initially identified as axonal guidance cues that determine the direction and migration of neurons during neurogenesis, cumulative evidence indicates that they have important functions in immune cell migration. Recently, secreted-type class 3 semaphorins Sema3A and Sema3E were shown to regulate immune cell migration by signaling through their receptors plexinA1 and plexinD1, respectively. Since it is emerging that CD4+ T cells trafficking into CNS play a critical role in the relapses of multiple sclerosis, we investigated the role of Sema3E in the migration of CD4+ T cells during the development of EAE.
We first examined the mRNA expression levels of Sema3E and plexinD1 in effector CD4+ T cells and found that both Sema3E and plexinD1 are markedly increased when naive CD4+ T cells were differentiated into TH17 cells. To investigate the role of Sema3E-plexinD1 interactions in T-cells, we crossed Lck-Cre transgenic mice with plexinD1flox mice and generated T-cell specific plexinD1deficient mice (LckCre+/plexinD1flox/flox). When we performed chemotaxis assays for CD4+ T cell by adding recombinant Sema3E to the upper chambers in the presence of chemokine, CCL21, chemotaxis of wild-type CD4+ T cell was enhanced by addition of Sema3E to the upper chamber, of which effects were canceled in LckCre+/PlexinD1flox/flox mice. When we induced EAE in Sema3E and T-cell specific plexin-D1 deficient mice by immunizing them with MOG-peptides, the clinical onsets of both mutant mice were significantly delayed compared with those of wild-type mice. However the maximum clinical scores of EAE in both mutant mice were not statistically different from those of wild-type mice.
Collectively, these data suggest that Sema3E participates in the pathogenesis of EAE by promoting CD4+ T cell migration into CNS.
Calcium signalling plays a crucial role in T cell activation and proliferation. The calcium response involves the influx of calcium through Orai1/CRACM1 channels located within the plasma membrane. Opening these channels requires continuous Ca2+ release from intracellular pools and is mediated by three different Ca2+ mobilizing second messengers, namely D-myo-inositol 1,4,5-trisphosphate (IP3), cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Due to a lack of biocompatible and specific inhibitors, the functional impact of each distinct Ca2+-mobilizing second messenger is poorly understood. Using a recently developed inhibitor that selectively interferes with NAADP-mediated Ca2+ release, we explored the role of NAADP in autoreactive effector T cells during experimental autoimmune encephalomyelitis (EAE) and neuritis (EAN), animal models for multiple sclerosis and Guillain Barré syndrome, respectively. Molecular analyses revealed that NAADP signaling influences T cell re-activation upon arrival in the nervous tissues: NAADP inhibitor treatment significantly reduced the levels of pro-inflammatory cytokines IFN and IL-17. Live two photon imaging showed that changes in T cell motility go together with these functional changes. In the EAE lesions effector T cells display characteristic locomotion patterns: 35% of the infiltrated T cells are stationary in the phase of antigen recognition whereas the remaining cells (65%) are motile and move with high speed through the CNS tissue. After NAADP inhibitor treatment a higher fraction (N80%) of T cells was motile and their invasive capacity was reduced. Accordingly, clinical symptoms of EAE and EAN were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. Remarkably, naïve and long-lived memory T cells were targeted significantly less by the NAADP inhibitor. The differential susceptibility correlated with the expression of RyR1, the putative receptor for NAADP-triggered Ca2+ signaling in T cells, which was up-regulated in effector T cells compared to that in naïve and memory T cells.
These data indicate that the NAADP/calcium signaling pathway is essential for the recruitment and activation of autoaggressive T-cells within their target organ. Interference with this pathway suppresses autoimmune inflammatory lesion formation and thus might qualify as a novel strategy for the treatment of T cell-mediated autoimmune diseases.
In vivo imaging of lymphocytes in the CNS reveals different behaviours of naive T cells in health and autoimmunity Autoimmune central nervous system (CNS) inflammation is supposed to be mediated by a perivascular accumulation of mononuclear cells preceding the actual infiltration of CNS parenchyma by myelin-specific T cells. The contribution of regulatory and naïve T cell phenotypes in these complex processes is still unclear. Therefore we investigated the navigation of naïve and regulatory T cells in vivo and in living brain tissue.
We could show by intravital two photon microscopy that in EAE naïve T cells are highly motile with a comparable mean track velocity of 0.16 ± 0.008 μm/s (N = 73) to lymphoid tissue.
To get a hint on what drives these naïve cells to travel through compact CNS parenchyma and which migratory signals exist in the brain for naïve cells we used the slice model since in healthy mice hardly any T cells can be find. In the slice model we circumvent BBB to see if naïve cells have migratory capacity per se in brain tissue but we could hardly find migratory cells contrasting our observations for effector T cells, where we found an CXCR4 dependent compartmentalisation around vessels (1) . Here, also regulatory T cells show a migration pattern with a highly dynamic nature in the perivascular area similar to Th1 and Th2 CD4 T cells, indicating a dominant role of this area for immuneregulation.
Our results visualize that even though naïve OT-2 T cells have migratory capacity in the lymph node they cannot find sufficient migratory signals in the CNS parenchyma under non-inflamed conditions but seem to undergo some kind of activation in the brain of EAE affected mice. From lymph nodes it is known that T cells migrate along reticular fibers that can be detected by their SHG signal. In the inflamed brain we could also observe reticular structures by their SHG signal but not in healthy brain and brain slices, suggesting that these inflammation induced structures may also provide the migratory signals needed for the naive T cells to migrate within in the CNS. The identification of the molecular origin of this reticular network and the signals navigating the cells should be an aim of future investigation. Reference 1. Siffrin, V., Brandt, A. U., Radbruch, H., Herz, J., Boldakowa, N., Leuenberger, T., Werr, J., Hahner, A., Schulze-Topphoff, U., Nitsch, R. et al. (2009) It is increasingly recognized that excess intracellular cations, particularly [Ca2+], contributes to neuronal and glial cell injury underpinning the pathology and development of long-term disability in multiple sclerosis (MS). Recent evidence from experimental autoimmune encephalomyelitis (EAE), an animal model of MS, suggests that the acid sensing ion channel 1 (ASIC1), that is activated under the acidotic tissue conditions found in inflammatory lesions and fluxes Na+/Ca2+, contributes to axonal injury. However, ASIC1 expression has not been studied in MS tissue, and the extent and cellular distribution of ASIC1 expression in neurons and glia in inflammatory lesions is unknown.
We show significant upregulation of ASIC1 in axons associated with injury within acute EAE and active MS lesions. We also demonstrate parallel findings in oligodendrocytes, and show that ASIC1 blockade is both myelo-protective and neuro-protective. Moreover, daily administration of amiloride, a licensed and clinically-safe blocker of ASICs, commenced at disease onset or at first relapse ameliorated disability in mice with chronic-relapsing EAE (CR-EAE).
Together these findings suggest that ASIC1 blockade offers novel dual neuro-and myelo-protective benefits justifying a trial of amiloride or other ASIC1 blockers in the treatment of patients with MS.
In vivo imaging of partially reversible TH17-induced neuronal dysfunction in the course of encephalomyelitis Neuronal damage in autoimmune neuroinflammation is the correlate for long-term disability in patients suffering from multiple sclerosis (MS). Although it is assumed that in autoimmune neuroinflammation, the primary immune response is directed against the myelin sheath, axonal and neuronal injury are already prominent in early disease stages and very likely determine the degree of long-term disability in patients. Here, we provide evidence that TH17 cells, which other studies have already shown to be associated with clinical deficit in EAE (Ivanov et al., 2006) , play a dominant role in direct neuronal injury during disease course.
We investigated the role of immune cells in early neuronal damage processes in animal models of MS by monitoring autoimmune neuroinflammation using two-photon microscopy of living anaesthetized mice. In the brainstem, we detected long-lasting interaction between immune cells and neuronal processes, especially during disease peak. Direct interaction of MOG-specific 2d2.tdRFP TH17 and neuronal cells in demyelinating lesions was associated with extensive axonal damage. Long-lasting contact led to severe, localized and partially reversible fluctuation in neuronal intracellular Ca2+ concentration, which is an important indicator of neuronal dysfunction, as demonstrated in B6.Thy1.CertnL15 mice. This Ca2+ increase could be blocked by NMDA-receptor antagonist MK-801, which indicates that excitotoxicity is a relevant feature in immune-mediated neuronal damage processes. Especially the TH17 cells were capable of inducing apoptosis in cultured neurons, irrespective of antigen specificity and in contact-dependent manor. Thus, our data indicate an important role of the TH17 effector phenotype for neuronal dysfunction and clinical deficit in chronic neuroinflammation.
Once arrived in the CNS, activated CD4+ T cells (here, in particular, TH17) are able to directly contact neurons as well as axons and induce Ca2+ changes in an antigen-independent manner in these target organ cells. Live-imaging during disease has characterized the neuronal dysfunction as early and potentially reversible, which suggests that immune-mediated disturbances of the neuronal compartment cause relapsing and remitting disability in patients with multiple sclerosis, in addition to conduction blocks as a result of changes to the myelin sheath. Devic's neuromyelitis optica (DNMO) is an inflammatory demyelinating disorder restricted to the optic nerves and spinal cord. Since the identification of a specific autoantibody (NMO-IgG) directed against aquaporin 4 (AQP4), an astrocytic water channel protein, DNMO has been considered an entity distinct from multiple sclerosis. Recent findings indicate that the NMO-IgG/AQP4 antibody has a pathogenic role through a complement-dependent astrocyte toxicity. However, the link with demyelination remains elusive. We hypothesized that the NMO-IgG/AQP4 antibody impairs astrocytic function and secondarily leads to demyelination.
Rat astrocytes and oligodendrocytes from primary cultures, and rat optic nerves, were exposed long-term (24 h) to IgG in the absence of complement. IgG was purified from the serum of DNMO patients who were either NMO-IgG/AQP4 antibody positive or negative, and from healthy controls. Flow cytometry analysis showed a reduction of membrane AQP4 and glutamate transporter GLT1 on astrocytes following contact with IgG purified from NMO-IgG/AQP4 antibody positive serum only. The activity of glutamine synthetase decreased in parallel, indicating astrocyte dysfunction. IgG from NMO-IgG/AQP4 antibody positive sera also reduced oligodendrocytic cell processes and approximately 40% died. This deleterious effect was confirmed ex vivo on isolated optic nerve. IgG from NMO-IgG/AQP4 antibody seronegative patients and from healthy controls had no similar effect. NMO-IgG/AQP4 antibody did not directly injure oligodendrocytes cultured without astrocytes. A toxic bystander effect of astrocytes damaged by NMO-IgG/AQP4 antibody on oligodendrocytes was identified. Progressive accumulation of glutamate in the culture medium of NMO-IgG/AQP4-antibody-treated glial cells supported the hypothesis of a glutamate-mediated excitotoxic death of oligodendrocytes in our models. Moreover, glutamate receptor NMDA subunits were detected on oligodendrocytes and co-treatment of glial cultures with NMO-IgG/AQP4 antibody and D+2-amino-5-phosphonopentanoic acid, a competitive antagonist at NMDA receptors, partially protected oligodendrocytes.
In our rat models, we demonstrate a direct effect of NMO-IgG/ AQP4 antibody on astrocytes independent of complement; an indirect effect on oligodendrocytes which is mediated by damaged astrocytes; and a possible glutamate-mediated mechanism of excitotoxicity to oligodendrocytes.
The C-C chemokine receptor CCR4 has an essential role in the development of autoimmune encephalomyelitis and its expression is required on dendritic cells, not on T cells The CC chemokine receptor 4 (CCR4) and its ligands, CCL17 and CCL22, contribute to the pathogenesis of allergic responses and infectious and autoimmune diseases by directing T lymphocyte trafficking to inflammatory sites. However, their role in CNS autoimmunity remains unknown.
Here we provide evidence that CCR4 is critically implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), since CCR4 knockout mice exhibit EAE resistance after immunization with myelin oligodendrocyte glycoprotein (MOG35-55). A cooperative role for both CCR4 ligands in EAE is supported by the fact that CNS immigrating dendritic cells expressed CCL17, while CCL22 was produced by dendritic cells in concert with macrophages/microglia. Adoptively transferred MOG35-55-reactive, CCR4 deficient T cells were still able to induce EAE in naïve mice indicating that CCR4 on T cells was dispensable for disease induction. Instead, disease susceptibility was restored in bone-marrow chimeras by expression of CCR4 on myeloid cells. Intracerebral microinjection of CCR4+ dendritic cells identified these cells as key mediators of EAE development.
Our data clearly demonstrate a novel and non-redundant role for CCR4 on dendritic cells in regulating the effector phase during EAE. Thus, therapies targeting CCR4 function in dendritic cells may represent an important strategy in the development of treatments for autoimmune CNS disease. We used transection model of SCI in which spinal cord was cut axially between Th9 and Th10 vertebra using C57BL/6 mice with wild type (WT) and interleukin-1 (IL-1) knock-out (KO) type. We examined motor function using Basso Mouse Scale (BMS) for locomotion during the 14th day post operatively (dpo). Sagittal sections of spinal cords obtained at 3rd, 7th, and 14th dpo were stained for immunohistochemistry. The area surrounded by GFAP, a marker of astrocyte, positive cells were measured as injured area by DP2-BSW software (Olympus). Homogenate samples of spinal cords obtained at the 1st, 3rd, 7th and 14th dpo were examined for chemistry using ELISA kit.
Motor function was significantly improved in IL-1 KO mice through the 3rd dpo compared with WT mice. The area surrounded by GFAP positive cells was significantly decreased in IL-1 KO mice at the 7th and 14th dpo compared with WT mice. IL-1β peaked on the 1st dpo and decreased gradually for the 14th dpo in WT mice. It was expressed on the Iba1, a marker of MG/MF, positive cells. TNF-a was also increased after SCI and kept high level through the 14th dpo in WT mice. It was significantly decreased in IL-1KO mice at the 1st, 3rd, 7th and 14th dpo compared with WT mice. Galectin-3 was significantly higher in IL-1 KO mice at the 14th dpo compared with WT mice. Galectin-3 positive cells tended to accumulate around injury site in IL-1 KO mice. In addition, it was expressed on F4/80, a marker of MG/MF, positive cells.
In conclusion of this study, endogenous IL-1 influenced the activation of MG/MF after SCI. Moreover, galectin-3 could take part in the difference of MG/MF activation after SCI. The blood-brain barrier (BBB) is composed of tightly bound endothelial cells (ECs) and perivascular astrocytes that regulate central nervous system (CNS) homeostasis. Recent studies indicate that components of the Hedgehog (Hh) pathway play an important role in vascular proliferation, differentiation and tissue repair in adult tissues. As BBB disruption is observed early in neuroinflammatory conditions such as multiple sclerosis (MS), this study aims to demonstrate that astrocyte-secreted Sonic Hh (Shh) contributes to the maintenance of BBB functions, including its immune quiescence.
Herein, we show that astrocytes express and secrete Sonic hedgehog (Shh) and that BBB-ECs bear Hh receptors and downstream transcription factors. In vitro and in vivo experiments show that while activation of the Hh pathway restricts BBB permeability, Hh neutralization impacts on BBB formation and stability during fetal development and adulthood. We further demonstrate that Shh promotes immune quiescence of BBB-ECs by decreasing the secretion of proinflammatory chemokines IL-8/CXCL8 and MCP-1/CCL2, the surface expression of cell adhesion molecules as well as the adhesion and migration of immune cells, a phenomenon that is dysregulated during neuroinflammation. In addition, in vitro stimulation of BBB-ECs with TNF-a and IFN-g deregulates the Hh signaling pathway and prevents the barrier stabilizing properties of Hh. Finally, Shh was found to impact on cytokine production and adhesion molecule expression in T helper (Th)1, Th2 and Th17 lymphocytes.
Our data suggest that the Hh pathway provides a barrierpromoting effect and an endogenous anti-inflammatory balance to CNS-directed immune attack, as occurs in MS. Chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and prion disease may evolve over many years prior to the presentation of clinical symptoms. In association with the accumulation of misfolded protein and the slow degeneration of neurons and their processes microglia, the resident macrophages of the CNS, take on an activated morphology and increase in number. How these cells of the innate immune system contribute to disease progression is at present unclear. In a murine model of prion disease we have demonstrated that in the presence of misfolded protein and slow degeneration of neurons the microglia are associated with an atypical anti-inflammatory profile dominated by TGFβ, PGE2 and CCL2. The microglia are a very long-lived population of tissue macrophages and thus retain an 'innate immune memory' of the ongoing tissue degeneration. This phenotype is switched to a robust pro-inflammatory phenotype by systemic inflammation, which in turn leads to exaggerated symptoms of sickness behaviours, increased neuronal degeneration and accelerated disease progression (Cunningham et al., 2005; 2009 ). Systemic inflammation has been shown to exacerbate neuronal degeneration in a number of other models of chronic neurodegeneration. Since systemic inflammation can switch the microglia phenotype in pre-clinical models we investigated whether systemic inflammation affects disease progression in patients with Alzheimer's disease. We find that patients with raised serum levels of tumour necrosis factor-alpha (TNF), indicative of chronic systemic inflammation, and those who contract an acute systemic infection show a greater rate of cognitive decline than patients with low levels of TNF and no systemic infections (Holmes et al., 2009) . We propose that systemic inflammation and infection contribute to the rate of progression of chronic neurodegenerative disease by switching microglia to a pro-inflammatory and tissue damaging phenotype. Microglial-mediated neuroinflammatory processes are involved in Alzheimer's disease (AD). In addition, recent reports have suggested a crucial role of invading blood-derived monocytic cells (BDMC) in the pathogenesis of cerebral amyloidosis in transgenic mice. We have established a novel protocol that allows for the conditional ablation of microglia by administrating ganciclovir (GCV) to transgenic CD11b-HSVTK (TK) mice. By crossing TK mice to various APP transgenic mice we have found that microglial ablation for up to four weeks has no effect on the formation or the maintenance of the amyloid pathology (Grathwohl et al., Nat Neurosci 2009) . More recently, we have again utilized the TK mice and established an irradiation-independent assay to repopulate the entire mouse brain with BDMC following microglia cell depletion. We have now taken advantage of these mouse models to determine the consequences of microglial ablation and BDMC invasion on normal brain function as well as the pathogenesis of the amyloid lesions in transgenic mouse models.
A possible role for the systemic environment in modulating brain aging and neurodegeneration
Growing evidence links neurodegeneration with altered immune responses not only in the brain but in the periphery as well. In addition, age is the main risk factor for sporadic forms of neurodegenerative diseases, and aging of peripheral organs may affect brain function. How the systemic environment affects brain health is largely unknown and while some of these interactions may involve cells entering the nervous tissue it is likely that many are mediated by soluble factors. We use antibody-based microarrays or Luminex technology to measure the relative levels of up to 700 secreted signaling proteins including cytokines and growth factors in plasma from humans and mice and correlate them with molecular and cellular changes in the brain. Using these methods we identified for example, proteins which are strongly neuroprotective when administered into mice systemically or age-related factors which regulate neurogenesis and neuroinflammation. Our findings point to systemic changes of immune responses and cellular signaling factors with aging and in neurodegeneration and may have relevance for our understanding and diagnosis of age-related neurodegeneration. They also support the use of our focused proteomic approach to study cellular signaling proteins which we collectively call the "communicome" as a means to understand pathophysiological and physiological changes in the brain. In my talk I want to summarize our recent insights into the pathogenesis of immune-mediated axon damage in vivo. Immunemediated axon damage plays a crucial role in inflammatory diseases of the central nervous system (CNS) like multiple sclerosis (MS). In MS, immune cells infiltrate brain and spinal cord and attack axons and their surrounding myelin sheets. We know by now that the number of axons damaged by immune cells critically determines the clinical disability of MS patients; however, we still understand very little about the process that leads to axon damage.
We have used an in vivo imaging approach to investigate the pathogenesis of immune-mediated axon damage in an animal model of multiple sclerosis. By time-lapse imaging of fluorescently labeled axons we could follow the slow and spatially restricted degeneration of axons in inflammatory CNS lesions. This "focal axonal degeneration" appears to be a novel type of axonal degeneration that can be differentiated from post-traumatic forms of axonal degeneration like Wallerian degeneration e.g. by its limited extension and slow speed of progression. We could further identify intermediate stages of "focal axonal degeneration" that can persist for several days and progress either to the degeneration or full recovery of the affected axons. Interestingly, demyelination does not appear to be a prerequisite for the induction of axon damage. In contrast, mitochondrial damage appears to be the earliest ultrastructural sign of axon damage. Further, early stages of "focal axonal degeneration" are often associated with persistent macrophage contacts suggesting that macrophage-derived mediators play a crucial role for the induction of this process. We are currently trying to identify the molecular mediators that induce the degeneration process and reveal the intra-axonal mechanism that leads to axon fragmentation.
We hope that this work will improve our understanding of immune-mediated tissue damage in multiple sclerosis and pave the way towards the development of targeted neuroprotective therapies.
In vivo imaging neuronal recovery in neuroinflammation Gan Wanbiao, Parkhurst Christopher, Hayes Scott, Gan Wen-Biao ⁎
Murine models of experimental autoimmune encephalomyelitis (EAE) have been widely used to study the effects of inflammation in the central nervous system (CNS). EAE mouse models have revealed neuropathology in the spinal cord, brainstem and cerebellum, but the impact of EAE on the cerebral cortex remains poorly understood. Using in vivo transcranial two-photon microscopy, we examined dendritic spine plasticity and microglial activation in the somatosensory cortex in a mouse model of EAE induced by immunization with myelin-oligodendrocyte-glycoprogein 35-55 (MOG). We found that mice with MOG-induced EAE displayed a significant increase in dendritic spine turnover at least 4-5 days before the onset of neurological symptoms. Concomitant with dendritic spine instability, microglia displayed a reactive phenotype including changes in morphology and distribution. Dendritic spine instability and microglial activation continued at the peak stage of EAE and subsided 2 months after the EAE-inducing MOG injection. These observations indicate that MOG-induced EAE profoundly disrupts synaptic connections in the cortex. They also suggest that microglial activation may play an important role in the pathogenesis of EAE. Several proinflammatory cytokines produced by active microglia, such as TNF-alpha and IL-1, are known to regulate synaptic plasticity. We are presently studying how microglial activation and increased proinflammatory cytokine production contribute to EAE-associated synaptic pathology and recovery in the cortex. Several novel autoantibodies have been discovered in the field of Neurology that, due to their outstanding significance for diagnosis and therapy, have been rapidly translated into clinical patient care. Among the most important markers are autoantibodies directed against aquaporin-4 and against NMDA-type glutamate receptors (also called NMDA receptors). Detection of these antibodies in the serum or cerebrospinal fluid can determine the fate of affected patients. Autoantibodies against aquaporin-4 are found in patients with neuromyelitis optica (NMO, also known as Devic's syndrome) and its subforms. Early and aggressive immunosuppressive therapy is decisive for the future disease course. Detection of anti-aquaporin-4 antibodies allows differentiation of NMO from multiple sclerosis, which is particularly important as NMO and MS are treated with different drugs. In addition, antibodies against aquaporin-4 discriminate a small but significant subgroup of patients with neuropsychiatric SLE and primary Sjögren's syndrome in whom therapeutic strategies such as for NMO need to be applied. Autoantibodies against NMDA receptors are characteristic of anti-NMDA receptor encephalitis, a currently still widely underdiagnosed autoimmune disease, which was first described in 2007 in young patients with ovarian teratoma. However, anti-NMDA receptor encephalitis is now also increasingly diagnosed in older female patients, in women without teratoma as well as in men and in children. The disease often starts with a flu-like preliminary stage, followed by psychiatric symptoms such as anxiety, excitement, strange behaviour, delusions and hallucinations. Therefore, a large proportion of these patients are initially admitted to psychiatric wards. Within a few weeks epileptic attacks and clouding of consciousness occur, which can eventually lead to coma. Final diagnosis of anti-NMDA receptor encephalitis is based on the detection of antibodies against glutamate receptors of type NMDA in the serum or CSF of patients. In principle, anti-NMDA receptor encephalitis is curable, even after long, intensive medical treatment and long-term ventilation. A prerequisite, however, is rapid diagnosis with early therapy and complete removal of the tumour, if present. Further novel and highly specific autoantibodies in patients with limbic encephalitis comprise antibodies directed against the GABAB-receptors and against novel antigenic targets previously attributed to VKKC that will also be discussed.
Response to interferon-beta treatment in multiple sclerosis: Pharmacogenomic studies Comabella Manuel ⁎ Centre d'Esclerosi Múltiple de Catalunya, CEM-Cat, Unitat de Neuroimmunologia Clínica, Hospital Universitari Vall d´Hebron, Barcelona, Spain
The mechanisms underlying heterogeneity in the response to treatment in multiple sclerosis (MS) are not completely understood, although genetic factors are most likely to be playing important roles. Moreover, given the complex nature of the disease, this heterogeneity is probably explained by the contribution of multiple genes. Disease modifying therapies (DMT) are the mainstay of treatment in relapsing-remitting MS and have demonstrated a beneficial effect on disease activity. However, DMT are partially effective, and their long-term impact on disease progression remains elusive. In addition, not all patients respond to current DMT.
The increasing number of new therapies for MS and the potential risk for a lack of response and/or serious adverse reactions make individualized therapy a high-priority for MS. Pharmacogenomics applies technologies such as gene expression profiling, single nucleotide polymorphism (SNP) screens, and proteomics in order to predict response to treatment and toxicity to drugs. Although pharmacogenomics holds great promise for individualized therapy in MS, big efforts should first be made to identify markers for treatment efficacy.
This talk will focus on the current status and future directions of pharmacogenomic studies in MS, mainly in relation with interferonbeta treatment. Multiple sclerosis (MS) is a heterogeneous and complex autoimmune demyelinating disease where tissue damage and repair process occurs simultaneously in the central nervous system. High-throughput analyses of genes, proteins and antibodies have been undertaken to elucidate the molecular signature of MS. Proteomic analysis and microarray studies of brain lesions, cerebrospinal fluid and immune cells of MS patients have revealed unexpected molecules and pathways involved in the disease pathogenesis and have identified new targets for therapy. However, each technique has limitations due to the half life of the target molecules, their compartmentalization within the cell and limitations of the platforms themselves. Moreover, direct comparison of transcriptomic and proteomic databases from different groups is complicated due to lack of standardization of platforms and due to the heterogeneity of tissue analyzed. Here, we present the different proteomic technologies utilized to study MS tissue, benefits and deficiencies of each approach and finding. We will also discuss a comparative proteomic and transcriptomic analysis of same tissues from multiple sclerosis brain lesions. Finally, we compare proteomics studies of MS tissue with a similar demeylination disease NMO. This combined, multi-level integrative approach illuminates molecular pathogenesis of MS and identifies potential targets for therapy.
The Immunology Lecture:
Conversations between tissues and T cells
NIH, Bethesda, MD, USA
The immune system has two questions it must answer when faced with a potential threat. 1) Shall I respond 2) What kind of response should I make?
For three quarters of a century immunologists trying to discover how the immune system answers the first question have based their theories and experiments on the fundamental belief that the immune system answers this question by discriminating between self and non-self. It was thought (and taught) that, if the system were perfect, it would attack everything that is non-self and be totally tolerant of anything that is self. I abandoned this belief and suggested instead that the immune system is more concerned with danger than with the distinction between self and non-self. The model starts with the idea that the immune system defines "danger" as anything that causes tissue stress or destruction. Under this model, antigen-presenting cells are activated by alarm signals from stressed or damaged tissues. Without this activation, no primary immune response can occur. I will show some of the recent evidence in its favor and discuss its implications.
I will also present some new ideas about the second question above. Thus far in immunology, there has been no concerted attempt to find a model for how the immune system determines the effector class for any particular response. Currently, it is widely taught that the effector class is tailored to the pathogen driving the immune response: for example, we make IgE to a worm infection, and killer cells against a virus. I no longer believe this. Although pathogens certainly have an influence on the immune responses made against them (usually finding ways to modify those responses to their liking), I believe that there is a more fundamental underlying system of control. I believe that the ultimate controllers of immunity are the tissues that the immune system was designed to protect. When injured, tissues emit alarm signals that initiate immune responses. When not injured, tissues present their own antigens, or release them to passing DCs to present to T cells in order to induce tolerance. Moreover, when an immune response does occur, the tissues send signals that direct the immune system to tailor that response to an effector class that can eliminate the pathogen without causing more damage to the tissue.
We must stop thinking of the immune system as an independent group of cells, patrolling the body to keep it free of foreigners. When we base our thinking on the view that the immune system is an extended family of constantly communicating cells and tissues that accept harmless (and beneficial) entities, while fighting harmful ones, we open new windows, suggest new experiments, and build a new understanding of the complexity and wonder of this powerful system. 633 CNS-immune system interactions: A dance that changes with development, age and experience
Neuroinflammation triggered by systemic inflammation and occurring during critical periods of CNS development is hypothesized to contribute to the development and/or pathogenesis of many neurodevelopmental disorders including cerebral palsy, schizophrenia and autism. In the adult CNS, the type and magnitude of microglial activation and macrophage influx triggered by systemic inflammation are well characterized. As yet, little is known about if and how microglial phenotype changes during critical periods of CNS development associated with synaptogenesis and oligodendrocyte development. Here, using flow cytometry and dual in situ hybridization/ immunohistochemistry we show that in the early postnatal murine brain, microglia display an activated phenotype that is not polarized toward either "classic" proinflammatory or "alternative" anti-inflammatory activation states. Furthermore, we find that at all ages examined, systemic inflammation induced by an intraperitoneal injection of LPS leads to widespread activation of microglia and a transient influx of peripheral macrophages into the murine CNS. However, the magnitude of macrophage influx into the CNS is developmentally modulated. In addition, CNS-infiltrating macrophages display a polarized pro-inflammatory phenotype. During this same developmental period, microglial activation is associated with high level expression of multiple alternative activation markers and induction of TREM2. We speculate that these microglial-specific forms of activation in part serve to compensate for the higher levels of proinflammatory macrophages that infiltrate the CNS during early development. Altogether our data contribute to the growing literature demonstrating that the brain may have age-specific susceptibilities to insults associated with the development and treatment of neurodevelopmental disorders.
Immune surveillance of the CNS: New concepts, new targets
Cleveland Clinic, OH, USA
We propose the hypothesis that, in health, the central nervous system (CNS) does not permit immune cell entry across the bloodbrain barrier, and immunosurveillance takes place within subarachnoid space, leaving the CNS parenchyma untouched. This concept is based on descriptive studies of human CSF and analytical studies of the subarachnoid space in murine EAE. Normal human CSF contains between 1.5 and 3 × 105 cells, the majority being CD4+/CD45RO+/ CD27+/CCR7+/CXCR3+/L-selectinhi central memory T cells, about half of which are CD69-positive. These cells routinely traverse the choroid plexus stromal vessels and epithelium, and enter and exit the CSF approximately twice per day, providing a continuous supply of lymphocytes with novel antigenic specificities. CSF T cells are enormously enriched for these activated CD4+ central memory T cells, as compared with circulating blood, where they constitute at most b5% of leukocytes. Lumbar and ventricular CSF of patients without CNS inflammation contain a similar cellular component, supporting the proposal that many CSF cells enter across the choroid plexus. All committed tissue specific memory T cells are represented in the CSF with an equal proportion as found in blood. In healthy mice, CD4+ T cells in the subarachnoid space perform serial 'scanning' encounters with MHC class II+ APCs. During EAE, antigen recognition and clonal expansion occur in the subarachnoid space, days before such events in the spinal cord parenchyma. We integrate these observations with experimental studies showing how subarachnoid space inflammation is coupled to parenchymal vascular activation, as well as invasion of the parenchyma by hematogenous cells. Finally, the novel finding of subpial demyelination early in the course of MS, suggests that the MS disease process may in part proceed 'from the outside in' with cortical demyelination and meningeal inflammation setting the stage for white matter lesions, and for the persistence of the disease process.
Whitacre Caroline, Williams J.L., Cox G.M., Smith K.M., Kithcart A., Shawler T., Satoskar A.
The Ohio State University, Columbus, OH, USA Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) affecting nearly 3 million people worldwide. It is known that autoreactive CD4+ T cells of the Th1 and Th17 subtypes are involved in MS pathogenesis. Currently licensed therapies for MS include forms of interferon beta as well as more specifically targeted agents directed at adhesion molecules and the antigen receptor complex. In the search for additional therapies, we have focused on hormonal, cytokine and genetic approaches to treatment of MS using the model system experimental autoimmune encephalomyelitis (EAE). The hormonal studies have their foundations in the observation that women with MS exhibit markedly fewer relapses during pregnancy, but show relapses of disease postpartum. Our studies have shown that EAE clinical signs are markedly reduced when pregnancy is induced during ongoing EAE, which is accompanied by a decrease in CNS histopathologic changes and suppression of inflammatory cytokine production. We have identified serum exosomes as the mediator of disease suppression and analyzed the contents of exosomes by differential gel electrophoresis and mass spectrometry. The results of those analyses suggest that exosomal proteins act to decrease inflammation as well as enhance neural regeneration.
Cytokine approaches to therapy of MS have focused on macrophage migration inhibitory factor (MIF) since levels of this cytokine have been shown to increase during relapses of disease. We have shown that MIF-deficient mice exhibit reduced clinical signs of EAE and CNS inflammatory infiltrates. Further, a small molecule inhibitor of MIF, designed by Cytokine Pharmasciences, has also been shown to be efficacious in the treatment of EAE, even when treatment is begun after the appearance of EAE clinical signs. We have shown that treatment of microglial cultures with MIF upregulates IL-1, IL-6, TNFα, iNOS and CCL2 levels. Moreover, microinjection of rMIF in vivo into the spinal cord has shown transient microglial reactivity and cellular accumulation within the CNS. Thus inhibition of MIF could well serve as an important therapeutic approach for CNS inflammation.
Genetic approaches to therapy have centered on microRNAs (miRs) which are small non-coding RNAs that negatively regulate post-transcriptional gene expression. We have identified miR29, which targets T-bet and interferon gamma (IFNγ), as important to study in MS. We reasoned that MS patients might have altered levels of miR 29 and indeed found that purified CD4+ T cells from MS patients had lower basal levels of miR-29 relative to control patients. These results identify miR-29 as a potential biomarker for MS and future candidate therapy.
This study was supported by NIH grants AI064320 and T32 AI055411 and National MS Society grant RG3272.
Stanford University, Stanford, United States; 2 Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Strubergasse, Salzburg, Austria; 3 Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, United States Stem cell activity in the brain decreases dramatically with age. Given perturbations to the systemic milieu of an organism, such as those induced by exercise, enhance neural stem cell frequency and improve learning and memory in aging mice, we hypothesized that systemic molecular changes could cause a decline in neurogenesis during aging.
To examine the effect of systemic factors on adult neurogenesis, we utilized a model of parabiosis in which a circulatory system is shared between young and old mice (heterochronic parabiosis). Exposure of a young brain to an aged systemic environment resulted in decreased neurogenesis, while exposure of an old brain to a young systemic environment increased neurogenesis. Using a targeted proteomic screen we identified a conserved subset of blood borne chemokinesincluding CCL2/MCP-1 and CCL11/Eotaxinwhose plasma levels correlate with reduced neurogenesis observed in normal aging and heterochronic parabiosis. Functionally, we demonstrate that mimicking an aged systemic environment by increasing the level of CCL11 in the periphery of young adult mice results in decreased neurogenesis in vivo. Additionally, we show that CCL2 and CCL11 decrease progenitor frequency and neuronal differentiation in vitro. Using a pharmacological approach we also demonstrate that the inhibitory effects of systemically administered CCL11 can be mitigated by neutralization either systemically or directly within the central nervous system.
Cumulatively, these results indicate that the decline in adult neurogenesis observed during aging can be attributed, in part, to changes in the levels of immune-related factors within the aging systemic milieu. Mesenchymal stem cells (MSCs) are promising vehicles for the treatment of neurodegenerative diseases such as multiple sclerosis (MS). This is in part due to their broad immunomodulatory properties and ability to home to sites of tissue inflammation and injury. We have previously investigated the therapeutic potential of human MSCs in ameliorating the MS-like disease in mice, experimental autoimmune encephalomyelitis (EAE). A comparison of MSCs from three different tissue sources revealed that the therapeutic efficacy of adipose-derived MSCs (Ad-MSCs) was greater than that of the other MSCs tested, in terms of suppression of clinical and pathological signs of disease. The anti-inflammatory cytokine interleukin-10 (IL-10) has been extensively studied in the EAE model and has been found to play an important immunoregulatory role. However, treatment of EAE with systemic administration of IL-10 has yielded contradictory results, suggesting that the timing and mode of delivery may be crucial to the effectiveness of IL-10 treatment. In the present study, we aimed to explore the use of Ad-MSCs as vehicles for delivery of IL-10 to mice with EAE.
We transduced Ad-MSCs with a lentiviral vector encoding human IL-10. To track these cells, vectors incorporated an enhanced green fluorescent protein (eGFP) sequence that was translated from an internal ribosome entry site sequence. As a control, cells were transduced with a vector engineered to express eGFP alone. Transgene (IL-10 and eGFP) expression was maintained over a number of passages as assessed by flow cytometry and ELISA. Importantly, the cell surface phenotype, differentiation potential and expression of chemokine receptors by Ad-MSCs were unaffected by the transduction process. In vivo transplantation studies showed that systemic administration of IL-10 expressing Ad-MSCs, either by intravenous or intraperitoneal injection, during the initiation phase of disease could prevent or significantly delay the development of myelin oligodendrocyte glycoprotein (MOG)-induced EAE. In protected mice, T-cell proliferative responses to MOG were found to be dramatically reduced in comparison to controls and unprotected mice. We are currently investigating possible mechanisms of action, including production of T regulatory cells in addition to suppression of antigen presenting cells and Th17 cells.
Our results show that MSCs modified to overexpress IL-10 can suppress EAE and that MSCs may be useful tools for delivering therapeutics molecules. Previously, our lab has shown that human bone marrow derived stem/progenitor cells (MSCs) enhance repair of the damaged brain following transient global cerebral ischemia in part through modulation of microglial activation. In this study, we aimed to confirm the finding that hMSCs inhibit activation of microglia and to identify the specific mechanisms underlying this effect.
To further assess the effects of inflammation on neuronal survival, both microglial cells and organotypic hippocampal slices (OHCs) were used for in vitro cocultures with MSCs. In these experiments, microglia were stimulated with the bacterial protein lipopolysaccharide (LPS). For treatment, MSCs were cocultured either directly with microglia or in transwell cultures with the slices. Our findings confirmed that MSCs attenuated activation of microglia in both cell and tissue cultures. In parallel experiments we treated activated microglia with recombinant stanniocalcin-1 (STC-1), an anti-inflammatory and anti-apoptotic protein expressed by MSCs. Here we show that STC-1 significantly inhibited the inflammatory response in microglia.
Our data indicate that MSCs can be activated to produce soluble anti-inflammatory factors capable of reducing microglia activation.
Roy Kristin ⁎ , Peitz Michael, Brüstle Oliver, Neumann Harald Institute of Reconstructive Neurobiology, University Hospital Bonn and Hertie foundation, University of Bonn, Bonn, Germany Microglia, the resident immune cells of the central nervous system, are responsible for the innate immune defence. Upon injury or inflammation they become activated and migrate to and within the lesion site. Microglia can cause ambivalent effects by releasing neurotrophic or neurotoxic factors as observed in different neurodegenerative diseases. Therefore, detailed studies concerning the function of microglia in neurodegenerative diseases are crucial. However, only a limited number of human microglia is possible to obtain what complicates investigations of new therapeutical approaches.
We now established a protocol for the differentiation of human induced pluripotent stem cells (hiPS) to microglial precursors (iPSdM). This method encompasses the differentiation of embryoid bodies to proliferating microglial precursors within a mixed culture of neurons, astrocytes and oligodendrocytes. The obtained iPSdM were positively stained for Iba1 and CD68, markers widely expressed on microglia. Moreover, flow cytometry confirmed the expression of the microglial markers CD11b, CD16, CD29, CD40, CD45 and CD49d whereas the cells were largely negative for the stem cell marker CD34. Furthermore, iPSdM displayed chemokine-directed migration towards fractalkine and phagocytosis of microsphere beads similar to primary murine microglia.
Thus, we were able to differentiate human iPS into microglial precursors with an efficiency suitable for further studies on the role of microglia under normal and neuroinflammatory conditions. Oxidative stress (OS) is caused by reactive oxygen species (ROS), for example hydrogen peroxide (H2O2), hydroxyl radical (HO• − ) and superoxide. OS can cause lipid peroxidation, DNA damage and cell death and is thought to be involved in pathogenesis of a variety of neurodegenerative diseases. Adult neural/progenitor cells (NPCs) are a putative source of regeneration during these conditions. The aim of our study was therefore to investigate if and how OS, here administered as H2O2, affects the properties of adult NPCs.
Primary NPC cultures isolated from brains of adult DA rats were propagated in vitro and after their second passage, exposed to H2O2 (50 μM or 100 μM) for different time periods. After exposure the cells were differentiated for 5 days, fixed and immunolabeled for β-IIItubulin (neurons) and GalC (oligodendrocytes). The percentage of various cell types was calculated. The proliferation was measured using tritium labeled thymidine. The expression of known fate determining genes in exposed and control NPC cultures was investigated using real-time RT-PCR.
Our results reveal a significant dose-dependent increase in the percentage of neurons in H2O2-exposed NPC cultures compared to control cultures. This effect was determined both by immunosytochemical analysis for the neuronal marker β-IIItubulin and by realtime RT PCR for neuronal genes (betaIItubulin) and pro-neronal genes (neurogenin 2). Interestingly, the H2O2 exposed cultures also had elevated gene expression of Vascular endothelial growth factor A (VEGF-A) and its receptor Vascular endothelial growth factor receptor 1 (VEGFR1). Both genes have been reported to be part of the NSC niche and also to affect NSC proliferation and differentiation.
Identifying the inflammation-induced gene expression pattern in adult neural stem cells Covacu Ruxandra ⁎ , Arvidsson Lisa, Perez Estrada Cynthia, Svensson A.
In multiple sclerosis (MS), the inflammatory lesions are often located in close vicinity to areas harboring neural stem cells (NSCs) such as the subventricular zone (SVZ) and the central canal of the spinal cord. The aim of our study was to investigate inflammationinduced changes on the gene expression profile in NSCs.
Dark Agouti rats were immunized to develop Experimental Autoimmune Encephalomyelitis (EAE) an MS-like disease model. Total RNA was extracted from fresh-frozen SVZ biopsies or from NSC cultures isolated from the SVZ and the spinal cord. The NSCs were propagated in vitro and were used for RNA isolation either directly after the second passage or after differentiation for five days. Each experimental group consisted of separate cultures/tissues from three animals. The purified and amplified RNA was applied on RAT gene 1.0 arrays from Affymetrix. Data was presented as fold change of EAE versus control and the statistical analysis used was unpaired t-test. Data sorting involved exclusion of p-values higher than 0.05 and removal of noise signals i.e. signals below 50. The analysis was performed using the Ingenuity Pathway analysis database.
The most prominent expression pattern was that of differentiation-related genes which displayed opposite changes in SVZ-derived cultures compared to spinal cord-derived ones. For example the differentiated SVZ cells showed upregulation of genes such as Stat1 (involved in astrogliogenesis), S100B (astroglial marker) and betaIIItubulin (neuronal marker) while the spinal cord cells showed downregulation of genes such as Gfap (astroglial marker), Acsl1/ Mash1 (proneuronal gene), Nkx2.2 (oligodendroglial differentiation) and Map2 (neuronal marker). Moreover, the expression pattern of Bmp4 displayed the same trend i.e. upregulation in SVZ cultures and downregulation in spinal cord cultures after differentiation. Similar changes were detected for the Bmp4 downstream signaling mediators Smad 4 and 5 and also Dlx5.
These results suggest that inflammation has different effects on different NSC pools in the central nervous system, which might imply that these NSC pools have different intrinsic properties.
Induced pluripotent stem cells as a potential source of autologous neural stem cells for multiple sclerosis therapy Vita-Salute San Raffaele University, Milan, Italy; 5 Stem Cells and Neurogenesis Unit Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy
Multiple sclerosis (MS) is an acquired inflammatory and neurodegenerative immune-mediated disorder of the central nervous system (CNS), characterized by inflammation, demyelination and axonal degeneration. In the most recent years, we and others accumulated pre-clinical data in experimental models of demyelination suggesting that therapies based on the transplantation of neural stem/precursor cells (NPCs) can contribute to prevent or repair CNS damage through immunomodulation and remyelination. To translate pre-clinical data into clinically useful therapeutic protocols a large numbers of expandable autologous human precursors should be obtained. Induced pluripotent stem (iPS) cellsa new source of pluripotent stem cells recently obtained by genetic reprogramming of somatic cellsmay represent the appropriate tool to reach the abovementioned aims. This project is aimed at obtaining NPCs and OPCs from both human and mouse iPS cells to test -first in vitro and then in vivo in animal models of MStheir therapeutic efficacy in promoting neural repair via immunomodulation and remyelination. Due to the fact that we are aiming at having an autologous source of NPCs and OPCs to cure MS, we will also assess the existence of any 'therapeutic' difference between iPS cells derived healthy controls vs. MS patients that may hamper their expansion capability and terminal differentiation.
Different iPS cell lines have been generated from MS patients' fibroblasts and healthy controls. Moreover, their pluripotent state has been confirmed by RT-PCR for non viral pluripotency associated genes, immunocytochemistry assays for pluripotency markers, the ES cell-like morphology and the capability to generate teratomas after injection in SCID mice. To induce iPS cells neural and oligoglial commitment, we have tested a variety of protocols which had been previously validated for ES cells, with some slight modifications. We have recently accumulated solid data showing the possibility to induce in vitro neural commitment (e.g. NPCs) of human as well as mouse iPS cells and to differentiate them into OPCs as confirmed by immunostaining experiments and RT-PCRbased analysis.
All in all, the development of a strategy to promote neural repair via remyelination and immunmodulation using iPS cells might represent a further step toward the human application of stem cellbased therapies in chronic demyelinating CNS disorders in which irreversible neurological damage occurs. Neuroimmunology Unit, Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Genoa, Italy; 2 University of Genoa, Genoa, Italy; 3 DISEM, Section of Neuropharmacology, University of Genoa, Italy
Mesenchymal stem cells (MSCs), a bone marrow-derived type of adult stem cells have been demonstrated to regulate immune responses and ameliorate experimental autoimmune encephalomyelitis. However, their ability to transdifferentiate in neurons upon in vivo administration is not proven. Thus, their use in neurodegenerative diseases is still debated. In this study, we intravenously ad-ministered MSCs in an SOD1 G93A transgenic model of amyotrophic lateral sclerosis (ALS).
Administration of C57B/6 mice derived MSCs at day 90, the time of the first appearance of clinical symptoms, about 20-30 days after the first signs of degeneration of motoneurons, results in a significant improvement of behavioral motor tests and increased survival compared to control animals.
A few Luciferase-positive MSCs could be detected in the CNS of SOD1 transgenic mice.
We demonstrated that MSCs significantly reduced astrogliosis and microglia activation in the central nervous system of the treated mice as depicted by a decreased expression in the treated mice of GFAP and IB4 respectively. We showed that MSC administration decreases the expression of stress-associated proteins associated with the oxidative insults in SOD1 mutant mice. Last, we observed a significant decrease in the release of glutamate by synaptosomes prepared from treated mice compared to controls. These results suggest that i.v. administration of MSCs is an amenable approach to treat a yet incurable disease such as ALS.
Mesenchymal stem cells switch microglia from a detrimental to a protective phenotype Giunti Debora ⁎ ,1 , Parodi Benedetta 1 , Vergani Laura 2 , Bruzzone Santina 3 , Usai Cesare 4 , Mancardi Gianluigi 1 , Uccelli Antonio 1 1 Neuroimmunology Unit, Department of Neuroscience, University of Genoa, Genova, Italy; 2 Department of Biology, Genova, Italy; 3 DISEM, Section of Biochemestry, Genova, Italy; 4 National Research Council, Section of Biophysics, Genova, Italy Inflammation in the CNS has been closely associated with the pathogenesis of neural damage resulting from cerebral ischemia and neurodegenerative diseases. Therefore, the effective control of microglial activation is regarded as an important therapeutic target in these neurological diseases.
Recent reports have shown that the neuroprotective effect of therapeutic Mesenchymal Stem Cells (MSCs) may be mediated not only by their ability to inhibit immune responses targeting the central nervous system but also by their capacity to produce trophic factors that may contribute to functional recovery, neuron survival and stimulation of endogenous neural precursors.
We studied the role of MSCs on the effector functions of resting and activated microglia.
We showed that MSCs, in transwell cultures, can inhibit activation of microglia reducing the production and the expression of TNFa, NO and iNOS, whereas MSCs did not have any effects on microglial proliferation. MSCs significantly increased microglia production of IL-10, the neurotrophin IGF-1 and the neuroprotective molecule Nurr1. We also observed that MSCs, following IFN stimulation, express high levels of CX3CL1, a chemokine mediating neuron-microglial interaction, synaptic transmission and neuronal protection from toxic insults. In the presence of MSCs, microglia increases the expression of CX3CR1, the CX3CL1 receptor, while the knock-down of CX3CL1 in MSCs abolishes such up-regulation. These results suggest that MSCs can exert a neuroprotective role on microglia augmenting CX3CR1 expression through the release of CX3CL1.
Finally, we showed that MSCs induce an increase, on microglia, in the expression of CD200R and TREM2, a receptor associated with an increased phagocytic activity. Indeed, MSCs induced an increase of intracellular calcium concentration leading to an enhanced phagocytosis.
These results suggest that MSCs affect microglia, a cell population of the innate immunity, inducing a switch from a detrimental to a neuroprotective phenotype. Mesenchymal stem cells (MSCs) are being considered as a new therapeutic option to treat autoimmune diseases including multiple sclerosis (MS). MSC have been shown to potently inhibit peripheral murine T cell activation, both by cell-cell contact and by production of soluble factors. Recently, we found that soluble products from human MSCs (hMSCs) decreased human Th1, but surprisingly, increased Th17 responses in vitro.
Our aim is to dissect the mechanism(s) by which human MSC reciprocally regulates Th1 and Th17 responses. We focused on PGE2, as this molecule in known to increase Th17 responses, and can be produced by MSCs.
hMSCs were purified from normal human bone marrow, expanded in vitro, and validated by phenotype and differentiation capacity. In parallel conditions MSCs were pre-activated with IL-1beta, a proinflammatory cytokine. MSC-conditioned supernatants were collected, and introduced into a human Th1/Th17 differentiation assay using normal subject peripheral blood mononuclear cells (PBMC) with anti-CD3/CD28, IL-23, and IL-4/IFNg blockade.
We found that polarized PBMC had a mixture of Th1 (IFNg+), Th17 (IL-17A+), and T cells expressing both cytokines, which we refer to as Th1/17. MSC-conditioned supernatants decreased the frequency of Th1 T cells and IFNg, but increased the frequency of Th17 T cells and IL-17A secretion (p = 0.007), without affecting the Th1/17 subset. Pre-treating MSC with IL-1beta caused a greater Th17 increase and Th1 decrease. We found that MSCs secreted large amounts of PGE2 (30 ng/mL; SE 10). IL-1beta pre-treated MSCs secreted 160% more PGE2 as compared to untreated MSCs (p b 0.001). Moreover, exogenous PGE2 caused reciprocal regulation of Th1 and Th17 responses, consistent with the observed MSC effect.
Our data implicates PGE2 secretion by hMSCs as a mechanism of Th17 enhancement. We propose that inhibiting PGE2 will minimize potentially harmful effects of Th17 enhancement by hMSC therapy. Mesenchymal stem cells (MSC) represent a promising therapeutic approach for autoimmune and degenerative diseases of the central nervous system (CNS), because they couple immune-suppressive properties with multi-differentiation potential. Previous studies have shown that preventive treatment with bone marrow-derived MSC (BM-MSC) reduces disease severity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis as well as in the familial model of amyotrophic lateral sclerosis (FALS) .
In this study we show that the intravenous administration of adiposederived MSC (ASC) in EAE animals with chronic established disease significantly ameliorates the disease course not only by reducing demyelination and axonal loss, but also by inducing Th2-type cytokine shift in antigen-specific T cells. A subset of ASC expresses activated a4 integrins and adheres to inflamed brain venules in intravital microscopy experiments. Bioluminescence imaging confirms that a4 integrins mediate ASC accumulation in inflamed CNS and after penetration, ASC induce a significant increase of the number of endogenous oligodendrocyte progenitors. As for the mechanisms responsible for such effect, we found that ASC cultures produce a number of neural growth factors (VEGF, IGF-I, bFGF, BDNF and PDGF-AB), all involved in the proliferation of both oligodendrocyte precursors and ASC. In conclusion, we show that ASC display clear therapeutic effect in animals with established disease by a bimodal mechanism, by suppressing the autoimmune response as well as by inducing local neuro-regeneration by activating endogenous progenitors.
We also evaluated the effect of the administration of ASC in FALS. Preliminary results showed that animals injected with ASC after disease onset had significant higher behavioral performance as compared to controls for almost 7 weeks. ASC penetrated and survived into spinal cord for up to 2 months. Since we observed no expression of neural and glial markers, we speculate that the neuroprotective role of ASC may be mediated by the release of neural trophic factors like VEGF, IGF-I, BDNF and FGF-2.
The convincing results obtained in EAE and the encouraging data from FALS, indicate that ASC may represent a valuable therapeutic approach not only for autoimmune but also for neurodegenerative disorders of the CNS and could be considered an elective strategy for future clinical trials in human patients affected with a wide range of neurological disorders. To investigate in vitro prostaglandin E2 (PGE2) production in different cell cultures and estimate PGE2 inhibitory effect on myelinstimulated T-cells proliferation following co-cultivation of autologous/allogenic bMSC with peripheral blood mononuclear cells (PBMC) from MS patients.
bMSCs from MS patients were well phenotypically characterized with confirming of their pluripotency. PBMC from MS patients were stimulated by PHA and myelin antigens (recombinant myelin oligodendrocyte glycoprotein (MOG) and synthetic peptides MBP68-82, MOG33-35 in the presence of MSC (10:1) and adding of supernatants from confluent MSC cultures and co-cultures of mitogen/antigens-stimulated PBMCs in the presence of MSC. For detecting CFDA SE labeling cells proliferation, flow cytometry was used. For the quantitation of PGE2 in cell culture supernatants, ELISA kit « ParameterTM » was used. Results: We did not find any significant differences between spontaneous PGE2 production by PBMCs from MS patients and following PBMC stimulation by PHA, myelin antigens and addition of supernatants from confluent MSC cultures. The presence of autologous as well as allogenic MSC with PBMC resulted in increased PGE2 production in co-cultures irrespectively of antigenic cultivation condition (p = 0.023). At the same time, we showed that MSCs from MS patients as well as supernatants from co-cultures of MSC and PBMC significantly reduced myelin-stimulated T-cells proliferation in vitro; the inhibition varied from 22 to 60% and highly correlated with PGE2 concentration in cell supernatants (R = 0.8, p b 0.01).
PGE2 is not constitutively produced by MSC and requires the paracrine signal from lymphocytes. Antigenic stimulation does not affect PGE2 production following co-cultivation of MSC and PBMC, and only the presence of MSC in cell culture leads to significant increase of PGE2 simultaneously with expressed suppression of myelin-stimulated T cell proliferation in MS patients. Understanding the mechanisms of MSC-mediated antiproliferative effect is crucial for further use of MSC in research and MS treatment. Neuromyelitis optica (NMO), also known as Devic's syndrome, is an immune-mediated neurologic disease that involves the spinal cord and optic nerves. A serum immunoglobulin G autoantibody (NMO IgG) has been shown to be a specific marker for NMO and the water channel aquaporin 4 (AQP4) has been identified as the target for NMO IgG. Measurement of autoantibodies (Ab) to AQP4 aids the diagnosis of neuromyelitis optica spectrum disorders (NMOSD) and we now described a new ELISA for AQP4Ab which is dependent on AQP4Ab acting divalently and forming a bridge between immobilized AQP4 coated on ELISA plate wells and liquid-phase AQP4-biotin.
In the assay, serum samples (50 μL) together with AQP4-biotin (25 μL) are incubated in AQP4-coated ELISA plate wells. After a wash step, AQP4-biotin bound is quantitated by addition of streptavidin peroxidase followed by the substrate tetramethylbenzidine. The higher the concentration of AQP4Ab in the test serum, the greater amount of AQP4-biotin bound resulting in an increase in OD450nm thus providing a quantitative AQP4Ab ELISA. AQP4Ab concentrations in patient sera are expressed as arbitrary units/mL using a calibration curve. With a cut off for positive of 5 units/mL, all 216 healthy blood donor sera tested were negative for AQP4Ab in the ELISA, while AQP4Ab were detected in 61/62 NMO sera positive for AQP4Ab by immunofluorescence (IF) (98%) and in 5/34 (15%) NMOSD sera negative by IF. AQP4Ab ranged from 5.2 units/mL to 160 units/mL for these 66 positive samples (mean 123 units/mL, median 160 units/ mL). Furthermore, none of 26 multiple sclerosis patients, nor 201 patients with various control autoimmune diseases were seropositive for AQP4Ab. Only one sample from a patient with Graves' disease (TRAb positive) was AQP4Ab positive by ELISA (6.1 units/mL). ELISA inter-assay coefficients of variations (n = 24) were 15.9%, 13.4% and 10.7% at 7.2, 32 and 115 units/mL respectively.
Overall, this convenient AQP4Ab ELISA shows comparable if not better performance to the 'gold standard' immunofluoresecence test. Also, it has good precision with easy handling characteristics suitable for routine laboratory use worldwide.
A subset of patients with multiple sclerosis develop a pathogenic autoantibody response to neurofascin Elliott Christina ⁎ ,1 , Arthur Ariel 1 , Derfuss Tobias 2 , Brennan Kathryn 1 , Meinl Edgar 2 , Olsson Thomas 3 , Jarius Sven 4 , Barnett Sue 1 , Linington Christopher 1 1 University of Glasgow, Glasgow, United Kingdom; 2 Max-Plank Institute of Neurobiology, Munich, Germany; 3 Karolinska Institute, Stockholm, Sweden; 4 University of Heidelberg, Heidelberg, Germany
Antibodies have long been implicated in the pathogenesis of multiple sclerosis (MS) however the specific targets remain illdefined and highly controversial. In order to investigate the pathogenic potential of autoantibodies present in patients with MS, we developed an in vitro screening strategy using a myelinating culture system derived from rat embryonic spinal cord in which we can quantify antibody mediated effects on both axons and myelin.
Plasma samples were taken from a cohort of patients with either MS (n = 9), peripheral neuropathies (n = 10) or healthy controls (n = 13). The total immunoglobulin G (IgG) fraction was purified from each patient sample via protein G affinity chromatography and added to highly myelinated cultures with fresh sera as a source of complement. This revealed that the majority of patients with MS had a demyelinating autoantibody response within the total IgG fraction. In contrast this demyelinating activity was not observed using IgG prepared from patients with peripheral neuropathies or from the healthy control group.
A potential specificity of these autoantibodies may be the axoglial protein neurofascin (Nfasc). To test this we purified Nfasc specific immunoglobulins by immunoaffinity chromatography from the MS and peripheral neuropathy control group. In the presence of complement all the Nfasc-specific autoantibody preparations obtained from MS patients were found to mediate the rapid and selective destruction of myelin in vitro and in addition half of the MS samples also mediated a significant loss of axons. This axopathic response was unique to the MS samples and demyelination was only observed in two of the ten peripheral neuropathy control samples. To investigate whether depleting plasma of the Nfasc specific antibody repertoire would diminish the observed pathogenic activity we purified IgG from the flow through after Nfasc antibody purification. This revealed that in one MS case Nfasc is the dominant autoantigen as removal of the Nfasc specific component abolished all pathogenic activity. Using this model system we have been able to formally demonstrate that MS is associated with a pathogenic autoantibody response to Nfasc that can mediate axonal injury and/or disrupt myelination indicating that this response may play a significant role in driving lesion formation in vivo.
Acetylcholine receptor and MuSK antibodies in mothers of babies with arthrogryposis Jacobson Leslie ⁎ , Leite Maria Isabel ⁎ , Vincent Angela ⁎ University of Oxford, Oxford, United Kingdom Myasthenia gravis (MG) is associated with antibodies to the acetylcholine receptor (AChR) or to muscle specific kinase (MuSK) which are both localized at the neuromuscular junction (NMJ). The AChR exists in two forms; during fetal life there is a fetalspecific gamma subunit which is replaced by the adult-specific epsilon subunit. Arthrogryposis multiplex congenital (AMC) is defined by multiple fixed joints, usually resulting from lack of movement in utero and caused by many different known and unknown genetic or environmental factors. A high level of maternal antibodies to fetal AChR was identified in some rare cases of AMC (Vincent et al., 1995) , often recurring in successive pregnancies, and not always associated with maternal evidence of MG.
Recently we have established more sensitive cell-based assays for antibodies to AChR or MuSK using human embryonic kidney cells transfected with plasmids encoding AChR subunits or MuSK (Leite et al., 2008) .
Here, we used these improved assays to look for antibodies binding specifically to the extracellular epitopes of fetal or adult AChRs or to MuSK. We studied a total of 20 sera: six maternal sera known to be AChR positive, seven from AChR antibody/MuSK antibody negative mothers of AMC babies previously studied (Dalton et al., 2006) , and seven from mothers where a maternal antibody was suspected. Human embryonic kidney cells were transfected with cDNAs for either the fetal or adult AChRs and clustered with the protein Rapsyn, or for MuSK. Maternal serum was tested at 1:20 and 1:250 dilution and binding of IgG detected with a secondary antibody conjugated to an Alexa Fluor 568 (red). Five of seven new cases were found to be AChR antibody positive by routine testing, and all were negative for MuSK antibodies by routine testing. All eleven AChR antibody positive maternal sera bound to the AChR by the cell-based assay, in most cases showing greater reactivity to fetal than to adult AChR. One of these mothers had no evidence of muscle weakness, and two had only minimal signs. Of the nine without AChR antibodies, one was positive for MuSK antibodies using the cell-based assay. The clinical data will be described.
The results confirm that maternal antibodies to AChR are a rare but potentially important cause of AMC. The more sensitive assays of potentially fetal-pathogenic antibodies will help to identify those mothers at risk. We generated N70 unique bivalent IgG1 monoclonal recombinant Abs (rAbs) by co-expressing the paired heavy-and light-chain V region sequences amplified from MS CSF plasma cells by single cell PCR in human tissue culture cells. In this study, rAbs were assayed for binding to glia and oligodendrocytes, including the 1321N1 astrocytoma cell line, fetal astrocytes and differentiating rat CG4 oligodendrocytes.
We identified a subset of MS rAbs that demonstrated a punctate staining of human astrocytes and 1321N1 cells. Although derived from a human astrocytoma, 1321N1 cells do not express GFAP, but instead express proteolipid protein and CNPase, markers more characteristic of an oligodendrocyte lineage. To further explore binding to oligodendrocytes, Abs were used to stain differentiating rat CG4 cells in conjunction with the stage-specific markers A2B5, O4 and MBP. Approximately 15 rAbs were identified that displayed clear vesicular staining of rat CG4 cells. For most immunopositive rAbs, staining was most pronounced in O4-and MBP-positive cells. None of the reactive MS CSF rAbs recognized proteins under denaturing conditions.
We have identified a subset of MS CSF rAbs that bind to antigens expressed by both fetal astrocytes and oligodendroglia. Based on the absence of staining to proteins in glial cell lysates, epitopes are likely conformational in nature. Epitopes may also be located within lipid rafts based on the pattern of staining, sensitivity to detergents, and studies showing the binding of some MS CSF rAbs to heteromeric myelin lipid complexes containing sulfatide and galactocerebroside (see poster presentation by Kathryn Brennan et al.). The molecular identity of antigens and their association with disease prevalence and pathogenesis are being pursued.
Antibodies to the voltage-gated potassium channel-associated complex: LGI1 and CASPR2 as antigenic targets in central and peripheral nervous system immunotherapy-responsive diseases Irani Sarosh R., Waters Patrick, Pettingill Philippa, Zuliani Luigi, Buckley Camilla, Lang Bethan, Vincent Angela
Patient sera that immunoprecipitate 125I-a-dendrotoxin-labelled VGKCs from digitonin extracts of rabbit brain tissue are defined as having voltage-gated potassium channel antibodies (VGKC-Abs). VGKC-Abs are associated with the peripheral nerve disease neuromyotonia (NMT), the central nervous system disease limbic encephalitis (LE) and with Morvan's syndrome (MoS), which involves both the peripheral and central nervous systems. These are all immunotherapy-responsive conditions. a-Dendrotoxin binds three VGKCs: Kv1.1, 1.2 and 1.6. Our aim was to determine which of the Kv1s were bound by each VGKC-Ab positive serum.
Initially, we extracted the Kv1-transfected HEK cells in digitonin and labelled the extract with 125I-a-dendrotoxin; however, only three of 104 sera precipitated significant levels of 125I-a-dendrotoxin-Kv1s. This suggested that the patient sera did not bind directly to the Kv1s but, instead, to a protein complexed with Kv1s in the digitonin-solubilised rabbit brain extracts. Indeed, when small amounts of a harsher detergent, SDS, were added to the rabbit brain digitonin extracts, the antigenic targets of the patient sera could be dissociated from the 125Ia-dendrotoxin-Kv1s (pb 0.0001 at 0.025% SDS).
We then tested immunoprecipitation of Kv1s from brain tissue digitonin extracts using commercial antibodies to proteins reported to complex with VGKCs. Antibodies to LGI1 and CASPR2, precipitated around 60% and 20% of the Kv1-complexes, respectively. We, therefore, expressed LGI1 and CASPR2 in HEK cells and incubated patient sera with these unpermeabilised cells. 46/104 (44%) and 28/ 104 (27%) sera bound the surface of LGI1 and CASPR2-transfected cells, respectively. These sera also bound the surface of live rat hippocampal neurons.
Finally, we found that CASPR2-antibodies were present in 85% of the MoS, 59% of the NMT and only 9% of the LE cases. In striking contrast, LGI1-antibodies were detected in 60% of the LE, only 15% of the MoS and none of the NMT cases.
These findings define new targets for immune-mediated neurological syndromes and suggest a molecular basis for the heterogeneity of the clinical syndromes.
Ngamli Fewou Simon, Rupp Angelika Frances, Nickolay Lauren Emma, Carrick Kathryn, Willison Hugh J.
Guillain-Barré syndrome (GBS) is the prototypic post-infectious disorder of the peripheral nervous system. GBS is often associated with pathogenic anti-ganglioside antibodies (AGAbs) that target specific peripheral nerve compartments such as the node of Ranvier and neuromuscular junction (NMJs). These sites are enriched in several ganglioside species and the distal motor nerve is especially vulnerable as it lacks a functional blood nerve barrier. Despite strong experimental evidence, little clinical data supports the vulnerability of NMJs in GBS. The severity of any injury should be determined by the concentration of membrane-bound AGAbs and we therefore hypothesized that antibody internalization at the pre-synaptic NMJ plasma membrane relatively desensitises this compartment compared with others from complement-mediated attack.
In this study, we have used mouse monoclonal anti-GM1, -GD1a, and -GD1b AGAbs and differentiated PC12 cells or mouse triangular sterni (TS) in vitro nerve muscle preparations to test this hypothesis. Pre-incubation of the AGAbs with PC12 cells at 37°C followed by exposure to normal human serum (NHS) as a complement source drastically reduces the cytotoxic effects mediated by complement. In TS preparations, plasma membrane ganglioside immunoreactivity was significantly reduced following AGAb incubation at 37°C, compared with 4°C. Likewise, immunohistological analysis showed a significant reduction of C5b-9 deposited at the NMJs of TS preparations incubated with AGAbs at 37°C, before exposure to the NHS. Similarly, the number of injured junctions was reduced when mouse sternomastoid muscles were labelled in vivo with AGAb, and then exposed to the NHS. Further studies using PC12 cells showed that internalized AGAbs were partially recycled at back to the cell surface and partially degraded in the lysosomes.
These results demonstrate that internalization of AGAbs attenuates NMJ injury in a murine experimental model of GBS. Myasthenia gravis (MG) is a B-cell mediated disease. B-cell activating factor (BAFF) plays an essential role in B-cell homeostasis. When over-expressed, BAFF protects B-cells from apoptosis, thereby contributing to autoimmunity. Patients with MG have higher levels of BAFF in the circulation compared to healthy subjects. BAFF-R and TACI (transmembrane activator and cyclophilin ligand interactor) are two cell-surface receptors that bind BAFF ligand. In naïve B-cells, the BAFF-R is the main receptor that mediates BAFF signals. When activated, B-cells downregulate BAFF-R and upregulate TACI expression. We postulated that the ratio of BAFF-R to TACI might be altered in patients with MG. Our aim was to determine the frequency of B-cells that express BAFF-R and TACI, and to determine the intensity of expression of each receptor on the B-cell surface.
MG patients (n = 15) were compared to healthy control subjects (n = 21). Cell-surface expression of BAFF-R and TACI was determined by immunofluorescent staining of peripheral blood mononuclear cells and flow cytometry analysis. We determined the frequency of B-cells that express BAFF-R and TACI, and the mean fluorescence intensity (MFI) of expression. The frequency of B-cells that express BAFF-R was 78.3 ± 2.2 in controls and 80.1 ± 3.6 in MG patients; the MFI of BAFF-R expression was 2440 ± 201 in controls and 2046 ± 424 in MG patients. The frequency of B-cells that express TACI was 15.0 ± 1.9 in controls and 24.3 ± 4.6 in MG patients; the MFI of TACI expression was 125 ± 14 in controls and 150 ± 31 in MG patients. The BAFF-R: TACI MFI ratio was 23.7 ± 3.0 for controls and 16.3 ± 2.6 for MG patients.
Our data show that the majority of circulating human B-cells express cell-surface BAFF-R. There is no difference between healthy subjects and MG patients in the frequency of B-cells that express BAFF-R. Some of the MG patients have a high frequency of B-cells that express TACI. Our data also show that, in patients with MG, the intensity of BAFF-R expression is lower while the intensity of TACI expression is higher than in healthy subjects. Both the frequency and intensity analyses show that there is a lower ratio of BAFF-R to TACI in MG patients. These data suggest that there is a higher frequency of activated B-cells in patients with MG. This study was supported by the Muscular Dystrophy Association (MDA).
Hertzenberg Deetje 1 , Betz Martina 1 , Lehmann-Horn Klaus 1 , Zamvil Scott 2 , Lalive Patrice 3 , Hemmer Bernhard 1 , Weber Martin ⁎ ,1 1 Technische Universitaet Muenchen, Munich, Germany; 2 University of California, San Francisco, San Francisco, United States; 3 University of Geneva, Geneva, Switzerland B cells may have a dual role in the pathogenesis of multiple sclerosis (MS) serving as source for antibody-secreting plasma cells as well as acting as antigen presenting cells (APC) for activation of selfreactive T-cells. Based on this assumption, B cell depletion has gained substantial interest as potential therapy. Evidence suggests however that certain B cell subsets have regulatory properties in centralnervous-system (CNS) autoimmunity. We investigated whether glatiramer acetate may specifically modulate pro-inflammatory B cell function in experimental autoimmune encephalomyelitis (EAE).
First, C57BL/6 mice were injected subcutaneously with 150 μg GA in PBS for 7 days followed by immunization with MOG p35-55. B cells were isolated from secondary lymphoid organs prior to or 12 days after immunization. In naïve and immunized GA-treated mice, B cells expressed lower levels of activation markers CD25 and CD69. Whereas expression of co-stimulatory molecules CD40 and CD80 was reduced, CD86 expression was enhanced; surface levels of MHC II remained unchanged. In vivo GA-treated B cells secreted less proinflammatory TNF and IL-6 and more anti-inflammatory IL-10. When used as APC, B cells isolated from GA-treated mice revealed a decreased capability to promote differentiation of naive MOG p35-55 specific T-cells into encephalitogenic Th1 and Th17 cells. Second, C57BL/6 mice were injected with B cell-depleting murine anti-CD20 or isotype prior to MOG p35-55 immunization. Upon disease onset, mice were treated daily with 150 μg GA. Independent of the pretreatment with anti-CD20 or isotype, GA treatment ameliorated EAE severity which was associated with development of regulatory (type II) monocytes secreting less TNF, IL-12 and more IL-10.
A) GA-treatment reduces the ability of B cells to promote development of Th1 and Th17 cells. Immune modulation of B cell APC function may be a valuable approach to selectively target a proinflammatory B cell property without abrogating B cell regulation. B) Despite its immunomodulatory effect on B cells, clinical benefit mediated by GA does not rely on the presence of CD20+ B cells. This finding suggests that combination of anti-CD20 and GA may be a promising therapeutic strategy which may also enhance sustainability of clinical benefit after cessation of therapeutic B cell depletion. Although most of the studies focused the attention on the parenchymal lesions (plaques), several evidence implied a primary role for the extraparenchymal structures, such as the meninges, to the central damage. A central issue related to the role of the recently described meningeal B cell aggregates in MS CNS is whether they relate to the B cell infiltrates found at the sites of parenchymal lesions in MS brain.
To address this question, we characterized the B cell repertoires derived from meningeal aggregates and the corresponding parenchymal lesions of three MS brains, by sequencing the variable region of the B cell receptor heavy chain (BCR VH region). In two MS brains, most of the expanded antigen-experienced B cells derived from meningeal aggregates were present in the plaques as well. We extended the analysis to the B cell repertoires derived from different areas of additional eleven MS brains, including meninges, plaques, NAWM and CSF. The majority of the B cell clones were exclusive to each location. However, several antigen experienced clones that were shared among different anatomical areas were detected.
This study highlighted the direct contribution of the extraparenchymal sites such as the meningeal aggregates to the parenchymal infiltrates. Moreover, we defined an extensive network of antigen experienced B cell populations within all the MS CNS areas. Further characterization of the molecular patterns involved in the migration of specific B cell subpopulations among the different sites of inflammation might provide crucial insights on the pathological mechanism of the disease.
Circulating immune complexes in patients treated for multiple sclerosis Iuliano Gerardo ⁎ , Cinquanta Luigi, Napoletano Rosa, Ferruzzi Annamaria
Circulating immune complexes (CIC) in patients with multiple sclarosis (MS) were studied with interesting but uncertain results. Their prevalence in acute, progressive, remission, and stable MS was calculated 33.3%, 30.2%, 26.1%, and 23.1% respectively. However, literature data are seasoned; there are no studies about immunomodulating treatments, few and contrasting on steroids and cyclophosphamide. In this paper the frequency of CIC elevation is assessed in MS patients with different therapies.
Our cohort of MS treated patients is tested yearly for autoantibodies, and other tests as complement and CIC. CIC were assayed by nephelometry; normal cutoff up to 3 mcg/ml. We collected 2005, 2006 and 2007 data, including 169 MS, 56 males, 110 females, age onset 11-59 years, mean 29.39; 136 with relapsing-remitting disease; 30 in secondary progressive phase.
Treatments in 2005 were: Intramuscular Interferon (IFN) b1a once a week: 37; subcutaneous full dose IFN b1a and 1b: 76; copolimer (COP): 18; mitoxantrone (MIX): 8; azathioprine (AZA): 5; IFN and AZA in association: 3; no therapy (NO): 15.
We calculated also "person-year" data, for a crude total of 466 "person-years".
Main outcome was the frequency of CIC in the patients grouped for sex, MS clinical parameters (relapses and EDSS score), MRI features, and therapy. Prevalences and incidences were calculated. Crosstabulations with odds ratios and chi-square were used to test frequencies.
Prevalence was calculated on the presence of one positivity in the three years followed; overall it was 48.19% (80 on 166); it was slightly higher in patients with COP (59%) and low dose IFN (57%); slightly lower in those with full dose IFN (43%); and significantly lower (10%) in those with immunosuppression (MIX).
Incidence was calculated on positivization of tests in the second year. Overall it was 19.5%. It was slightly higher in females (21.4%), and in patients with COP (37.5%). Patients with MIX had no incidence.
Year-person method enhanced statistics; though, only immnunosuppressives (MIX, AZA or combined IFN-AZA) showed significantly lower risk (between 0.15 and 0.25).
The high prevalence of CIC confirms the impression that humoral immunity plays a key role in MS. The differences among therapies could be interpreted in relation to specific pharmacodynamics: immunosuppression for mythoxantrone, fake antigen for copolymer, and different decreases for different dosed interferons. Myasthenia gravis (MG) is a heterogenous anti-receptor autoimmune disease characterized by autoantibodies (Ab) to nicotinic acetylcholine receptor (AChR) of the neuromuscular junction in 85-90% of patients (AChR Ab positive: AP) or Ab against musclespecific kinase (MuSK) (MuSK positive: MP) in up to 40% of anti-AChR negative patients (Seronegative: SN). Autoantibody related mechanisms lead to impaired neuromuscular transmission and muscle weakness in different subgroups of disease. Differential cytokine and CD40L expression in MG is compared with rheumatoid arthritis (RA) patients and healthy controls (CON) in this study.
CD19+B and CD4+T cells were magnetically isolated (MACS) from peripheral blood of 63 MG (Median age (Ma): 37 years, 41 women) (27 AP, 18 MP and 18 negative for both Ab (SN)), 13 RA (Ma: 52 years) patients and 17 CON (Ma: 44 years). 56% of the MG patients were on corticosteroids. Expression of IL10, IL12A (IL-12p35) and IL6 in B, and IL10, IFNG and CD40L (CD154) in T cells was determined by using semi-quantitative RT-PCR (Sybr Green). Expression levels were evaluated with GAPDH gene by comparing 2 -DD CT and analyzed by non-parametric tests.
CD40L gene expression of CD4+T cells was lower in MG (10.0, p = 0.003) and in RA (7.7, p = 0.0001) than in CON (15.5) and this finding also held true in patients without corticosteroid treatments (11.8, p = 0.023 and 6.8, p = 0.0001, respectively). All subgroups of MG (AP, MP and SN) had lower CD40L gene expression than CON (p = 0.002, p = 0.005 and p = 0.024, respectively), with no difference from each other. On the other hand, MG patients had relatively higher levels of CD40L gene expression than the RA group (p = 0.046). No other B and T cell cytokine gene expression was different between the groups.
Relatively lower expression of CD40L in T cells without differences in IL10 or IFNG makes the differential effects of T cells in regulating Ab production unlikely in MG and its subgroups.
This study is supported by TUBITAK.
Epilepsy -An autoimmune disease?
Brenner Tanja To investigate whether potentially pathogenic autoantibodies are present in certain forms of epilepsy, sera were collected from newlydiagnosed epilepsy patients, taken before the start of a clinical treatment trial (n = 185) and from a cohort of consecutive epilepsy patients attending specialist clinics (n = 236).
All sera were screened for antibodies against voltage-gated potassium channels (VGKC), voltage-gated calcium channels (VGCC), glutamic acid decarboxylase (GAD), N-methyl-D-aspartate receptor (NMDAR), and glycine receptor (GlyR) and compared to sera from healthy and disease controls (n = 160).
Increased VGKC antibody titres were found in 6.5% of newlydiagnosed patients, compared to 3.1% in the consecutive patient group and 1% of controls. VGCC antibodies were not detected in either cohort, whereas high titre GAD antibodies (N100 U/ml) were found in 1.5% in both patient groups. Interestingly, the proportion of patients with NMDAR antibodies was markedly higher in the newly diagnosed patients (7%) compared to the consecutive patients (2.5%). The response of the newly-diagnosed epilepsy patients, with or without antibodies, to treatment with antiepileptic drugs (AED) was compared. The results show a tendency for the antibody-positive patients to do less well on AED treatment than the sero-negative patients. However due to the low number of patients, the results did not reach significance (Chi-squared p = 0.059).
The results suggest an immune-mediated component in some forms of epilepsy, although further studies will be required to establish whether the antibodies are directly pathogenic or merely markers of a secondary immune response.
Further characterization of the B-cell phenotype in ectopic follicle-like structures in the multiple sclerosis brain Magliozzi Roberta ⁎ ,1 , Serafini Barbara 1 , Rosicarelli Barbara 1 , Reynolds Rychard 2 , Aloisi Francesca 1 1 Istituto Superiore di Sanità, Rome, Italy; 2 Imperial College, London, United Kingdom B-cell follicle-like structures have been detected in the postmortem cerebral meninges of approximately 45% of the examined patients with secondary progressive multiple sclerosis (SPMS) and their presence has been associated with more extensive cortical damage and more severe disease clinical course. Although these structures show some features of germinal centers (GC), such as follicular dendritic cells, proliferating B-cells, plasma cells and expression of activation-induced cytidine deaminase, their role in supporting a true GC reaction is still unclear. Furthermore, the finding that these ectopic lymphoid structures comprise a high frequency of B cells infected with Epstein-Barr virus (EBV) raises the suspicion that their formation might result from the ability of the virus to induce B-cell proliferation and maturation through latency proteins, independently of antigen and T-cell help.
In this study we selected brain tissue blocks from 10 SPMS cases containing meningeal B-cell follicle-like structures and analyzed them for expression of CD10 using immunohistochemical techniques. CD10 first appears on pro-B cells during lymphocyte differentiation, is lost during maturation to naïve B cells and reappears on the cell surface during antigen-dependent GC maturation. We found that CD10 was abundantly expressed in lymph node GC but was not detected or was expressed in few cells in B-cell follicle-like structures in MS cerebral meninges. In parallel, we demonstrated that most intrafollicular B cells in the MS brain express the memory marker CD27.
Despite expression of some GC features, ectopic B-cell follicle-like structures developing in the MS brain mainly comprise antigenexperienced B cells. These findings suggest that the mechanism responsible for the formation and maintenance of such atypical structures might be EBV-associated and not canonical, antigen-driven B-cell activation. This work was supported by the EU FP6 Neuropro-MiSe and Italian Multiple Sclerosis Foundation. MOG is localized on the surface of myelin sheaths and therefore accessible for antibody binding. Anti-MOG antibodies have been linked to myelin damage in MS lesions and reported to be higher in the serum and CSF of MS patients relative to controls. In addition, anti-MOG antibodies are capable of inducing demyelination both in vitro and in vivo.
To define the genetic regulation of the anti-MOG IgG EAE is a starting point for dissecting causative disease pathways that involve antibody response.
Three different populations derived from susceptible DA and resistant PVG rats, were used. 421 (DAxPVG)F1xDA, 471 (DAxPVG) F1xPVG and 894 (DAxPVG) G10 advanced intercross line (AIL) rats were induced with EAE. Genotyping has been performed using microsatellite markers. Serum anti-MOG IgG isotypes, total IgG, IgG1, IgG2b and IgG2c, were evaluated at the onset and late stage of disease using ELISA method.
Linkage analysis in backcrosses revealed multiple IgG-regulating loci demonstrating polygenic control of anti-MOG antibody response. We also observed distinct loci that regulate IgG levels at the onset of disease compared to late stages. For example IgG2b production at the onset is regulated by a locus on chromosome 8, whereas a region on chromosome 10 determines its late stage response. A striking influence on almost all of IgG isotypes is conferred by a locus on chromosome 4 with PVG allele driving higher IgG levels. Using the AIL we significantly reduced the confidence interval of this locus, from 25 Mb to 2 Mb, containing only few genes including antigen presenting lectin-like type receptor complex. A congenic strain, that carries PVG alleles at this locus on the susceptible DA background, confirmed that the locus regulates EAE, B cell numbers and IgG levels. IgG2b and IgG levels were higher in congenic rats compared to DA controls. Interestingly, this correlated with less severe disease.
There is a distinct genetic regulation on the production of the different IgG isotypes at the onset and late phase of disease and genes on chromosome 4 are the major regulators of anti-MOG IgG levels. We examined a large cohort of patients, who included 47 from the University of Heidelberg with diagnoses of stiff person syndrome (SPS), PERM or hyperekplexia (HypEX), 145 sera referred for routine analysis from patients with a possible diagnosis of PERM or SPS, and in 224 healthy or disease controls. We used a cell-based indirect immunofluorescence assay (CBA), using GlyR alpha-1-expressing HEK cells. Sera (diluted 1:20) and or CSF (measured undiluted) were tested and the results scored according to visual score as described previously for AChR antibodies (Leite et al., 2008) .
GlyR-Abs in healthy individuals (n = 14) and neurological diseases (n = 224 including 10 MS, 90 AQP4-Ab+, 15 MG, 12 VGKC-Ab+, 12 TSP and 85 NMDA-Ab+) were negative. From the 47 patients, positive serum values were detected in 12 patients with diagnoses of either PERM, hyperekplexia, or SPS. One patient had both GlyR and GAD antibodies. Most of the patients with available sera and CSFs produced similar scores (serum diluted and CSF undiluted), but in five individuals the undiluted CSFs were positive at a time when the sera at 1:20 were negative. Positive results were also found in 25 of the 145 sera from patients with symptoms suggestive of PERM or SPS, or of other syndromes (retinal, brainstem or spinal cord disturbance); these patients had histories suggesting an acquired autoimmune disorder. We found that GlyR-Abs showed variable but often very high intrathecal synthesis. Clinical manifestations and response to immunotherapies were compared between patients with or without GlyR-Abs.
We conclude that GlyR-Abs are positive in some patients not only with PERM, stiff person syndrome, or acquired hyperekplexia but also in some other conditions in which GlyRs may be implicated. GlyR-Abs are now part of the growing number of (predominantly) non-paraneoplastic antibodies to CNS channels or receptors in potentially immunotherapy-responsive neurological disorders. At the moment, there is an ongoing need for early diagnosis of multiple sclerosis (MS). This may be accomplished by identification of MS-specific biomarkers. The aim of this study is to identify a panel of early MS-associated antigenic targets suitable as biomarkers. Since cerebrospinal fluid (CSF) of MS patients is characterized by the presence of oligoclonal band antibodies, it is a suitable source for this purpose.
To identify markers for early disease, autoantibody profiling was performed with a phage display technique, called serological antigen selection (SAS), on pooled CSF from Clinically Isolated Syndrome (CIS) patients (n = 4), who developed definite MS, early Relapsing Remitting (RR) MS (n = 6) and Primary Progressive (PP) MS (n = 6) patients. This procedure is based on expression of a cDNA library fused to filamentous phage protein VI and interactions between exposed proteins and Immunoglobulin G (IgG) antibodies. We produced two cDNA display libraries: first a normalized cDNA library derived from active chronic MS plaques, with varying degrees of demyelination and inflammatory activity (10 6 primary recombinants) was used. The second cDNA library used, is constructed from normal brain with a primary diversity of 0.9 × 10 6 recombinants. cDNA inserts from each library were cloned into our pVI phage display vector system, resulting in a library size of 1.1 × 10 7 colony forming units (cfu) and 5.3 × 10 6 cfu, respectively. The results of the SAS procedures were characterized with PCR and restriction digestion analysis. Subsequently, enriched putative antigenic targets displayed on phage were identified with sequence analysis. This led to the identification of 18 CSF candidate antigens for CIS, 11 for RRMS and 13 for PPMS. Some of the enriched clones identified were identical, although derived from separate SAS procedures with the two cDNA libraries, indicating a specific antibody response. Clones that have been identified show also homology with MS-related proteins. However, no antibody reactivity against these targets has been described so far.
We have identified several candidate markers for MS and to confirm their specific immunoreactivity, phage ELISA will be performed on CSF and serum of individual patients used in the SAS procedure, additional MS patients and controls with other (neurological) diseases. Eventually, the identification of these MS-associated antigenic targets may help in early diagnosis and predict future disease progression.
Yu Xiaoli ⁎ , Green Miyoko, Lam Chiwah, Bautista Katherine, Gilden Donald, Bennett Jeffrey L.
Neuromyelitis optica (NMO) is a severe inflammatory demyelinating disease that predominantly affects the optic nerves and spinal cord. It is the first CNS demyelinating disease in which an antibody directed against autoantigen aquaporin 4 (AQP4) plays a role in the pathogenesis of disease. NMO IgG autoantibodies bind to extracellular epitopes on the AQP4 protein. We previously demonstrated that more than 50% of recombinant antibodies (rAbs) generated from clonally expanded plasma cells in the cerebrospinal fluid (CSF) of an AQP4 seropositive NMO patient were directed against AQP4, and reproduced NMO pathology in the EAE model. Our goal in the present study was to identify epitopes/mimotopes of AQP4-specific CSF NMO rAbs.
We screened linear (12-mer) and circular (7-mer) phagedisplayed random peptide libraries with eight NMO rAbs generated from a single patient. Potential target peptides were isolated from phage peptide libraries by three to five rounds of panning on NMO rAb bound to Protein A-coated microtiter plates. Three NMO rAbs selected a total of 7 high affinity phage linear peptides, and one rAb identified 7 circular peptides. The affinity of each selected peptide was further defined by dose-response and inhibition immunoassays. Western blots demonstrated that peptides recognized by two NMO rAbs represented linear epitopes, and sequence alignment results showed that they shared sequence homology with NMO autoantigen AQP4 in the extracellular Loop-A region.
We have identified several peptides that bind to AQP4-specific rAbs generated from clonally expanded NMO CSF plasma cells. Some of the peptides have homology to protein sequence on the extracellular surface of AQP4. Peptide epitopes/mimotopes may have potential uses for disease prognosis, monitoring, and therapy. Peripheral blood mononuclear cells (PBMC), isolated peripheral immunoglobulin G+ (IgG+) B cells or CSF cells were infected with Epstein-Barr virus (EBV) in the presence of irradiated allogeneic PBMC and B cell stimulating factors to obtain continually dividing B cell lines. B cell immortalization was verified by screening the culture supernatant for antibody production using dot blotting. Clonality of the immortalized B cell lines was verified using a B cell spectratyping procedure. To screen for autoreactivity, binding of the produced antibodies to a human oligodendroglioma (HOG) cell line, PBMC and an epithelial alveolar carcinoma cell line (A549) were analyzed by flow cytometric analysis.
We obtained 288 immortalized B cell lines from 15 MS patients, 29 B cell lines from 8 patients with a non-or other inflammatory neurological disease (NIND/OIND) and 37 B cell lines from 3 patients with a clinically isolated syndrome (CIS). Most B cell lines were obtained from the PBMC although 7 B cell lines were isolated from the CSF of 1 MS patient and 2 from 1 NIND patient. B cell spectratyping analysis indicated that more than 82% of the immortalized B cell lines were monoclonal, eliminating the need for cloning. HOG-specific intracellular binding was shown for antibodies from 5 immortalized B cell lines, all generated from PBMC of MS patients. Intracellular binding to both HOG and A549 cells but not to PBMC was demonstrated for 37 other B cell lines, while binding to all examined cell types was found in 37 immortalized B cell lines. Further, intracellular binding to HOG cells was confirmed for several of the immortalized B cell lines by immunocytochemistry.
B cell immortalization has proved to be a useful method for the production of antibodies. Autoreactivity of the generated monoclonal antibodies has been demonstrated and is now further examined by detecting antibody binding to healthy and diseased brain tissue from human and rhesus monkey. Moreover, characterization of the obtained B cell lines based on diversity is currently in progress by sequencing the immunoglobulin heavy chain genes. Hashimoto's encephalopathy (HE) is a steroid-responsive neurological disorder associated with elevated serum thyroid antibody (Thy Ab) concentrations, and previous reports have suggested the presence of antibodies to neuronal proteins which could potentially be pathogenic. To assess the relevance of anti-neuronal antibodies in HE, we investigated the sera of five acute onset encephalopathy patients with elevated serum Thy Abs and no identifiable etiology. Sera of Thy Ab positive and negative patients with acute and chronic neurological diseases, including multiple sclerosis, and of Thy Ab positive and negative healthy individuals served as controls.
All sera were examined by immunohistochemistry using frozen rat brain sections, immunocytochemistry using cultured rat hippocampal cells, radioimmunoassay for voltage-gated potassium channel (VGKC), voltage-gated calcium channel and glutamic acid decarboxylase antibodies, a cell based assay for N-methyl-Daspartate receptor antibodies and paraneoplastic antibodies using a commercial kit. A strikingly high proportion (24/34) of the Thy Ab positive patients (18/25) and the Thy Ab positive healthy individuals (6/9) had anti-neuronal antibodies as opposed to the Thy Ab negative cases (3/56) by immunohistochemistry. We failed to identify a distinctive immunoreactivity pattern that was unique to HE patients. Three of the five HE cases showed evidence of antibodies to cell-surface antigens which could be pathogenic, although only one was against a defined antigen (VGKC). None of the sera examined showed antibodies to other defined cell surface or paraneoplastic antigens.
The antibodies found in the serum of HE patients may merely reflect an increased susceptibility to anti-neuronal autoimmunity in patients with susceptibility to autoimmune thyroid disease. Further work is required to try to identify specific neuronal targets in this condition, and to see whether there is an antibody specific for the disease. Alternatively, it may be that HE does merely represent a heterogenous group of encephalopathies mediated by diverse antibody-mediated mechanisms. Clinical trials with the B-cell depleting antibody rituximab support a critical pathogenic role of CD20+ B-cells in multiple sclerosis (MS). However, the immunopathogenic mechanism in which B-cells are involved is currently unknown. We have investigated the effect of Bcell depletion on central nervous system (CNS) pathology and cellular and humoral immune mechanisms in a preclinical MS model, i.e. experimental autoimmune encephalomyelitis (EAE) in the common marmoset.
We used a human-anti-human CD20 IgG1 antibody that crossreacts with marmoset CD20. EAE was induced in thirteen marmosets by immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA. After 21 days, seven animals received an intravenous loading dose of 20 mg/kg anti-CD20 antibody, followed by a weekly intravenous dose of 5 mg/kg. The antibody induced profound and long-lasting B-cell depletion from PBMC and lymphoid organs throughout the observation period of 105 days. Whereas all placebo-treated control monkeys developed clinically evident EAE, overt neurological deficits were substantially reduced in three antibody treated monkeys and four antibody treated monkeys remained completely asymptomatic. B-cell depletion reduced inflammation and demyelination in the brain, spinal cord, and optic nerve. Strikingly, none of the antibody-treated animals contained grey matter lesions, in contrast to five of the six control animals. Immunoglobulin levels specific for rhMOG were reduced in treated monkeys. Furthermore, B-cell depletion led to an altered distribution of monocytes and activated T-cells, i.e. increased number of monocytes in blood, increased T-cell proliferation in blood, and reduced Tcell proliferation and IL 17A production in lymphoid organs.
In conclusion, B-cell depletion prevents the development of clinical and pathological signs of EAE, which is associated with altered location and activity of MOG-reactive T-cells.
Novel highly sensitive laboratory diagnosis of myasthenia gravis and neuromyelitis optica in routine use Tzartos Socrates, Trakas Nikolaos, Tzartos John ⁎ , Stergiou Christos, Zisimopoulou Paraskevi
The detection of autoantibodies against acetylcholine receptor (AChR) or Muscle Specific Kinase (MuSK) by radioimmunoprecipitation assays is probably the best available diagnosis for myasthenia gravis. Similarly, detection of autoantibodies against aquaporin-4 (by various approaches) is strongly indicative for the presence of neuromyelitis optica (NMO). However, no such autoantibodies are detected in some MG or NMO patients' sera, probably due to the limited sensitivity of the available techniques and to the binding of most produced autoantibodies on the target autoantigen, thus leaving too few autoantibodies in the circulation.
Despite the fact that radioimmunoprecipitation assays are quite sensitive, there is a limitation derived from the fact that only small amounts of test sera (~5 μl) may be used. Increasing the serum volumes usually results in excessive background radioactivity. We overcame this problem by a novel two-step radioimmunoprecipitation assay, which allows the use of high volumes of patients' sera, while the background radioactivity remains low. By this method, the sensitivity increased more than 10-fold compared with that of regular assays. Sera with previously ambiguous titers (bound radioactivity values too low to be significant), for anti-AChR, anti-MuSK or anti-aquaporin-4 antibodies, were found clearly either positive or negative. Moreover, sera that have been previously characterized as negative were found positive with this 2-step assay, important for the treatment of these patients.
These highly sensitive radioimmunoassays should be invaluable for the diagnosis of MG and NMO in patients with minute amounts of circulating autoantibodies. These assays are currently in routine use in our Institute.
Pathogenic antibodies in neuromyelitis optica: Increased sensitivity of cell-based assay with clustered aquaporin-4
Waters Patrick ⁎ , Vincent Angela, Leite M. Isabel
Cell-based assays in which native antigens are expressed on the surface of transfected cell lines (often human embryonic kidney HEK cells) are proving to be the best approach to detect potentially pathogenic autoantibodies to membrane proteins. Neuromyelitis optica (NMO) is a serious inflammatory demyelinating disease of the central nervous system (CNS). Antibodies against the predominant water channel of the CNS, aquaporin-4 (AQP4), are implicated in the disease pathogenesis and have been detected, by a variety of assays (Waters et al., 2008) , in around 70% of clinically-definite NMO patients. We previously showed that N50 sera from myasthenia gravis patients negative for binding to acetylcholine receptors in solution, could bind strongly when the acetylcholine receptor was clustered on the surface of HEK cells (Leite et al., 2008) . Here we asked whether clustering of AQP4 would also increase the sensitivity of testing for antibodies in NMO.
AQP4 is expressed as two major isoforms, M1 and M23. Both form tetramers, but only the M23 isoform produces large orthogonal arrays of hundreds of tetramers, both in vivo and in transfected HEK cells. We found that antibodies to AQP4 can be detected in many NMO sera using either M1 or M23, but FACS analysis of binding to the clustered M23 isoform was substantially increased compared with binding to M1, (MFI 423.9 v 194.2; control sera −2.4 v − 4.1). As a result, we found an increase in the number of positive results from 23 to 32 in one month of routine serological testing. Seven of eight sera that were only positive on M23 cells were from patients with NMO-spectrum disorders and one had been diagnosed as ADEM/MS. The clinical diagnosis on the ninth is not yet available.
Thus use of M23 transfected cells provides a more sensitive assay for anti-AQP4 antibodies, and clustered antigens are likely to be helpful in detection of other autoantibodies to specific membrane proteins (e.g. NMDA receptor antibodies).
Plasma cell-like B cells produce aquaporin4 autoantibody in neuromyelitis optica Norio Chihara ⁎ ,2 , Toshimasa Aranami 2 , Wakiro Sato 2 , Yusei Miyazaki 2 , Sachiko Miyake 2 , Tomoko Okamoto 3 , Masafumi Ogawa 3 , Tatsushi Toda 1 , Takashi Yamamura 2 1 Kobe University Graduate School of Medicine, Kobe, Japan; 2 National Institute of Neuroscience, NCNP, Tokyo, Japan; 3 National Center Hospital, NCNP, Tokyo, Japan Neuromyelitis optica (NMO) is an inflammatory disease primarily affecting the optic nerve and spinal cord. Serum autoantibody against aquaporin-4 (AQP4-Ab) has been shown to be a disease-specific NMO marker. Increasing evidence suggests that this autoantibody may play a pathogenic role in NMO. The source of this autoantibody can be a therapeutic target of the disease. However, it has not been identified yet. Because a proportion of NMO patients are reported to have other autoantibodies such as anti-nuclear or anti-SS-A/SS-B antibodies, NMO might share autoantibody producing mechanism with other autoimmune diseases. B cell lacking CD180 is reported as a producer of autoantibodies in other autoimmune diseases. CD180 is a member of the leucine-rich repeat family of molecules that is highly expressed by naïve and memory B cells, but not by plasma cells. The purpose of this study is to investigate the characteristics of an AQP-Ab producer in NMO.
Twenty-four patients seropositive for AQP4-Abs, 20 healthy subjects (HS), and 11 conventional multiple sclerosis (CMS) patients were enrolled in this study. Each patient's B cell subpopulation in the peripheral blood was enumerated by flow cytometry and purified with a cell sorter for assessment of B cell associated gene expression and AQP4-Ab production. AQP4-Abs titers in the supernatant of purified B cell subpopulations were measured by an immunofluorescence assay using human AQP4 transfectants. Effects of the cytokines on survival and activation of AQP4-Abs producing cells were analyzed in vitro.
We report here a small B cell subpopulation, which lacked CD180 and exhibited several similarities with plasma cells, significantly increased in the peripheral blood of AQP4-Abs seropositive patients compared to HS and CMS patients (p b 0.05). The frequency of these plasma cell-like B cells (PCB) remarkably increased during relapse of NMO patients. Most notably, PCB spontaneously produced AQP4-Abs, whereas other B cell subpopulations did not. We also found that interleukin 6, a proinflammatory cytokine increased in NMO patients, enhanced the survival of PCB and their AQP4-Ab secretion.
These results provide a novel therapeutic strategy for NMO that specifically targets PCB. Voltage-gated potassium channel antibody (VGKC-Ab)-associated limbic encephalitis (LE) is an immune-responsive, largely nonparaneoplastic disease. About 20% of cases have malignant tumours, usually thymoma or lung cancer. Here we describe a case of VGKC-Ab-LE associated with thymic hyperplasia.
A 46-year-old woman presented with a subacute history of shortterm memory loss, temporal lobe seizures and psychotic features. Brain MRI showed T2-weighted bilateral mediotemporal lobe hyperintensities while screening for occult tumours, including metabolic total body PET scan, and onconeural abs proved negative. VGKC-Abs were highly raised at 3330 pM. Treatment with high dose steroids followed by monthly plasma-exchanges in association with oral immunosuppressants resulted in a marked improvement. Nine months after the onset, VGKC-Ab titre had declined to 1481 pM, but a sample obtained 15 months after the onset surprisingly revealed an increase in the antibody titre (2531 pM) even though the patient remained in clinical remission. A further screening revealed an enlarged thymus. The patient underwent thymectomy and pathology revealed a thymic hyperplasia with B-cell-containing germinal centres.
VGKC-LE can associate not only with thymomas, but also with a hyperplastic thymus, similarly to the well-known finding in myasthenia gravis. The persistence of high VGKC-abs titre should prompt the search for an enlarged thymus, and thymectomy should be considered to prevent a possible malignant evolution. Further cases collection and pathology studies will possibly reveal the role of the thymus in VGKC-Ab production.
Reactivity to human antigens of antibodies produced in multiple sclerosis plaques Burgoon Mark P. ⁎ , Anderson Sarah W., Miller Breanna, Yu Xiaoli, Bennett Jeffrey L., Gilden Don, Owens Gregory P.
The antigenic targets of the intrathecal B cell response in patients with multiple sclerosis (MS) are unknown. To examine this response and the potential role of B cells in the pathogenesis of MS, we characterized the reactivities of individual clonally expanded antibodies that were expressed in the demyelinating lesions of MS brain.
Laser capture microdissection was used to isolate single CD38+ plasma cells from demyelinating lesions of a postmortem relapsingremitting MS brain. Heavy and light chains of IgG expressed by 52 plasma cells were identified by RT-PCR. Sequence analysis revealed restricted expression of clonally expanded clones that exhibited somatic mutation from germline and molecular features of an antigen-driven response. Four of the expanded clones were subcloned into mammalian expression vectors to produce bivalent functional IgG and analyzed for specificity in immunoassays. Several recombinant antibodies (rAbs) immunostained one or more human cell lines derived from astrocytes, oligodendrocytes, neurons, fibroblasts, B cells or embryonic kidney cells. One rAb nonspecifically stained all cell lines, while several others specifically stained distinct cell lines of CNS and non-CNS origin. To compare the breadth of this response to the specificity of antibodies from an inflammatory CNS disease of known cause, we also performed the identical immunoassays with rAbs produced from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Three of the 11 SSPE rAbs that were examined stained human cell lines derived from astrocytes, oligodendrocytes or fibroblasts. Two of these three SSPE rAbs also recognized measles virus, the causative agent of SSPE.
These results reveal that a subset of intrathecally synthesized antibodies in MS plaques as well as in SSPE brain react with autoantigens in the CNS. The University of Queensland, Brisbane, Australia
We have previously described an atypical form of experimental autoimmune encephalomyelitis (EAE), which can be actively induced in C3H/HeJ mice by injection of myelin proteolipid protein (PLP) peptide PLP190-209 in adjuvant, and which results in development of lesions that are restricted predominantly to the brainstem and cerebellum of the mice. Subsequently, we have used PLP190-209specific T cells to passively induce EAE. To our surprise, these mice consistently developed lesions in the brainstem, but never developed lesions in the cerebellum. Since the mice with actively induced EAE produce both PLP190-209-specific T cells and antibodies, we have investigated the role of antibody in the development of cerebellar lesions in this model. EAE was induced by passive transfer of PLP190-209-specific T cells ± immunoaffinity-purified PLP190-209 specific antibody or an irrelevant antibody as control. Within 2 days of onset of neurological signs, mice were perfused. One micron sections were cut every 10 μm through the brainstem and cerebellum and stained with cresyl violet to assess lesions. Whole cross-sections were photographed and the percentage area of each cerebellum covered by lesions was determined using NIH Image software. To assess the role of complement, EAE was actively induced, and then mice were treated daily with injections of cobra venom factor (CVF), which inhibits complement. These mice developed EAE and were assessed as above. CVF has a short half-life in serum, and therefore, in some mice, CVF treatment was stopped at EAE onset, and 4 days later mice were assessed for lesions in the cerebellum.
Mice with passively transferred EAE induced by T cells alone (n = 30) all developed EAE with lesions in the brainstem, but not the cerebellum. In contrast, 60% of mice receiving T cells + PLP190-209specific antibody developed lesions in the cerebellum. Actively immunized mice receiving CVF developed no lesions in the cerebellum; however, 4 days after stopping CVF treatment, lesions could once more be observed in the cerebellum.
These findings strongly suggest that PLP-specific antibody plays a role in determining the location of lesions in this animal model of MS, and that the antibody effects are complement-mediated. We suggest that retargeting of lesion distribution by antibody could be a potentially clinically important and previously unappreciated element of antibody action in MS and, as such, require further investigation.
Signaling through kainate receptors enhances murine B cell proliferation and Ig production Chaimowitz Natalia ⁎ , Daniel Conrad
Abundant evidence indicates an interplay between the immune and nervous systems. Altered plasma glutamate levels, for example, have been strongly correlated with immunodeficiency states, such as AIDS and malignancies, suggesting that glutamate is capable of acting as an immunomodulator. While glutamate receptor signaling has been examined in some immune cells (e.g. T cells), the effects of glutamatergic stimuli on B lymphocytes remain unknown. To determine if glutamate is capable of modulating B cell function, we first examined mouse B cells for glutamate receptor expression. Furthermore, mouse splenocytes were cultured in glutamateenriched media; proliferation and antibody production were determined. FACS analysis shows that splenic B2 B cells express the receptor subunits that comprise the kainate receptor (KAR), a specific subtype of ionotropic glutamate receptor. KAR surface expression was found to be upregulated upon B cell stimulation. Moreover, when splenocytes and B cells were stimulated with IL-4 and CD40 ligand in glutamate enriched media, there was a significant increase in cellular proliferation and antibody (IgG1 and IgE) production. The KAR specific antagonist NS-102 is being used to confirm that these effects are mediated by KARs. The data also suggests that ADAM10 is required for the proliferative enhancing activity of glutamate. These results suggest that glutamate is capable of enhancing B cell function. In a previous study aimed at the high-throughput profiling of the autoantibodies present within multiple sclerosis (MS) cerebrospinal fluid (CSF), we identified a novel MS CSF autoantibody target, sperm associated antigen 16 isoform 2 (SPAG16-2). As tissue expression, protein function and disease-contributing role of SPAG16-2 are unknown, further investigation into this candidate MS autoantigen was warranted. In this study, we aimed to further characterize SPAG16-2 as a novel candidate MS autoantigen.
To analyze the presence and biomarker potential of anti-SPAG16-2 antibodies in MS serum, a recombinant protein ELISA was used for immunoreactivity screening in sera from MS patients (n = 69) and controls (healthy controls, n = 43; inflammatory and non-inflammatory neurological diseases, n = 47). Elevated serum antibodies against SPAG16-2 were detected in a significantly larger proportion of MS patients compared to controls (P = 0.0004). At a specificity level of 100% (0/90 controls), elevated anti-SPAG16-2 antibody levels were demonstrated in 9/69 MS patients, giving rise to a sensitivity of 13%.
In addition, to investigate the biological relevance of SPAG16-2 in MS, the tissue expression of the antigen in MS and control brain was analyzed by immunohistochemical approaches. We demonstrated increased expression of SPAG16-2 in the center of MS brain lesions (n = 2), while no detectable staining was seen within the normal appearing white matter of MS and control brain tissue (n = 2).
Moreover, passive antibody-transfer experiments with anti-SPAG16-2 monoclonal antibodies in MOG-peptide induced experimental autoimmune encephalomyelitis (EAE), the animal model for MS, indicated a disease-exacerbating effect of these antibodies. Mice receiving anti-SPAG16-2 antibodies (n = 5) had a higher disease burden indicated by significantly higher disease scores compared to animals injected with isotype control antibodies (n = 4) (P b 0.01). Spinal cords from mice injected with anti-SPAG16-2 antibodies were characterized by increased macrophage infiltration without any obvious effect on demyelination. We are currently further investigating how anti-SPAG16-2 antibodies exert their pathogenic effects.
In conclusion, the findings in this study indicate that SPAG16 isoform 2 constitutes a novel candidate MS autoantigen. Future experiments are aimed at the elucidation of the function of this protein and the role of autoantibody reactivity against it in MS.
Brenner Tanja ⁎ ,1 , Suleiman Jehan 2 , Gill Deepak 3 , Brilot Fabienne 2 , Antony Jayne 3 , Dale Russell C. 2 In previous studies of adult patients with epilepsy and encephalitic disorders, antibodies were reported to voltage-gated potassium channels (VGKC) as well as to other CNS targets, e.g. N-methyl-Daspartate receptor (NMDAR) and glutamic acid decarboxylase (GAD). VGKC antibodies are associated with limbic encephalitis in adults (Vincent et al., 2004) , but childhood cases have not been reported.
We tested samples from 37 children with status epilepticus (mean age 6.8 years, range 7 months-15 years), including 14 with encephalitis and 23 with other aetiologies, for antibodies to VGKC, NMDAR and GAD, by routine serological techniques. The results were compared to 69 paediatric controls (including healthy controls, encephalitis lethargica, NMDAR encephalitis and others). 24% (9/37) of the children with status epilepticus (SE) had a positive VGKC antibody titre of N100 pM, the cutoff routinely used in adult patients, compared with only 1/69 of the healthy and disease controls. Of the 14 SE patients with encephalitis, 5 tested positive for VGKC antibodiesthey were previously normal and had acute encephalopathy, refractory seizures and cognitive impairment (limbic encephalitis). Of the 23 patients with other aetiologies, 4 were positive for VGKC antibodies. Immunotherapies were variable and the outcomes included complete recovery, cognitive impairment, refractory epilepsy and mesial temporal sclerosis. No antibodies to NMDAR or GAD were detected in any of the 37 SE patients.
The high proportion of raised VGKC antibodies in children with status epilepticus suggests that VGKC-antibody limbic encephalitis is present in the paediatric population and, as in adults, can sometimes be associated with hippocampal sclerosis (Bien et al., 2007) . The poor recovery following central nervous system (CNS) injury has generally been attributed to the formation of the glial scar as well as to the infiltration and activation of immune cells. However, if well controlled, these two phenomena are actually essential for the repair process. Here, we aimed to study the reciprocal relationship between the blood-derived macrophages, one of the predominant immune populations in the lesion site, and the glial scar matrix.
For that goal, we employed in-vivo manipulations that separately targeted each one of these processes. By inhibiting the production of chondroitin sulfate proteoglycan (CSPG), a major component of the glial scar, we demonstrated that this commonly viewed obstacle to regeneration is a necessary template that dictates the antiinflammatory nature of the infiltrating monocytes that encounter it, thereby promoting resolution of the local inflammation. Reciprocally, by conditional ablation of the monocyte-derived macrophages with diphtheria toxin we showed that the same macrophages that use the scar for their own education are the ones that eventually degrade it.
Although insufficient for complete repair, the blood-derived macrophages and the glial scar matrix, comprise a feedback loop that enhances their beneficial properties while restricting their potentially harmful effects. Therefore, refinement of this endogenous crosstalk and potentially self-containing response may open new therapeutic avenues in the treatment of spinal cord injury and other CNS pathologies.
A new role for adenosine A3 receptors in chronic neuroinflammation van der Putten Celine ⁎ ,1 , Zuiderwijk-Sick Ella A. 1 Microglia activation is a prominent feature in many neuroinflammatory disorders. Since unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration, regulatory mechanisms are essential. Extracellular adenosine can modulate cellular activation through adenosine receptor (ADORA)-mediated signaling. Four ADORA subtypes have been described that can either increase (A2A and A2B receptors) or decrease (A1 and A3 receptors) intracellular cyclic AMP levels. The cellular response to extracellular adenosine is thus orchestrated by the expression pattern of the different ADORA subtypes.
In this study we investigated how ADORA-mediated signaling modulates microglial TLR-induced cytokine production during chronic inflammatory conditions. Using primary rhesus monkey microglia, we characterized the expression profile of all ADORA subtypes in unstimulated and TLR-activated microglia. TLR-mediated activation of microglia resulted in a dramatically altered ADORA expression profile, characterized by the simultaneously up-regulation of A2AR and down-regulation of A3R expression levels. As a consequence of this altered expression profile, activated microglia were highly sensitized to adenosine-mediated inhibition of subsequent TLR-induced cytokine responses. This enhanced inhibitory effect could be attributed to an increase of A2AR-mediated signaling, and we further demonstrate that this involves the suppression of NF-kappaB activation. Importantly, by using combinations of subtypespecific agonists and antagonists, we show that in unstimulated microglia, A2AR-mediated inhibitory signaling was effectively counteracted by A3Rmediated signaling. In activated microglia, the decrease in A3R-mediated signaling shifted the balance towards A2AR-mediated signaling, sensitizing the cells to inhibitory signaling.
In conclusion, we report a differential, activation state-specific expression of ADORA in microglia and uncover a new role for A3R as dynamically regulated suppressors of A2AR-mediated inhibition of TLR-induced pro-inflammatory cytokine responses. Therefore, our data would suggest exploration of combinations of A2AR agonists and A3R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.
Activation of innate immune responses is a hallmark of the active lesion in multiple sclerosis Multiple sclerosis (MS) is a chronic neuro-inflammatory disease of the central nervous system (CNS). Numerous infectious triggers have been implicated in MS pathogenesis, but conclusive evidence is missing.
Here we show that interferon-alpha (IFN-alpha), which is an innate cytokine produced upon viral infection, is over-expressed in active areas of MS lesions but not in inactive MS lesions, normal appearing white matter or healthy control brains. We detected IFNalpha specifically expressed by MHC-class II expressing cells, e.g. microglia and macrophages within perivascular and parenchymal areas of MS lesions. IFN-alpha is present predominantly in active MS lesions/borders suggestive of local production as part of the acute inflammatory process. Interestingly, we found IFN-alpha expression in HEK cell cultures transfected with Epstein-Barr virus-encoded small RNAs (EBERs), which are highly abundant in EBV infected cells. We also detected EBERs in these preselected active MS lesions by in situ hybridization in areas with IFN-alpha over-expression and in other neurological control cases.
We hypothesize that IFN-alpha production within the CNS might be driven by viral RNAs such as EBERs leading to activation of innate immune responses creating an anti-viral state but also igniting neuroinflammation. Multiple sclerosis (MS) is a chronic neuro-inflammatory disease of the central nervous system (CNS). Numerous infectious triggers have been implicated in MS pathogenesis, but conclusive evidence is missing.
Here we show that interferon-alpha (IFN-alpha), which is an innate cytokine produced upon viral infection, is over-expressed in active areas of post-mortem MS lesions but not in inactive MS lesions, normal appearing white matter or healthy control brains. We detected IFN-alpha specifically expressed by MHC-class II expressing cells, e.g. microglia and macrophages within perivascular and parenchymal areas of MS lesions. IFN-alpha is present predominantly in active MS lesions/borders suggestive of local production as part of the acute inflammatory process. Interestingly, we found IFN-alpha expression in HEK cell cultures upon transfection with Epstein-Barr virus-encoded small RNAs (EBERs), which are highly abundant in EBV infected cells. We also detected EBERs in these preselected active MS lesions by in situ hybridization in areas with IFN-alpha overexpression and in other neurological control cases.
We hypothesize that IFN-alpha production within the CNS might be driven by viral RNAs such as EBERs leading to activation of innate immune responses creating an anti-viral state but also contributing to neuro-inflammation.
Aging and ApoE4 on innate immunity in non-demented and Alzheimer disease brains Cribbs David, Berchtold Nicole, Cotman Carl University of California, Irvine, Irvine, United States
Our previous microarray analysis revealed that genes related to immune/inflammatory function undergo prominent change in the brain in cognitively normal aging. Here we extend these findings to AD, to analyze how immune-related gene expression is modulated by age, AD, and ApoE, an important AD risk factor, using data from a genome-wide transcriptome study.
Human tissue samples from three brain regions affected in AD (hippocampus, entorhinal cortex, and superior frontal gyrus) and one region that is relatively spared in AD (post-central somatosensory gyrus) were obtained at autopsy from 54 normal controls aged 20-99 years, and 26 AD subjects (75-99 years of age). Four brain regions were assessed, three of which are vulnerable to decline and accumulation of pathology with age and AD (hippocampus, entorhinal cortex, and superior frontal gyrus), and one region that develops minimal pathology (postcentral gyrus).
Aging was associated with a robust gene response across brain regions, with 55% of the immune-related probesets showing altered expression with advanced age, predominantly undergoing upregulated expression. The 6th-7th decades showed the most gene change, with relatively little change in the preceding or subsequent decades. Surprisingly, the transition to AD induced far less extensive change in immune-related genes than was observed with aging (~8% of the 729 probesets). ApoE genotype strongly affected the extent of the gene response in both cognitively normal aging and AD, with ApoE4 associated with a more extensive response, particularly in aging. Regional analysis revealed that the brain regions vulnerable in AD (hippocampus, entorhinal cortex and superior frontal gyrus) underwent age-related increases in immune-gene involvement in aging and AD, while the post-central gyrus showed extensive age-related changes but no further change in AD.
Aging and ApoE4, the two most powerful predictors for developing AD, induce profound changes in expression of genes related to innate immunity. More extensive change in this class of genes occurs in the course of cognitively normal aging than in the transition to AD. These data may provide insight to why epidemiological studies consistently find a protective effect of anti-inflammatory drugs only when taken during normal aging, while such drugs in AD patients have typically failed. We previously described a blood-brain barrier (BBB) breakdown leading to serum-protein leakage in the hippocampus of patients with intractable temporal lobe epilepsy (TLE). A BBB disruption induces an accumulation of immunoglobulins G (IgGs) in parenchyma and surprisingly, in pyramidal neurons. We postulated that IgG leakage contributes by immune activation or by toxicity to epileptogenesis. In this study, we correlated the BBB damage with serum protein leakage in epileptic and non epileptic tissues.
Epileptic (E) tissue: hippocampi and temporal cortex were obtained from surgery of patients suffering from intractable TLE.
Control (Ct): pieces of temporal cortex were obtained from autopsy of patients without history of seizures or other neurological diseases who died from multiple injuries.
Samples were collected with consent of patients and in accordance with Helsinki declaration.
By immunohistochemistry we evaluated the tight junction integrity (expression of ZO1 protein), the BBB permeability (IgG leakage) and complement activation (expression of C3c, C5b-C9 and HLA proteins).
In E tissue, we observed: i) a BBB disruption and IgG diffusion into the parenchyma, ii) an IgG accumulation in pyramidal neurons and in few astrocytes, iii) an abundant immunoreactivity for HLA proteins in macrophage-like cells, localized around blood vessels, iv) the presence of C3c positive perivascular infiltrates, and v) C5b-C9 (MAC) deposits in cells displaying a microglia/macrophage morphology.
In Ct tissue, BBB presented any damage but IgGs were found in some neurons.
This study described in chronic epileptic focus different degrees of BBB permeability, probably induced by angiogenic processes reported in TLE by our team (Rigau et al., 2007) . In Ct tissue BBB was preserved in spite of IgGs' presence in parenchyma and in some neurons suggesting a transient disruption of tight junctions. The presence of biological active fragments of C3 indicates the activation of the complement cascade and C5b-C9 expression observed in the cells with the morphology of microglia/macrophage shows activation of the terminal pathway, with a formation of complement membrane attack complex (MAC), which is toxic for neurons (Xiong et al., 2003) . Future investigations of immune dysregulation should determine by which mechanisms IgG uptake participates in epileptogenesis.
Characterizing genetic regulation and function of complement activation in the traumatized CNS The complement system plays an important role in the innate immune defence and is also believed to be active in neurodegenerative conditions. Recently, a role for complement in synaptic stripping during development was also suggested. In this study we have addressed the genetic influence on local expression of complement factors in the central nervous system (CNS) using a well characterized nerve injury model in the rat and its potential effect on synaptic plasticity occurring after injury.
First, the temporal profile of expression of C1q and C3 was characterized in the inbred DA and PVG strains, showing significantly higher expression in the DA rat. In contrast, expression CD59, a membrane attack complex (MAC) inhibitor, was similar or lower in the DA strain. There was no evidence of MAC activation, or of neutrophil infiltration, using immunohistochemistry. We next assessed degree of synaptic loss by measuring synaptophysin immunolabeling, which demonstrated increased loss of terminals in the ventral horn of DA rats, suggesting a role for complement activation in elimination of synapses after injury. Finally, in order to map expression differences for C1q, C3 and other locally expressed complement components on a whole genome level, a microarray study was done in a F2(DAxPVG) cohort. Multiple expression quantitative trait loci (eQTLs) were identified, revealing specific genetic regions regulating the expression of several of the important complement proteins, including C1qa, C1qb, C3 and C9, as well as a strong genetic influence on the expression of the complement receptor 2.
This data constitutes an important finding in the understanding of the potential effects of complement activation and how complement is regulated in the traumatized CNS. It also has implications for the interpretation of the role of the complement system in relation to whole genome scans for neurodegenerative disease. Microglia are the professional phagocytes for the clearance of microbes or apoptotic cells within the central nervous system (CNS). Different types of microglial phagocytic receptors are involved in microglial clearance function like the triggering receptor expressed on myeloid cells-2 (TREM2) and its associated adaptor molecule DAP12. Another phagocytic receptor that has been suggested to signal via DAP12 is the complement receptor-3 (CD11b/CD18). We now investigated the involvement of complement receptor-3 in the complement mediated phagocytosis by microglia.
The first protein of the classical complement cascade is C1q. The expression of its RNA by both, microglia and neurons, was demonstrated by RT-PCR. Immunohistochemical analysis revealed binding of the C1q protein to neurites after the enzymatic removal of sialic acid residues on the glycocalyx of neurons. Moreover, microglianeuron co-culture experiments showed that neurons displaying less sialic acid on their glycocalyx led to removal by microglia. While intact processes remained untouched, neuronal processes marked with C1q were phagocytozed by microglia within 24 h. First blocking experiments with antibodies directed against CD11b now indicated the involvement of the mircoglial complement receptor-3 in this process.
Our data illustrate that neuronal structures opsonized by complement C1q binding to an asialydated glycocalyx are recognized and removed by microglia. bridges between innate and adaptive immunity is becoming increasingly evident. Each DC subset shows a specific and distinctive pattern of expression of Toll-like receptors (TLR) and effector functions vary according to the host's status of health or disease. Recent studies have demonstrated that improper activation of TLRs can lead to local or systemic autoimmune processes. In addition, in some circumstances, functional and numerical impairment of specific DC subsets can be associated to autoimmunity. The role of DC in the pathogenesis of multiple sclerosis (MS) has recently received great attention since these cells have been found to be increased in perivascular cuffs, active lesions and cerebrospinal fluid of MS patients. On the view of such scenario, we are searching for abnormalities in DCs of MS patients compared to healthy subjects.
We evaluatedthe absolute count of the major circulating BDCA1+, BDCA3+ and CD16+ myeloid and CD123+ plasmacytoid DC by means of nine-colour flow cytometry. Our single-platform assay, based on published clinical guidelines for the absolute count of rare populations, allows to measure the absolute number of the different DC subsets in a single tube, avoiding sample processing variability and so ensuring accuracy in the determination of the balance between subsets. We also set out to evaluate their cytokine profile upon stimulation with different TLR agonists. Multiparametric flow cytometry allowed us to evaluate simultaneously in different DC subsets up to three cytokines in response to TLR agonists. Our data showed that the frequency (calculated as absolute number per μl of whole blood) of plasmacytoid DCs and myeloid DC subsets is slightly reduced in the blood of MS patients compared to healthy individuals. Plasmacytoid DCs obtained from MS patients also showed decreased ability in the production of interferon alpha in response to TLR7 compared to healthy donors.
In conclusion our results suggest an altered DC function in multiple sclerosis. Overall we believe that our analysis could help to understand the big picture regarding the innate immune response and MS immunopathogenesis.
Differential responses of human microglia and blood derived myeloid cells to FTY720 Durafourt Bryce A., Lambert Caroline, Johnson Trina A., Blain Manon, Bar-Or Amit, Antel Jack P.
Myeloid cells within the central nervous system (CNS), comprised of the resident parenchymal microglia and the blood-derived dendritic cells (DCs) and macrophages, serve immune regulatory and effector functions that contribute to the course of multiple sclerosis (MS). As the sphingosine-1-phosphate (S1P) receptor agonist FTY720 crosses the blood brain barrier, we examined its potential to impact on the properties of these myeloid cells.
Using real-time polymerase chain reaction (RT-PCR), we observed that human adult and fetal CNS-derived microglia expressed similar patterns of S1P receptor (S1PR) expression as monocyte-derived DCs and macrophages. S1P receptor 1 (S1P1) mRNA levels were higher while S1P3 and S1P4 were lower compared to monocytes. S1P5 mRNA was not detected in macrophages or fetal microglia and low-intermediate transcript levels were detected in the other cell types. Monocytes from MS patients undergoing treatment with fingolimod (FTY720) showed no alteration in the relative expression pattern of S1PRs compared to untreated healthy donors. DCs and macrophages generated in vitro in the presence of the active phosphorylated form of FTY720 (FTY720-P) expressed similar S1PR patterns as vehicle-treated cells.
Despite similarities in receptor expression, we observed distinct intracellular signaling events in the different cell subtypes following short-term in vitro FTY720-P exposure. Treatment of macrophages and DCs with FTY720-P led to decreased phosphorylation of ERK and a concomitant induction of myosin light chain (MLC) II phosphorylation, whereas this response was absent in microglia. In contrast, FTY720-P induced robust ERK phosphorylation in human fetal astrocytes. The ability of FTY720-P to modulate cytokine production in the myeloid cells was also examined. FTY720-P treatment inhibited production of IL-12p70 by DCs and macrophages as induced by CD40L. In microglia, levels of secreted IL-12p40 as induced by poly I:C were not modulated by FTY720-P. Levels of secreted IL-10, TNF-alpha, and IL-6 remained unaffected by FTY720-P treatment in all cell types.
We demonstrate that the distinct myeloid cell populations present in the human CNS, under steady-state or inflammatory conditions, exhibit similar patterns of S1PR expression but distinct signaling and cytokine modulation in response to FTY720-P. Our results suggest that FTY720 may have the potential to selectively bias the inflammatory microenvironment in the CNS. Background: Interleukin (IL)-27 is a cytokine produced by antigen presenting cells with multiple roles in the induction as well as in regulation of the immune responses. However, the effect of IL-27 on natural killer (NK) cells is not well defined. NK cells, particularly the CD56bright subset, play a significant role in the immune regulation and might be playing a protective function during Autoimmunity. Objective: To investigate the possible effects of IL-27 on human CD56bright and CD56dim NK cell phenotype and function. Methods: NK cells were isolated from buffy coats of healthy individuals using Mitenyi Beads, sorted into CD56bright and CD56dim NK cells and cultured under different conditions with or without addition of IL-27 to study the potential effects of IL-27 on proliferation, expression of surface markers, transcription factors and cytokine secretion of treated NK cells. IL-27 stimulated NK cells were co-cultured with autologous T cells to investigate the regulatory function of treated NK cells. In addition, IL-27 stimulated NK cells were studied for induction of cytotoxic function against target cell line. Results: Phenotypic characterization suggests that IL-27R is expressed by both ex-vivo isolated CD56bright and CD56dim NK cell subsets, with a markedly increased expression among CD56bright NK cells. IL-27 treatment decreased the proliferation of both CD56bright and CD56dim NK cells. This decreased proliferation of NK subsets was correlated with an increased expression of IL-10 in stimulated NK cells subsets. Moreover, IL-27 treated CD56bright NK were suppressive against autologous CD4 T cell proliferation in a contact dependent mechanism. This suppressive ability seems to be dependent upon expression of perforin among treated NK cells. In contrast, IL-27 treatment of CD56dim NK cells did not change their cytotoxic or suppressive function.
IL-27-treated NK cells gain immunoregulatory functions.
Enhanced stimulation of phagocytosis of E. coli by murine microglia with a combination of muramyl dipeptide and one TLR agonist Ribes Sandra ⁎ ,1 , Adam Nina 1 , Ebert Sandra 1 , Nau Roland 2 1 Department of Neurology, University of Göttingen, Göttingen, Germany; 2 Department of Geriatrics, Evangelisches Krankenhaus Göttingen-
Parenchymal microglia (MG) express Toll-like receptors (TLRs) and Nod-like (Nod) receptors that recognize microbial stimuli and mediate responses contributing to the clearance of the infectious agent from the brain. Here, we hypothesized that the combination of Nod and TLR agonists might stimulate MG thereby increasing their ability to phagocytose E. coli.
Primary cultures of mouse MG were exposed to muramyl dipeptide (MDP; Nod2), polyinosine-polycytidylic acid [poly(I:C); TLR3], endotoxin (LPS; TLR4) and oligonucleotides containing unmethylated cytosin-guanosin motifs (CpG; TLR9). A control group of unstimulated cells was included in all experiments. After stimulation, cells were challenged with E. coli K1 at a ratio of 100 bacteria per cell. Phagocytosis was left to proceed for 30 and 90 min at 37°C. For phagocytosis inhibition studies cytochalasin D (CD, final concentration 10 μM) was added to the cell cultures 30 min prior to bacterial addition and remained present throughout the experiment. To monitor intracellular killing inside MG, cells were allowed to phagocytose bacteria for 90 min. After bacterial exposure, MG were washed and incubated in a medium containing gentamicin (200 mg/l) to kill extracellular bacteria. Thereafter, cells were washed and lysed with distilled water. Viable intracellular bacteria were enumerated by quantitative plating of serial 10-fold dilutions. ANOVA (followed by Bonferroni's multiple comparisons test) was performed to analyse the differences between groups (n ≥ 12); p b 0.05 was considered statistically significant. The interplay between MDP (Nod2 ligand) and one TLR agonist enhances the ability of MG to phagocytose and intracellularly kill a pathogenic strain of E. coli. This approach could improve the brain resistance of immunocompromised patients against infections caused by these Gram-negative bacteria.
Jiang Wenzheng, Bielekova Bibiana ⁎
MS is T-cell mediated, demyelinating disease of the central nervous system. While most of MS research focuses on adaptive immune responses, it is becoming clear that functional abnormalities of the innate immune system can lead to dysregulated adaptive immunity, e.g. activated NK cells can inhibit autologous T cells during viral infections and in multiple models of autoimmunity. Moreover, NK cell deficiencies have been reported in many human autoimmune diseases, including MS. Previous data from our laboratory demonstrated that therapeutic effect of daclizumab, humanized mAb against CD25, is linked to drug-induced expansion of CD56bright NK cells and that these cells can kill autologous activated T cells. The goal of the current study was to identify the mechanism utilized by CD56bright NK cells in this cytotoxicity.
Polyclonally activated T cells were effectively killed by autologous NK cells at physiological ratios (1:10 E:T ratio) in modified flowcytometry based killing assay, which also demonstrated predominant degranulation of CD56bright, as opposed to CD56dim NK cells. The NKmediated killing was strongly inhibited by EGDA, which blocks degranulation of NK cells but not by pan-caspase inhibitor Z-VAD-FMK, or Fas-or TRAIL-inhibiting reagents. Consistent with granule exocytosis-dependent pathway of cytotoxicity, we observed by confocal microscopy transfer of granzymes (GzmKNANB) from NK cells to activated T cells. The amount of transferred granzymes to target cells was quantified by flow cytometry and was found to be greatest for GzmK. Moreover, the efficacy of GzmK-mediated transfer to target cells was significantly greater when NK cells were isolated from daclizumab-treated patients as compared to untreated MS controls. Because GzmK (and GzmA) kill by inducing mitochondrial dysfunction and reactive oxygen species (ROS), we measured mitochondrial transmembrane potential and intracellular ROS formation and found both significantly impaired in the activated (but not resting) T cells upon co-culture with NK cells. Finally, we confirmed that NK-mediated killing of activated T cells is potently inhibited by anti-oxidants.
GzmK has limited expression in the human immune cells, but is constitutively expressed in CD56bright (but not on CD56dim) NK cells. To our knowledge, these data represent first physiological evidence for the important role of GzmK in human immune system.
Macrophage recruitment to the brain following traumatic brain injury is partially-dependent on CCR2
Hsieh Christine ⁎ , Niemi Erene, Nakamura Mary, Seaman William
Traumatic brain injury (TBI) is a major health problem, and it is the number one cause of death in children in the United States. The initial injury to the brain is followed by an inflammatory immune response. While inflammation may have protective qualities, particularly against infection, it may also worsen brain injury, increasing neuronal cell death. The nature of the inflammatory response, however, is still poorly defined, and it has been difficult to distinguish the roles of macrophages from those of resident immune cells, especially microglia, and from other inflammatory cells, such as neutrophils. Furthermore, in light of evidence suggesting that immune cells, including macrophages, may promote recovery after CNS injury, it is important to understand the role of these cells in TBI.
We have studied the inflammatory response in mice following TBI induced by controlled cortical impact. By using multi-color flow cytometry, we have characterized macrophages, microglia, and neutrophils. We find that macrophages on the injured half of the brain increase 10-fold by four days following TBI. These macrophages express CD86 (B7.2) and MHCII, indicating that they are activated and have the capacity for antigen presentation. In contrast, microglia express little to none of these antigen presentation molecules.
We also find that almost all macrophages in the injured brain express the chemokine receptor CCR2, and that CCR2-deficient mice have a significant (~50%) reduction in the number of brain macrophages following TBI, indicating that macrophages are recruited to the injured brain at least in part by CCR2. Interestingly, our preliminary studies also reveal a subset of infiltrating macrophages that expresses arginase-1, a marker of alternatively activated macrophages, which have been described as important for tissue repair and recovery.
In sum, TBI in our model is followed by a large influx of activated peripheral macrophages. The recruitment of macrophages is in part dependent on CCR2, and at least a subset of these may be involved in brain repair.
Molecular basis for the induction of protective type I interferon within the central nervous system Several neurotropic viruses such as vesicular stomatitis virus (VSV) induce peripheral neutralizing antibody responses and still can infect cells within the central nervous system (CNS). Recently we showed that intranasal VSV instillation of mice selectively lacking the type I interferon receptor (IFNAR) on neuroectodermal cells of the CNS resulted in severe encephalitis and death whereas control mice did not show signs of disease (Detje et al., 2009) . Although under such conditions IFNAR triggering was instrumental in constraining virus spread within the CNS, it remained unclear whether, and if how, protective type I interferon (IFN-I) was induced locally within the brain.
Upon intranasal VSV challenge with 103 plaque-forming units (pfu), a dose that is well tolerated by wild-type mice, Interferon-β deficient mice (IFN-β−/−) succumbed to the infection within 10 days. Although up to 3 days after infection, IFN-β−/− and wildtype mice showed similar virus clearance, from days 5-6 on virus levels continuously increased in the CNS of IFN-β−/− mice. Of note, IFN-β−/− mice showed a similar resistance to intravenous VSV challenge as compared to wild-type mice. These data suggest that IFNβ alone was critically required to promote virus arrest within the CNS, whereas in periphery IFN-β and IFN-α can complement each other. Analysis of IFN-β reporter mice expressing luciferase under control of the IFN-β promoter (IFN-β+/Δβ-luc) showed that 48 h post intranasal infection IFN-β expression was found in the nasal area and in the olfactory bulb.
Collectively these data are compatible with the model that upon intranasal and intravenous infection protective IFN-I is differentially induced. This notion is further supported by the observation that similar to IFN-β deficient mice, animals devoid of Toll-like receptor (TLR)-signaling (MyD88−/−Trif−/−) or RIG-I-like receptor (RLR)signaling (CARDIF−/−) succumb to intranasal infection, whereas these strains well tolerated intravenous infection. Currently we are testing whether TLR and RLR signaling have to be active in order to mediate protection within the CNS and whether both mechanisms can complement each other in periphery. Netrins are a family of secreted proteins that direct the migration of cells and axon growth cones during neural development, where neuronal migration is modulated by a balance between chemoattractive and chemorepulsive signals. Besides its neuronal guidance involvement, other functional aspects of netrin-1 may include antiinflammation. The aim of this study was to examine the expression and localization of netrin-1 in the CNS with EAE and irradiation injury.
Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (pb 0.01). Netrin-1 was maintained at the recovery stage of EAE. Immunohistochemistry detected the netrin-1 protein in neurons, gliocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was increased mainly in infiltrating inflammatory cells at the peak stage. Netrin-1 was also normally and intensely immunostained in gray matter neurons and white matter gliocytes. The changes of netrin-1 in irradiation injury were also compared.
These results suggest that endogenous netrin-1 is increased in CNS inflammation including EAE and radiation, where it contributes to the modulation of CNS inflammation. *This research was performed under the program of Basic Atomic Energy Research Institute (BAERI) which is the Nuclear R&D Programs funded by the Ministry of Science & Technology of Korea (KOSEF).
Neuroprotective function of the microglial immunoreceptor tyrosine-based inhibitory motif-signaling receptor Siglec-11
Wang Yiner, Claude Janine, Linnartz Bettina, Neumann Harald ⁎
Sialic acid-binding immunoglobulin superfamily lectins (Siglecs) are members of the immunoglobulin superfamily that recognize sialic acid residues, which form a cap on the glycocalyx. Siglec-11 is a recently identified microglial human-specific CD33-related Siglec without counterpart in mice that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Polysialylated neuronal cell adhesion molecule (PSA-NCAM) is a putative ligand of Siglec-11.
We observed gene transcription and protein expression of Siglec-11 splice variant 2 in human brain tissue samples by RT-PCR and Western blot. Siglec-11 was specifically expressed on microglia in human brain tissue as determined by immunohistochemistry. Cultured murine microglial cells were genetically modified to express human Siglec-11 splice variant 2. Stimulation of Siglec-11 by crosslinking antibodies suppressed the lipopolysaccharide (LPS) induced gene transcription of the pro-inflammatory mediators interleukin-1beta (IL1) and nitric oxide synthase-2 (NOS2) in microglia. Moreover, phagocytosis of apoptotic neuronal material and fibrillary amyloid-beta was reduced in Siglec-11 transduced microglia. Expression of PSA-NCAM was detected on microglia and neurons by immunohistochemistry and RT-PCR. Coculture of microglia transduced with Siglec-11 and neurons demonstrated neuroprotective function of Siglec-11, which was dependent on polysialic acid (PSA) residues on neurons, but independent on PSA on microglia.
Data demonstrate that human Siglec-11 expressed on murine microglia interacts with the glycocalyx of neurons, reduces microglial LPS-induced gene transcription of pro-inflammatory mediators, impairs phagocytosis and prevents microglial neurotoxicity. Multiple sclerosis (MS) is regarded as an autoimmune disorder of the central nervous system. While extensive data supports a role of adaptive immune mechanisms in MS pathogenesis, evidence for an involvement of innate immunity and particularly neutrophils are still scarce. The goal of this project is to provide a systematic analysis of phenotypic and functional markers of neutrophils in MS patients.
Whole blood from MS patients and healthy controls was analyzed by flow cytometry for expression of the following phenotypic-, functionaland activation markers: CD11a, CD11b, CD14, CD16b, CD31, CD43, CD49d, CD62L, CD63, CD64, CD80, CD86, CD162, CD181, CD217, TLR1, TLR2, TLR4, fMLPR and MHC-class II. In parallel, whole blood counts were obtained by Coulter counting. Up to this point, flow cytometric data from 26 healthy controls and 83 untreated MS patients are available (17 CIS, 30 RRMS in remission, 16 RRMS in relapse, 9 SPMS and 11 PPMS) . Current analyses show that most of the surface markers did not differ significantly between the groups. In general, neutrophils from RRMS patients showed a trend towards higher activation compared to healthy controls, with upregulation of CD64 and TLR4. In addition, neutrophils from RRMS patients tended to demonstrate increased expression of molecules involved in antigen presentation (CD86 and HLA-class II). Granulocyte counts were significantly higher in RRMS patients during relapse as compared to patients during remission (p= 0.02, Mann-Whitney test) and SPMS and PPMS patients compared to healthy donors (p= 0.03 and p = 0.001 respectively).
In conclusion, neutrophils from MS patients do not seem to differ significantly with respect to surface phenotype when compared with healthy controls. The elevated granulocyte counts might reflect ongoing inflammatory activity. In our current studies, we pursue the putative functional roles of class II and CD86 expression on neutrophils as well as the factors underlying the granulophilia observed in MS patients. The innate immune system detects the onset of an infection by the presence of diverse sensing molecules termed pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patters (PAMPs), as well as endogenous ligands derived from the host itself. PRR stimulation induces the expression of proinflammatory cytokines, interferons (IFNs) and costimulatory molecules that connect the innate and acquired immunity.
Toll-like receptor 3 (TLR3) and the helicase RIG-I are transmembrane and cytosolic PRRs respectively, which detect dsRNA and RNA virus such as Cytomegalovirus and Epstein-Barr virus among others. TLR3 and RIG-I engagement not only activates both NFkappaB and AP-1 transcription factors inducing the production of inflammatory cytokines, but it also activates IRF3 which leads to the type I IFN production. Rheumatoid arthritis (RA) is developed as a result of a complex interplay of genetic, environmental factors and a deregulated immune response. Exogenous ligands of TLR3 and RIG-I, such as Cytomegalovirus, Epstein-Barr virus and Parvovirus, have been described in the joint of RA patients, as well as endogenous ligands such as host RNA derived from necrotic cells of the damaged joint.
Vasoactive intestinal peptide (VIP) and its receptors VPAC1 and VPAC2 are expressed in fibroblast-like synoviocytes (FLS) from osteoarthrosis (OA) and RA patients, with a differential expression in both pathologies. VIP exerts anti-inflammatory effects both in animal models and in human RA.
The aim of the present study is to test the presence of dsRNA receptors in FLS and to study the effect of VIP. Our results demonstrate the basal expression of both RNA sensors, TLR3 and RIG-I at mRNA and protein level. Both sensors are significantly more expressed in RA-FLS than in OA-FLS. We have studied the effect of VIP on the signalling pathways of TLR3 and RIG-I after stimulation with the dsRNA synthetic ligand PolyI:C. VIP prevents the translocation to the nucleus of IRF3 in OA-and RA-FLS whereas NFkappaB (p65) activation is only prevented by VIP in OA-FLS.
These results validate the role of VIP as a negative regulator of proinflammatory and type I IFN pathways and corroborate its potential as therapeutic agent.
Statins enhance TLR-induced pro-inflammatory responses of rhesus monkey microglia van der Putten Celine ⁎ ,1 , Kuipers Hedwich F. 2 Statins are inhibitors of the endogenous mevalonate pathway which directs the biosynthesis of cholesterol and isoprenoids. Since statins also have immunomodulatory capacities they are being considered (co)therapy in inflammatory disorders including multiple sclerosis (MS). However, different studies have reported contradicting findings on how statins influence responses of the innate immune system, in particular Toll-like receptor (TLR)-mediated responses. These discrepancies could at least in part be attributable to the use of different types of statins, different cell types or species used in these studies.
In this study we investigated the effects of different statins on TLRmediated responses in microglia, and compared these effects to bonemarrow (BM) derived macrophages of the same donor. Unexpectedly, we found that simvastatin, lovastatin and atorvastatin all strongly enhanced microglial TLR1/2-induced pro-inflammatory cytokine production. TLR1/2, 3, 4 and 8-induced responses were all enhanced in statin-treated microglia, and analysis of 11 cytokines revealed that TLRinduced production of IL6, IL12 and GMCSF were most strongly enhanced, whereas the induced expression of immunomodulatory molecules as IL1R antagonist were unaffected. Secondly, we compared the effect of statins on microglia to that on BM-derived macrophages of the same donor. Interestingly, microglia were found to be more sensitive to statin-mediated pro-inflammatory effects. Experiments using combinations of specific inhibitors downstream of the pathways affected by statins revealed that inhibition of cholesterol biosynthesis was mainly responsible for the observed effects, both in microglia and in BM-derived macrophages. Preliminary results from real-time rtPCR analyses suggest that microglia express lower intrinsic levels of key enzymes involved in the mevalonate pathway than BM-derived macrophages which might well underlie the enhanced sensitivity of microglia to statins.
In conclusion, this study reports that TLR-induced pro-inflammatory responses of microglia are similarly enhanced by different types of statins, and that microglia are particularly sensitive to statinmediated effects when compared to other macrophages. These observations suggest that statins might aggravate rather than dampen innate immune responses, particularly in the CNS. They could thereby possibly contribute to adverse effects when MS patients that use statins would suffer from bacterial or viral infections.
The multiple sclerosis susceptibility allele in TNFRSF1A creates a new RNA isoform that results in a truncated TNFRSF1A protein Multiple sclerosis is an inflammatory and neurodegenerative disease with an important genetic component. Our meta-analysis of genome-wide association scans (GWAS) in MS identified a novel susceptibility locus in TNFRSF1A (rs1800693, P = 1.6 × 10-11). Disruption of TNF signaling severely affects human and murine demyelinating neuroinflammatory diseases.
The rs1800693 polymorphism is intronic and is located near the exon-intron 6 boundary. We found that this polymorphism gives rise to a new splice variant isoform of TNFRSF1A which lacks exon 6 and is predicted to produce a truncated protein deficient in its transmembrane and intracellular component. The level of RNA expression of the truncated isoform (delta 6) was measured in PBMCs of 84 genotyped healthy subjects from the PhenoGenetic Project and displays a robust linear correlation with the frequency of the susceptible allele (using the PLINK analysis toolkit): while the full-length isoform is present in all individuals, the delta 6 isoform is only found in the presence of the MS susceptibility allele. The level of expression of this novel isoform is also upregulated in PBMC of subjects with one or two copies of the risk allele after PMA stimulation. The delta 6 isoform of TNFRSF1A does not appear to affect either (1) the levels of soluble TNFRSF1A and TNFRSF1B in serum samples from healthy (n =265) and MS subjects (n = 282) or (2) the surface expression of TNFRSF1A on monocytes and granulocytes as assessed by flow cytometry. In vitro experiments show cytoplasmic intracellular expression and secretion of the delta 6 isoform of TNFRSF1A.
Here, we present evidence of one functional consequence of a high frequency (45%) MS susceptibility allele within TNFRSF1A: this variant leads to the production of a novel TNFRSF1A RNA isoform and production of a truncated protein.
The role of activated microglia in LPS-induced neuroprotection in an aseptic brain injury model Chen Zhihong ⁎ , Jalabi Walid, Dutta Ranjan, Trapp Bruce
Therapeutics that protect against neuronal death will have many applications for treating acute and chronic diseases of the central nervous system (CNS) such as neurodegenerative diseases that include multiple sclerosis (MS). We have previously demonstrated that multiple low dose i.p. injections of LPS (lipopolysaccharide) in mice lead to global activation of microglia, reduced inhibitory GABAergic pre-synaptic components, and the induction of the prosurvival signaling pathways in neurons. Here, by using an aseptic cryogenic brain injury model, we study the contribution of this neuroprotective effect offered by microglia.
By immunohistochemistry, we show that LPS-activated microglial cells gather around the penumbra of the lesions caused by experimental brain injury. These cells demonstrate phagocytic capability and actively participate in clearing of hemorrhagic debris, which are characteristics of anti-inflammatory tissue-repairing "M2" phenotype macrophages. A further analysis of the dataset obtained using DNA microarray technology has revealed that a cluster of genes that are related to the M2 subtype has indeed increased their transcription after LPS injections. In addition, LPS preconditioned mice have reduced volume of lesion size after brain injury; and these animals correspondingly perform better on "rotarod", the equipment used to test locomotor behavioral functions.
Conclusively, we demonstrate that LPS preconditioning leads to increased M2-microglia and decreased neuronal loss after brain damage. The molecular events that elicit this neuroprotective action possibly proffered by activated microglia are now under investigation. Actively induced Lewis rat Experimental Autoimmune Encephalomyelitis (EAE) is a highly reproducible model for investigating the cellular and molecular mechanisms involved in leukocyte central nervous system invasion. The disease is induced by the subcutaneous injection of guinea pig Myelin Basic Protein (gpMBP) resuspended in incomplete Freund's adjuvant added with Mycobacterium Tuberculosis (CFA). The disease becomes clinically evident 9-10 days post EAE induction (p.i.) and neurological signs peak on days 13-14 with extensive leukocyte inflammation mainly located in the spinal cord (S.C.).
On day 14 p.i. we previously observed by flow cytometry the massive presence of a cellular population other than lymphocytes in the S.C. (i.e. the target organ) as well as in the spleen (i.e. imunoregulatory organ) of EAE rats.
We hypothesized that this population could be composed of polymorphonuclear cells and we wondered if its presence was related to CFA use or if it had to be referred to the disease itself. Thus, we focused our attention on the characterization of the immune system cellular components present in the S.C. and in the spleen of CFA and EAE animals on day 14 p.i.
We demonstrated that the cell population observed in the S.C. was mainly composed of neutrophils which invaded the central nervous system together with lymphocytes only in EAE animals; in fact, no leukocyte infiltration was observed in CFA treated rats.
The remaining cellular changes could be observed to the same extent in both CFA and EAE animal spleen. In fact, in both CFA and EAE spleen we observed a red pulp hyperplasia together with a white pulp lymphoid hypoplasia while the flow cytometry confirmed a decrease in T cell and that the relevant cellular population previously observed was mainly composed of neutrophils.
We also observed that CFA administration and EAE induction similarly increased neutrophils and monocytes absolute number vs controls, while a significant reduction in circulating lymphocytes was reported in EAE animals vs. CFA treated ones.
CFA was clearly responsible for spleen cellular changes as well as neutrophil and monocyte number increase among circulating cells. Nevertheless, the presence of infiltrating leukocytes in the spinal cord and the difference in circulating lymphocytes between CFA and EAE animals were both peculiar events occurring in EAE.
The investigation on neutrophil functional role in EAE development is our future endpoint. Naturally occurring CD4+CD25+FOXP3+ regulatory T cells (Tregs) are thought to suppress the activity of pathogenic T cells in autoimmune diseases such as multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system. Tolllike receptors (TLRs) are key components of the innate immune system which have been detected on CD4+C25+ Tregs. Recent data point to the direct or indirect involvement of TLRs in modulating the function of Tregs. We tested the hypothesis that TLR2 stimulation reduces the suppressive functions of human Tregs.
We show that TLR2 is preferentially expressed on human CD4+ CD25+ Tregs as compared to effector cells. We also show that the triacetylated lipopeptide TLR2 ligand Pam3CSK4, which requires the TLR1-TLR2 heterodimer for recognition, reduces the suppressive activity of human Tregs. In contrast, the suppressive functions of Tregs are not reduced by the diacylated TLR2 ligand FSL-1, which is recognised by the TLR2-TLR6 heterodimer. Preliminary data show that Pam3CSK4 enhances IL-6 secretion by Tregs and Tresp. In addition, stimulation by this agonist enhances TGF-β expression by Tregs, suggesting that TLR2 activation may promote Th17 cell differentiation.
These data support the hypothesis that in MS and potentially other immune-mediated diseases, infections may lead to exacerbation of disease activity by causing reduction of Treg suppression and differentiation of pathogenic Th17 cells. We report that TLR7 stimulation by Imiquimod, a synthetic analog of ssRNA, suppresses disease severity in a chronic murine EAE model. Preliminary studies have also found that stimulation with TLR9 agonist CpG type A also results in suppression of chronic EAE. In both cases disease suppression is associated with an expansion of plasmacytoid dendritic cells (pDCs) in the CNS as shown by expression of PDCA-1, B220 and CD11c. In TLR7 experiments we have found increased production of IFN-beta in spleens of mice treated with Imiquimod as measured by RT-PCR. We have also generated pDCs in vitro and found that Imiquimod induces the expansion of pDCs and also the production of IFN-beta. The expansion of pDCs in response to imiquimod is dependent on the type I interferon receptor (IFNAR) as mice lacking the receptor did not expand pDCs. In vivo depletion of pDCs has been found to reduce EAE severity. Interestingly, preliminary experiments have shown that Imiquimod exacerbates EAE in the absence of pDCs which indicates that the protective effect of Imiquimod is dependent on pDCs.
Our data demonstrate that TLR7 agonist Imiquimod and TLR9 agonist CpG type A reduce EAE disease severity and there is a potential for immunotherapy in MS by inducing the expansion of plasmacytoid dendritic cells which then produce IFN-beta which is currently used as a treatment of MS. Theiler's murine encephalomyelitis virus (TMEV), a singlestranded RNA virus, causes a chronic progressive CD4+ T cell mediated induced demyelinating disease (TMEV-IDD) in susceptible mouse strains, serving as a virally-induced murine model of the human disease multiple sclerosis. The innate immune system serves as the first line of host defense against pathogens such as viruses. Toll-like receptors (TLRs), members of the pattern recognition receptor (PRR) family, recognize pathogen associated molecular patterns (PAMPs), and ligand binding activates proinflammatory and antiviral responses on APCs. Central nervous system (CNS)-resident cells, microglia isolated from human and neonatal murine tissue, express multiple TLRs in vitro and in situ. Therefore, we examined TLR-stimulated microglia and macrophage immune functions in the murine CNS in response to TMEV infection.
Following TMEV infection, TLR2, TLR3, TLR7, TLR8, and TLR9 expression are significantly upregulated at day 7 post-infection (p.i.), suggesting that TLRs are dynamically regulated during acute Theiler's virus infection. Using quantitative real-time PCR, TLR3 expression (which recognizes viral double-stranded RNA) is highly inducible on FACS-sorted microglia (CD11b+CD45LO) in response to TMEV-infection at day 7 p.i. These data suggest that viruses may selectively upregulate expression of TLR3 for mounting antiviral responses, which is necessary for TMEV viral clearance in the CNS. To further elucidate the role of TLR3 in TMEV-IDD, we examined the development of induced demyelinating disease in its absence (TLR3−/− SJL). Compared to wildtype SJL mice, TLR3−/− SJL mice are hypersusceptible to disease and exhibit increased demyelination, suggesting that TLR3 is protective against TMEV-IDD. Additionally, TLR3−/− SJL mice are defective in TMEV clearance in the CNS, as measured by increased viral titers in TLR3−/− mice compared to wild-type controls. TLR3−/− SJL mice are deficient in the total CNS cell numbers at day 7 p.i., which is also reflected in reduced total CD8+ T cell percentage and cell number, which would affect TMEV clearance.
In the future, experiments will be focused on understanding the role of TLR3 on the effector functions of CNS-resident cells during acute infection. This study was supported by T32 AI060523 and 1F31NS061621-01A1. Reactive astrocytes regulate inflammation and promote tissue repair in injured CNS. The neuroprotective action can be mediated by neurotrophins (NT), such as Brain Derived Neurotrophic Factor (BDNF) which can act on neurons expressing the BDNF receptor TrkB.
The aim of this work was to understand whether NT promote the neuroinflammatory or the neuroprotective side of astrocytic reactivity.
We observed that the BDNF-TrkB axis was the predominant NT pathway in cultured human astrocytes and that TrkB was strongly upregulated on reactive astrocytes in multiple sclerosis (MS) lesions.
Astrocytic responses to BDNF and functional consequences for neuronal cells were investigated in vitro. We observed that similarly to the inflammatory mediator IL1, BDNF activated astrocytes to release soluble factors that induced degeneration of rat spinal neurons. Appropriate experimental settings led to the identification of the released factors.
Finally, we analysed clinical expression of experimental autoimmune encephalomyelitis (EAE) in double transgenic mice, where TrkB had been selectively depleted in GFAP positive cells. We demonstrated that blockade of the BDNF-TrkB axis in astrocytes protected animals from EAE development.
Overall, we identified a novel neurodegenerative pathway triggered by astrocytes in response to BDNF and contributing to MS pathology and experimental neuroinflammation.
This study was supported by FISM (Fondazione Italiana Sclerosi Multipla). Triggering receptor expressed on myeloid cells-2 (TREM2) is a microglial cell surface receptor, which signals via the immunoreceptor tyrosine-based activation motif (ITAM) of the associated common adaptor molecule, DAP-12. In humans, loss of function mutation of either TREM2 or DAP12 leads to chronic neurodegeneration and formation of bone cysts, both hallmarks of the Nasu-Hakola disease.
We now demonstrate that TREM2 deficient (KO) mice, even in the absence of any treatment, present mild signs of increased microglial activation in several brain regions (ventral tegmental area, substantia nigra, hypothalamus, and cortex) and dopaminergic neuronal degeneration in the nigrostriatal system. Since chronic systemic inflammation has been postulated to promote neurodegenerative processes, we challenged mice intraperitoneally with lipopolysaccharides (LPS). After four intraperitoneal LPS challenges over four days, TREM2 KO mice showed significant (p b 0.001) up-regulation of microglial marker proteins in the ventral tegmental area, substantia nigra, hypothalamus and cortex compared to untreated TREM2 KO mice. Consequently, signs of dopaminergic neuron degeneration were increased in the substantia nigra of the LPS challenged TREM2 KO mice compared with the untreated TREM2 KO mice.
Our data revealed mild spontaneous neuroinflammation and neurodegeneration in TREM2 deficient mice, which were amplified by systemic inflammatory challenge with LPS. We have recently demonstrated that monocyte-derived macrophages support motor function recovery following spinal cord injury. Healing following damage to the central nervous system (CNS) in general, and to the inner retina in particular, depends on rescue of neurons that escape the lesion, regeneration and cell renewal. We therefore hypothesized that blood-borne macrophages are directly involved in such healing processes, and hence are essential following an insult to the visual system that relies on functional retinal ganglion cells (RGCs).
Here, by using bone marrow chimeric mice, we found that bloodborne macrophages infiltrated the damaged ganglion cell layer only following glutamate intoxication. Ablation of monocytes in the peripheral blood resulted in impaired survival of RGCs, and reduced retinal progenitor cell (RPC) renewal in the ciliary body. In accordance, enhancement of the monocytic population augmented the survival of RGCs and increased RPC proliferation. Using these experimental tools, we found that monocyte-derived macrophages contributed to the anti-inflammatory and neuroprotective milieu of the injured retina.
Thus, our findings identify a novel fundamental role of monocytederived macrophages in regulating the immune response following retinal injury while contributing to neuroprotection and cell renewal. The neuroprotective potential of these cells might have far-reaching implications regarding retinal neuropathies and other neurodegenerative disorders of the CNS. The pathogenesis of Alzheimer's disease (AD) is generally attributed to the increased production and accumulation of β-amyloid protein that results in direct neuronal toxicity and in microglial activation which, through the production of inflammatory mediators, contributes to neuronal damage.
Amyloid plaques contain an increasing number of molecules, whose significance has not been clearly characterized. Among these, deposition of β-amyloid in autoptic AD brains is strictly associated with chitin, an insoluble polymer of N-acetyl-glucosamine, which is detectable by Calcofluor staining both in amyloid plaques and within the cytoplasm of surrounding microglia.
The aim of our study was to assess whether chitin could contribute to the pathogenesis of AD, investigating its biological effects on neurons and microglia.
We first demonstrated the production by N9 microglial cultures of chitotriosidase (the enzyme that degrades chitin), whose levels were increased by both β-amyloid and inflammatory stimuli (LPS, IFNg and/or TNFa). When N9 or primary microglial cultures were exposed to exogenous chitin, both cells were able to phagocyte small-sized chitin deposits through the mannose receptor, and the process was significantly inhibited by β-amyloid. Similarly to what was described with β-amyloid, phagocyted chitin induced a marked proliferation and activation of microglial cells, with consequent production of proinflammatory cytokines and reactive oxygen species. This activating effect of chitin was significantly attenuated by TGF-β.
A central point concerns the production of chitin by mammalian cells, which lack chitin synthase. We demonstrated that in the presence of an excess of N-acetyl-glucosamine, murine microglial cells, but not fibroblasts, are able to produce Calcofluor-positive chitin-like deposits, which are then released in the medium.
Our results indicate that in particular conditions of altered glucose metabolism and hexosamine pathway microglial cells produce chitinlike deposits which are then released in the extracellular space. Both endogenous and exogenous chitins activate microglia, a process that may contribute to neuronal toxicity. β-Amyloid and inflammatory mediators can modulate chitin pathway by influencing its up-take and degradation. These data suggest that chitin may play a role in the pathogenesis of AD, and microglia is fundamental in its production and degradation.
Early microglial activation and blood-retinal barrier breakdown during autoimmune optic neuritis Fairless Richard ⁎ , Williams Sarah, Hoffmann Dorit, Diem Ricarda
Optic neuritis is a common, early symptom in patients with multiple sclerosis (MS). It also occurs as one of the symptoms of experimental autoimmune encephalomyelitis (EAE) in brown Norway (BN) rats immunised with myelin oligodendrocyte glycoprotein (MOG), resulting in axonal degeneration, demyelination and inflammatory infiltration of the optic nerves. However, in this model, prior to the onset of clinical EAE symptoms and infiltration of immune cells into the spinal cord and optic nerves, we have reported early degeneration of retinal ganglion cells (RGCs). The aim of this project was to investigate early changes in the retina and optic nerve during onset of optic neuritis with respect to activation of the innate immune system, in order to identify causes of this early neurodegeneration.
To monitor neurodegeneration, RGCs were retrogradely-labelled with Fluorogold and retinas excised during the induction phase of optic neuritis. RGCs begin to degenerate from day 5 post-immunisation (p.i.), whereas histopathological analyses of optic nerves confirmed that optic neuritis did not begin until approximately day 14 p.i. In addition, frozen sections from retinas and optic nerves were analysed by immunohistochemistry for evidence of microglial activation, and the integrity of the blood-brain and blood-retinal barriers were investigated by immunofluorescence and Evans Blue extravasation. Using these methods, we found that microglial activation occurred in both retina and optic nerves from as early as day 5 p. i. In addition, although the blood-retinal barrier was disturbed from the same time-point, the blood-optic nerve barrier remained intact until the onset of optic neuritis. However, in agreement with other studies, we found that the blood-brain barrier of the optic nerve head was incomplete, in both healthy and immunised animals. In addition, activated microglia were distributed throughout the optic nerve with a frequency inversely proportional to the distance from the optic nerve head.
We propose that following immunisation of BN rats with MOG, the vulnerability of the blood-retinal barrier, and permeability of the optic nerve head may act as entry points for microglial-activating and potential neurotoxic factors from the periphery. In turn, early microglial activation in this model may be a factor in explaining the early neurodegeneration of RGCs, and may also explain why visual disturbances are a common, early symptom in patients who go on to develop MS.
Expression of IL-1β and TNF-a in Alzheimer's pathology in mice before and after endotoxin challenge Ilkjaer Laura ⁎ , Babcock Alicia Anne, Finsen Bente
Chronic activation of microglia, possibly by amyloid beta (Aβ) plaques, is significant in Alzheimer's disease (AD), and might play a central role in its pathogenesis. Activated microglia produce potentially neurotoxic substances including the cytokines tumour necrosis factor a (TNF-a) and interleukin-1β (IL-1β). Here, we investigated the mRNA levels of TNF-a and IL-1β in the neocortex in an APP/PS1 double transgenic (Tg) mouse model for AD and in wild type (WT) mice in the age groups 3, 6, 9, 12, 15, 18, 21 and 24 months by qPCR. Also, the number and percentages of TNF-a and IL-1β producing microglia and macrophages in 12 month old WT and Tg mice were investigated by flow cytometry. Since systemic infection/inflammation is believed to aggravate AD pathology, we also investigated the effect of treating 9 and 11 month old mice weekly, with an endotoxin, for 3 and 1 month respectively. Age dependent amyloid plaque accumulation in APP/PS1 Tg mice was investigated by stereology in 3, 6, 9, 12 , and 15 month old mice.
Stainings of CD11b+ microglia/macrophages showed an age-and transgene-dependent activation of microglia which was consistent with FACS data from 12 month old mice and appeared to correlate with an age-dependent increase in amyloid plaque density. The levels of TNF-a mRNA increased significantly with age in APP/PS1 Tg mice, whereas we observed close to baseline levels of IL-1β mRNA. FACS data showed a significant increase in the number of TNF-a and IL-1βexpressing microglia in Tg mice. Following treatment with an endotoxin, levels of IL-1β mRNA in Tg mice rose significantly compared to PBS treated mice (~2 fold) and also compared to WT mice receiving treatment. TNF-a mRNA levels were also increased after endotoxin treatment but not to the same extent.
In conclusion, the results point that the continuous plaque deposition induces a chronic low-grade microglial-driven inflammatory reaction in the aged APP/PS1 Tg mice and that a long-term inflammatory challenge might induce and enhance the production of potentially neurotoxic substances such as TNF-a and IL-1β. Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Several neurodegenerative disorders are associated with microglial accumulation and activation. Microglia have both protective and toxic effects for neurons through production of various cytokines, chemokines, and the other soluble factors. Glutamate-mediated excitotoxicity contributes to neuronal damage in various neurological disorders. Degenerating neurons produce various signaling molecules which regulate microglial function.
In this study, we identified a role of the CX3C-chemokine fractalkine (FKN) between degenerated neuron and microglia, particularly in the perspective of neuronal survival and microglial phagocytosis.
Secreted soluble form of FKN from mouse primary microglia and cortical neurons were measured by ELISA. Effect of FKN treatment in the expression of various cytokines, phagocytosis-related factors, and antioxidant enzyme heme oxygenase-1 (HO-1) in microglia was assessed by RT-PCR and ELISA. Phagocytosis of neuronal debirs was evaluated uptake of fluorescent dye-stained mouse primary cortical neurons by microglia. Using several specific inhibitors and Western blotting, intracellular signaling pathways involving clearance of degenerated neurons and HO-1 production were examined.
FKN was secreted from neurons affected by glutamate toxicity, and promoted microglial phagocytosis of neuronal debris through release of MFG-E8, also known as a mediator of apoptotic cell clearance. FKN induced antioxidant enzyme HO-1 in microglia without producing neurotoxic molecules such as nitric oxide, tumor necrosis factor, and glutamate. Consequently, treatment of FKN significantly repressed neuronal cell death by glutamate in primary neuron-microglia cocultures. Using several specific MAPK inhibitors, FKN-induced HO-1 expression and neuroprotective action were mediated through extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathway. Moreover, the HO-1 induction is associated with activation of NF-E2-related factor 2, the transcription factor involving antioxidant response element pathway.
Secretion of FKN from glutamate-exposed neurons may act as a help-me signal and neuroprotectant through activation of phagocytosis of unwanted debris and expression of the antioxidant HO-1 by microglia. Cerebral ischemia is accompanied by an acute inflammation, involving the activation of microglia and the infiltration of neutrophil granulocytes and monocytes into the brain. How these different immune cell types contribute to the neuronal outcome after cerebral ischemia is still under discussion. Various significant actions of immune cells are just to reveal by imaging them either in vitro/ex vivo or in vivo. We developed a postischemic ex vivo model of immune cell (fluorescently labeled) application on hippocampal slices with eYFP expression in neurons. We observed two significant mechanisms how microglia protect neurons after ischemia. On the one hand microglia were found ischemic induced in close proximity or in physical cell-cell contact to the neurons and on the other hand microglia eliminated infiltrating neutrophil granulocytes very fast and efficient. Blocking both properties yielded in an exacerbation of neuronal damage. To test our hypothesis in vivo we generated a mouse transgenic for neutrophils (Lys-EGFP) and microglia (CX3CR1-EGFP). For experimental cerebral ischemia we used a model of permanent middle cerebral artery occlusion combined with an occlusion of the common carotid arteries for 20 min. With intracranial two-photon microscopy (TPM) we are able to image these cells to a depth of 300 μm in vivo after ischemic lesions. To date we observed a rapid infiltration of neutrophils and a very fast response of microglia to damaged vessels after ischemia. In more detail neutrophil granulocytes adhere promptly to the vessels and subsequently invade the cerebral parenchyma. Furthermore microglia seemed to shield inflamed vessels by sending their processes. This approach is suitable to answer a wide range of questions on how immune cells respond to cerebral ischemic events and might contribute to gain intelligence for developing suitable therapeutic strategies. Cuban multiple sclerosis (MS) patients were evaluated to Intrathecal measles (M)-rubella (R)-and varicella zoster (Z)antibody synthesis and NFL-AI, since these have not been reported in our country, yet. A comparative distinction was also reported considering the different epidemiology in the tropical and untropical regions.
23 Cuban MS patients with a representative age distribution and gender ratio were analysed for CSF neurotrophic virus and protein neurofilament light subunit (NFL) antibody reaction, using an ELISA assay was evaluated as specific Antibody Index. The normal values of AI were between 0.7 and 1.2 (median AI = 0.95). Lower frequency of intrathecal rubella antibody synthesis (rubella-AI = 1.5) was observed in Cuban patients (30% of the cases, gender ratio of rubella-AI positives m:f = 1:6) compared with German patients (60%, gender ratio m:f =1:1.8) which could be explained by low incidence of rubella infections in Cuba: NFL-AI, was increased (AI ≥ 1.5) a low number of cases with a range of AI = 1.6-13.9 (median AI = 2.9), nevertheless, a significant correlation was observed between NFL-AI and relapsing in MS patients (p b 0.05). A few numbers of MS cases investigated had an increased NFL-AI combined with one or more antibody species intrathecally synthesized to neurotrophic virus frequently seen in MS.
Intrathecal antibody synthesis in MS correlates with the fraction of seropositives in the population. Natural infection or vaccination may be a precondition for the intrathecal antibody synthesis against neurotropic viruses in MS. The difference in NFL autoantibody levels observed in this group of MS patients confirmed the results of some previous reports in relationship to the utility of NFLP-AI as axonal damage markers. In addition, the significant relationship observed with relapsing in this subgroup of patients, promotes the probable relevance of these markers as subrogate markers for MS.
In vivo imaging after stroke investigated by intracranial 2-photon microscopy
Riek-Burchardt Monika ⁎ ,1 , Neumann Jens 2 , Orlando Gabriella 3 , Gunzer Matthias 3 , Reymann Klaus G. 1,4 1 Leibniz Institute for Neurobiology, Project Group Neuropharmacology, Magdeburg, Germany; 2 Otto-von-Guericke-University Magdeburg, Department of Neurology, Magdeburg, Germany; 3 Otto-von-Guericke-University Magdeburg, Institute for Molecular and Clinical Immunology, Magdeburg, Germany; 4 Institute for Applied Neurosciences gGmbH, Magdeburg, Germany
The CNS damage caused by stroke is accompanied by an acute inflammatory response. The inflammatory process during stroke consists of activation of resident brain microglia and recruitment of leucocytes, namely neutrophils and monocytes/macrophages. However, just what is being sampled by these microglial processes has not been demonstrated in vivo, and the nature and function of any interactions between microglia and the invading immune cells are incompletely understood. Recently, we could identify a new neuroprotective mechanism of the CNS whereby microglia guard neurons by engulfment of toxic neutrophil granulocytes invading brain slices in an in situ stroke model. In our current work we investigate the in vivo relevance of these findings.
For induction of cerebral ischemia we use a model of permanent middle cerebral artery occlusion combined with an occlusion of the common carotid arteries for 20 min. Crossing Lys-EGFP (green fluorescent neutrophils) mice with CX3CR1-EGFP (green fluorescent microglia) mice allows the visualization of both cell types in the same animal, using large differences in morphology and migration characteristics as a marker for the identification despite similar colour. We investigate the post-ischemic changes of microglia/neutrophil morphology and behaviour using intracranial live imaging via twophoton microscopy at different time points (6, 12, 24 and 48 h) after the insult. The technical possibilities of two-photon fluorescence microscopy enable a look, up to 500 μm into live tissue.
Our examinations demonstrate that the described method is very powerful to investigate the in vivo situation of post-ischemic microglial activation and neuro-immune cross-talk. We observed an early extravasation of neutrophils after ischemia. In the first 24 h local microglia changed their morphology dramatically. We will present the first post-ischemic in vivo studies from different microglial activation phases with time-lapse movies directly from the infarct area.
Intracranial in vivo 2-photon microscopy is a useful method to investigate immune cell interactions directly within vital tissue. These imply a new quality and new possibility to examine the inflammation processes in the brain after cerebral ischemia.
Stroke is characterized by inflammation with intense lymphocyte infiltration. A lot of recent studies suggest that this lymphocyte recruitment, in particular CD4+ T cells, is fundamental for the progression of cerebral ischemia lesion. However, the role of the different T helper cell subsets (Th1, Th2 and Th17) in stroke is unknown.
We use organotypic hippocampal slice cultures (OHC), which contain the different CNS cell types and retain the complex 3-D organization of the nervous tissue. For induction of experimental stroke the OHC were exposed to oxygen and glucose deprivation (OGD). In this in vitro model we have discovered the effects of T helper cell specific cytokines (IL-4, IL-17 and IFN-gamma) on neuronal cell death after OGD. Our results show that 24 h after OGD the neuronal cell death was significantly decreased after treatment with 20 nM IL-4. The application of 10 nM IL-17 and 10 nM IFN-gamma was not neuroprotective.
These findings indicate that the main cytokine of the T helper cell type 2 (IL-4) may have beneficial effects in ischemic brain injury. We therefore suppose Th2 cells as a potential supporter for neuroprotective effects after stroke.
Monitoring of degeneration of the retinal nerve fiber layer by optical coherence tomography in rats with autoimmune optic neuritis Hein Katharina ⁎ ,1 , Gadjanski Ivana 1 , Sättler Muriel 1 , Kretzschmar Benedikt 1 , Diem Ricarda 2 , Bähr Mathias 1 1 Dept. of Neurology, Göttingen, Germany; 2 Dept. of Neurology, Homburg/ Saar, Germany
Multiple sclerosis is a chronic autoimmune disease which frequently manifests with optic neuritis. Axonal loss is thought to be the major pathological feature of permanent disability in this disorder. Since the retinal nerve fiber layer (RNFL) is composed largely of unmyelinated axons of retinal ganglion cells measuring RNFL thickness represents a promising tool of monitoring axonal loss in patient with optic neuritis. In the present study, we evaluated the accuracy of spectral domain OCT in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis.
RNFL thickness measurements were performed by periodically imaging during the disease development and progression. Our results showed that RNFL thickness declined significantly before clinical manifestation of the disease and progressed continuously during the disease course. RNFL thickness measured by OCT had good repeatability and reproducibility and also corresponded with histomorphometric measurements. Furthermore, high correlation of decreased RNFL and retinal ganglion cells density was found.
In summary, we showed for the first time that OCT can reliably monitor neurodegeneration in an experimental model of autoimmune optic neuritis in rodents. Moreover, comparing RNFL thickness decline with histopathological analysis of the optic nerve our results suggest an early and in part inflammation independent process of RNFL degeneration in autoimmune optic neuritis. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) and remyelination in MS ultimately fails. Although strategies to promote myelin repair are eagerly sought, mechanisms underlying remyelination in vivo have been elusive. CXCR2 is expressed on neutrophils and oligodendrocyte lineage cells in the central nervous system (CNS). CXCR2 positive neutrophils facilitate inflammatory demyelination in demyelination models such as experimental autoimmune encephalomyelitis (EAE) and cuprizone intoxication. Systemic injection of a small molecule CXCR2 antagonist at the onset of EAE decreased demyelinated lesions. These results left the cellular target of the CXCR2 antagonist uncertain, and did not clarify whether CXCR2 blockade prevented demyelination or promoted remyelination. Here, we show that the actions of CXCR2 on nonhematopoietic cells unexpectedly delay myelin repair. Bone marrow chimeric mice (Cxcr2+/ → Cxcr2/ and Cxcr2+/ → Cxcr2+/+) were subjected to two distinct models of myelin injury. In all cases, myelin repair was more efficient in Cxcr2+/ → Cxcr2/ animals. Oligodendrocyte progenitor cells (OPCs) in demyelinated lesions of Cxcr2+/ → Cxcr2/ mice proliferated earlier and more vigorously than in tissues from Cxcr2+/ → Cxcr2+/+ animals. In vitro demyelinated CNS slice cultures also showed better myelin repair when CXCR2 was blocked with neutralizing antibodies, or was genetically deleted. Our results suggest that CXCR2 inactivation permits optimal spatiotemporal positioning of OPCs in demyelinating lesions to receive local proliferative and differentiating signals. Given that CXCR2 exerts dual functions which promote demyelination and decrease remyelination by actions towards hematopoietic cells and non-hematopoietic cells respectively, our findings identify CXCR2 as a promising drug target for clinical demyelinating disorders. We suggest that the small moleculemildronate [3-(2,2,2trimethylhydrazinium) propionate dehydrate], a representative of aza-butyrobetaine classmay be suitable for the regulation of crosstalk between the immune and nervous system processes impaired in Parkinson's disease (PD). Previously we have found its mitochondria-regulating activity (Pupure et al., 2008) and neuroprotective action in anti-HIV drug azidothymidine neurotoxicity model in mice (Pupure et al., 2010) .
In the present study, we modeled PD in rats by unilateral intrastriatal injection of 20 μg of 6-hydroxydopamine (6-OHDA). Rats were pretreated by mildronate (50 and 100 mg/kg, i.p., daily, for two weeks). After evaluation of behavior (apomorphine rotations), rats were sacrificed, and frozen striatum (STR) and substantia nigra (SN) were cut in 10 μm sections and examined immunohistochemically by assessing markers for neuronal and macro/microglial cells: tyrosine hydroxylase (TH), a neuronal marker; ubiquitin (regulatory peptide involved in ubiquitin-proteasome degradation system); iba-1 (ionized calcium binding adaptor molecule 1); glial fibrillary acidic protein (GFAP); inducible nitric oxide synthase (iNOS); and glial cell line-derived neurotrophic factor (GDNF).
In 6-OHDA-lesioned STR the following results were obtained: loss of TH positive cells, an increase in the expression of ubiquitin, iNOS, iba-1 and GFAP; GDNF expression was not influenced. Mildronate in 6-OHDA-lesioned STR, completely reversed to the control (CSF instead of 6-OHDA) level the number of TH positive cells, prevented overexpression of ubiquitin, GFAP and iNOS; a tendency to elevate GDNF (STR) and to decrease iba-1 (SN) expression was found.
These data convincingly demonstrate an ability of mildronate to protect nigrostriatal dopamine cells against 6-OHDA destructive action (TH normalization). Mildronate is also capable of regulating/ improving the ubiquitin-proteasome pathway, and reducing inflammation (iNOS) and reactive macroglia/astrocytosis (normalized GFAP expression). One may suggest that structural peculiarities (charged moieties) allow to refer mildronate to the privileged small molecules with a regulatory profile, providing the multi-faceted neuroimmunoregulating and neuroprotective. University of the Saarland, Homburg/Saar, Germany; 2 Twincore-Center for Experimental and Clinical Infection Research, Hannover, Germany; 3 Georg-August University Goettingen, Goettingen, Germany
Although the pathological role of the prion protein in transmissible spongiform encephalopathies has been widely investigated, the physiological role of the cellular prion protein (PrPc) remains elusive. Amongst the many functions attributed to PrPc, there is increasing evidence that PrPc is involved in cell survival and mediates neuroprotection. In addition, a potential role in the immune response has also been suggested. The aim of this study was to determine how these two functions interplay during autoimmune disease.
Firstly, we induced experimental autoimmune encephalomyelitis (EAE), a commonly used model of multiple sclerosis, in C57Bl/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG). In over 90% of these mice, optic neuritis, as characterised by demyelination, inflammatory infiltration and axonal degeneration of the optic nerves, was present. Through Western blotting, we observed an up-regulation of PrPc protein in all CNS compartments analysed throughout the disease course. Next, we induced EAE in transgenic mice both deficient for and over-expressing PrPc. Mice deficient for PrPc were found to have an exacerbated EAE disease course in comparison to wild type mice. However, histopathological analyses revealed that optic neuritis was exacerbated in both mouse strains compared to wild type animals, both showing increased numbers of infiltrating cells, demyelination and axonal degeneration. In order to dissect the role of PrPc in neurodegeneration in these animals, Fluorogold labelling of retinal ganglion cells (RGCs), the neurons whose axons form the optic nerve, was performed prior to immunisation. Quantification of surviving RGCs in animals with EAE revealed significant neuroprotection of RGCs in PrPc over-expressing mice, whereas mice lacking PrPc had reduced numbers.
Our data show a strong correlation between the level of PrPc expression and the survival of RGCs. Furthermore, this neuroprotective function of PrPc was shown to be independent of its role in regulating the immune response. The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is activated in neurons and glia in response to oxidative DNA damage resulting from cerebral ischemia/reperfusion. Since PARP-1 is compartmentalized to the nucleus, a critical unanswered question is, how does nuclear PARP-1 send death signals to mitochondria? Despite some intriguing recent findings, this signaling pathway remains poorly understood. Bnip3, a Bcl-2 family pro-apoptotic protein, causes neuron death in a manner sharing several common features with PARP-1-induced neuron death. Studying a link between PARP-1 and Bnip3 is attractive based on these commonalities.
To test our hypothesis that PARP-1 activity increases Bnip3 expression and neuron death two models were used: 1) Bnip3 activation paradigm (hypoxia, 4-48 h) to determine whether PARP-1 is required for Bnip3 upregulation, and 2) an established method of activating PARP-1 with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a DNA-alkylating agent that potently activates PARP-1 and elicits PARP-1-dependent cytotoxicity. Primary mouse cortical neuron cultures from wild-type and PARP-1−/− mice were exposed to hypoxia (4-48 h) , and to MNNG (50 μM, 30 min). Neuron viability was visualized using the calcein/ethidium assay for live and dead cells. Hypoxia caused significant neuron death by 17 h and reduced survival to less than 10% by 48 h. Cell death was PARP-1-dependent as survival was significantly enhanced by both the PARP-1−/− genotype and the PARP inhibitor, N- Ndimethylacetamide.HCl (PJ34) . Hypoxia (48 h) also enhanced Bnip3 protein expression nearly 3-fold in a manner abolished by PARP-1 deletion and PJ34 (10 μM) treatment, indicating that hypoxic Bnip3 expression is dependent on PARP-1. Using direct PARP-1 activation with MNNG in normal oxygen, we found reduced neuronal survival that was concentration and time-dependent. Importantly, significant neuron death, first seen between 4 and 6 h after MNNG, was preceded by a 3-fold increase in Bnip3 protein expression at 4 h that was blocked by genetic PARP-1 deletion.
These experiments show that (1) hypoxic Bnip3 expression is PARP-1-dependent, and (2) Beclin 1 is a protein that plays a critical role in the generation of mature autophagic vesicles and is subsequently involved in the clearance of protein aggregates. Beclin 1 is reduced in affected brain regions of Alzheimer's disease (AD) patients and is associated with an accumulation of amyloid aggregates. Recent studies suggest reduced Beclin 1 levels in neurons impairs autophagy and promotes amyloid accumulation. However, microglia are also capable of regulating the clearance of these aggregates through phagocytosis. Despite emerging links between autophagy and phagosome maturation, the role of Beclin 1 in microglia remains unclear. Therefore we sought to determine whether Beclin 1 regulates microglial phagocytosis.
Here we use lentivirus encoding Beclin 1 shRNA to reduce Beclin 1 levels in microglial cells (i.e., BV-2 cells) and assess whether reduced Beclin 1 impairs phagocytosis or amyloid beta (AB) clearance. To measure phagocytosis we utilize latex beads, which are rapidly phagocytosed when added to microglial cultures. Using this model we find that reduced Beclin 1 levels in microglia decreases both the number of cells that phagocytose beads and the number of internalized beads per cell. Impaired internalization of beads in Beclin 1 deficient microglia appears to specifically result from defects in phagocytosis since endocytosis of transferrin receptors is not impaired. Importantly, phagocytic defects in Beclin 1 deficient microglia can be reversed by rescuing Beclin 1 levels via a Beclin 1 overexpressing lentivirus. Using an ex-vivo amyloid precursor protein (APP) brain slice model, we also find that Beclin 1 deficient microglia are less efficient at degrading cortical and hippocampal AB. Impaired AB clearance by Beclin 1 deficient microglia cannot be explained by deficits in lysosomal function or cellular migration, both of which were comparable with control cells, suggesting that Beclin 1 regulates intrinsic cellular machinery required for phagocytosis.
Together these data suggest that reduced Beclin 1 levels in AD brains may allow for enhanced disease progression by impairing microglial phagocytosis. Therefore, strategies that enhance Beclin 1 levels may provide a novel approach for the treatment of AD. Protein members of the granin family exert important roles in sorting and aggregation of secretory products and in the subsequent granule biogenesis of the regulated secretory pathway. In addition to their intracellular functions, intact granins and their proteolyticderived peptides mediate cell-to-cell signaling, including homeostatic processes, inflammatory reactions, and the innate immunity. SgIII is a little known granin expressed by endocrine cells, platele