CORD-19:27a606f3c89e21ba2b287011593ed80b86a32113 / 45775-45781
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Abstract
John Newsom-Davis (JND) was a major international figure in the field of neurology and neuroimmunology. He combined clinical expertise with commitment to scientific understanding of disease mechanisms and their implications for treatment. He influenced a generation of neurologists and neuroscientists and his exceptional personal qualities of intelligence, tolerance, energy and modesty are greatly missed by all.
JND brought clinical observations to the laboratory and vice versa. With experience of both muscle spindle electrophysiology and intensive care medicine, in 1973 he started collaborating with Ricardo Miledi at the University College London in work on myasthenia gravis (MG). The discovery of antibodies to acetylcholine receptors in serum of these patients by Jon Lindstrom and colleagues, and the demonstration of passive transfer of disease to mice by injection of patients' IgG by Klaus Toyka and Dan Drachman, led JND to test the effects of plasma exchange as a treatment. The remarkable efficacy of this therapywhich lasted for several weeksshowed convincingly that myasthenia was indeed caused by humoral immunity. The discoveries that followed in JND's and others' laboratories owe much to the implications of this simple clinical experiment. Over the next 20 years, he and his team in London and then Oxford systematically investigated the role of the thymus and involvement of T cell immunity in MG, defined the existence of humoral immunity to voltage-gated calcium channels in the Lambert Eaton syndrome, and showed similarly that some cases of acquired neuromyotonia were associated with antibodies to voltage-gated potassium channels (VGKC).
With retirement in 1998, JND stepped aside from the laboratory and, while continuing to see MG patients and "Editing" Brain, he began to establish a major international trial of thymectomy in MG with Gil Wolfe, Gary Cutter and many others; this was eventually funded by the NIH. Meanwhile his team has continued to identify new antibody-associated conditions, such as other forms of MG and central nervous system disorders, ensuring that his legacythe diagnosis and treatment of immunotherapyresponsive conditionswill persist. This talk will briefly review his achievements and how these have been taken forward over the last ten years.
Josep Dalmau, MD, PhD Limbic encephalitis refers to an inflammatory process involving the hippocampi, amygdala and less frequently frontobasal and insular regions. This disorder used to be considered extremely rare, invariably associated with cancer, and unresponsive to treatment. However, recent studies indicate that limbic encephalitis is more frequent than previously thought, and a substantial number of patients may recover. This increased recognition of limbic encephalitis is due in part to the development of clinical diagnostic criteria and the identification of antibodies directed against two broad categories of antigens: 1) intracellular or classical paraneoplastic antigens, including Hu, Ma2, and CV2/CRMP5, among others, and 2) cell surface antigens including, voltage-gated potassium channels (VGKC), N-methyl-D-aspartate receptor (NMDAR), and others expressed in the neuropil of the hippocampus. While the disorders related to the first category of antibodies associate with cancer (lung, testis and other), prominent brain infiltrates of T-cells, and limited response to treatment, the disorders related to the second category of antibodies associate less frequently with tumors, appear to be antibody-mediated, and respond better to immunotherapy. While studying limbic encephalitis, we identified a large group of allied disorders in which the limbic system is often involved, but other areas of the brain are also affected. Of particular interest is the disorder that associates with antibodies to extracellular epitopes of NR1/NR2 heteromers of the NMDA receptor. Patients with this syndrome are often young women with ovarian teratoma, although the disorder can also affect men and children with or without tumors. Most patients present with severe psychiatric symptoms and are first seen by psychiatrists. Subsequently patients develop seizures, dyskinesias, autonomic instability or hypoventilation. There is a correlation between antibody titers and outcome. The antibodies are directed against the NR1 subunit of the NMDA receptor, differentiating this disease from a number of disorders attributed to antibodies against NR2 subunits. In vitro studies indicate that patients' NR1 antibodies can modulate the number of NMDA receptors. There are other autoimmune encephalitides for which the target antigens are on the cell surface of neurons and neuronal processes of the hippocampus. The phenotype of these patients is different from that of anti-NMDA receptor encephalitis and resembles more a classical limbic encephalitis with frequent relapsing-remitting seizures and memory deficits. Work in progress suggests that the autoantigens are ion channels or receptors critically involved in processes of learning and memory. In all, the study of these disorders provides a link between immunologic processes and neuronal events involved in memory, cognition, seizures, and neuronal degeneration.
Interferons (IFNs)-inducible myxovirus resistance protein A (MxA) has recently been used as an indirect marker of neutralizing antibody against IFN in patients with multiple sclerosis (MS). MxA also inhibits viral replication by modifying cellular function including the apoptotic pathway. Moreover, a recent study has suggested that haplotypes of SNPs on the MxA promoter region affect the response to IFN-beta treatment in MS. Our goal is to investigate the genetic and pathological role of MxA in patients with MS.
We examined SNPs of the MxA promoter region in 67 MS patients and 103 normal controls. All MS patients fulfilled McDonald's criteria. Moreover, to elucidate the functional roles of SNPs, we conducted Luciferase assay with pGL3-basic vector including patient-derived or artificially mutated MxA promoter region. We also examined MxA protein expression in peripheral blood monocytes (PBMC) by quantitative RT-PCR.
We found a significantly higher frequency of the haplotype with −88T and −123A in MS patients which correlates with an overexpression of MxA and has been reported to be associated with a worse response to IFN-treatment. Moreover, we obtained novel findings showing that nt-88 played a leading part with type I IFNs while nt-123 played a similar role independently unrelated to type I IFNs. Quantitative RT-PCR demonstrated higher expression of MxA in MS patients than in normal control.
SNPs on the MxA promoter region may play an important role in the pathophysiology of MS, and hyper expression of MxA is associated with MS. Further studies of MxA may lead to novel therapeutic strategies for MS.
3 -The plasmacytoid cell: A clue to the pathogenesis of multiple sclerosis?
Crawford Colin L. 1 1 Imperial College of Medicine, Charing Cross Hospital, London, UK In secondary progressive multiple sclerosis (SPMS), lymphoid follicles containing B and plasma cells have been observed. However, these plasma cells stained with the specific marker CD138 were confined to the perivascular spaces and were not found in the brain parenchyma. In multiple sclerosis (MS) patients, cells in the brain parenchyma, considered to be plasma cells, contain subplasmalemmal linear densities (SPLDs) and are frequently binucleate. Both these features are not present in plasma cells. Instead, these cells could be plasmacytoid cells, which are the precursors of epithelioid cells in sarcoidosis and have also been found to contain SPLDs. In sarcoidosis, giant cells due to fusion are common, so that the binucleate cells in MS could also arise by cell fusion rather than division. The plasmacytoid cell has now been identified as a type of dendritic cell. These cells are increased tenfold in the cerebrospinal fluid of patients with early MS.
Non-lepromatous leprosy is a granulomatous disorder similar to sarcoidosis. In order to produce an autoimmune animal model of nonlepromatous leprosy, Dutch Bantam rabbits were injected with a suspension of human sensory peripheral nerve (sural nerve and dorsal roots) plus adjuvant. Some of these rabbits developed a state of granulomatous hypersensitivity. ie an epithelioid cell granuloma was produced at skin test sites using dilute suspensions of peripheral nerve antigen. When various non-myelin fractions were used at these sites, cells whose cytoplasm contained abundant rough endoplasmic reticulum with SPLDs were observed. A cell was observed indistinguishable from a plasma cell containing a small thickening on the plasma membrane. Plasma-like cells were also found in the endoneurium. The most active fraction producing cells with SPLDs was a deoxycholate-extracted membrane fraction, in doses of 1 μg, produced from the low speed nuclear pellet after the myelin had been removed. This fraction also induced plasma-like cells and axonal damage in the endoneurium.
Plasmacytoid dendritic cells could be identified in MS tissue using the specific marker CD123. If confirmed, a specific non-myelin autoantigen may be implicated in the pathogenesis of MS. Furthermore, as these plasmacytoid dendritic cells secrete large amounts of interferon alpha, this cytokine could cause axonal damage in MS patients. The implication of CD8 T cells in the pathogenesis of multiple sclerosis (MS) and its animal models is increasingly being appraised. CD8 T cells in the central nervous system (CNS) of MS patients have been shown to be highly activated with an effector memory phenotype. Contribution of CNS factors to the acquisition of such phenotype by CD8 T cells has not been elucidated. Interleukin-15 (IL-15) is a pro-inflammatory cytokine that is crucial for the generation and maintenance of memory CD8 T cells. IL-15 produced by cells in the tissue microenvironment of other inflammatory diseases has been revealed as a potent activator of infiltrating CD8 T cells leading to an enhanced cytotoxicity towards target cells. Our goals are i) to investigate whether IL-15 is present in the CNS in the context of MS ii) to determine whether IL-15 in situ can enhance deleterious CD8 T cell responses against CNS target cells thus contributing to the observed tissue damage.
Primary cultures of human astrocytes significantly increased their IL-15 expression upon activation with pro-inflammatory stimuli (IFNgamma, TNF-alpha, IL-1beta) (flow cytometry). In order to determine if such cells could be a functional IL-15 source, they were cultured with myelin specific human CD8 T cell lines. Upon co-culture with activated astrocytes, CD8 T cells acquired an effector memory phenotype with increased perforin and granzyme B levels (flow cytometry). Moreover, these CD8 T cells were more cytotoxic towards primary cultures of human adult oligodendrocytes (51Cr release). These astrocyte-mediated functional enhancements of CD8 T cell function were all abrogated by anti-IL-15 blocking antibodies. Immunohistochemistry performed on active MS lesions showed an increased proportion of astrocytes expressing IL-15, demonstrating the physiological relevance of our in vitro observations.
Our results underline the contribution of CNS resident cells, namely astrocytes, to a microenvironment favoring cytotoxic CD8 T cell responses via sufficient amounts of IL-15. In situ IL-15 has already been shown to promote autoimmune effector CD8 T cell responses in other autoimmune diseases such as rheumatoid arthritis and celiac disease. IL-15 bioavailability in the CNS may be implicated in the exacerbation of tissue damage targeting oligodendrocytes during MS pathogenesis. Diagnosing Multiple Sclerosis (MS) is not always easy because MS and other demyelinating diseases such as neuromyelitis optica (NMO) may be indistinguishable clinically. Although anti-Aquaporin (AQP) 4 antibody can differentiate NMO from MS clinically, the possible link between the two demyelinating diseases remains a mystery. Recent advances in the proteomic technology in search of biomarker or biomarker signature that accurately identifies the clinical syndrome would allow for improved diagnosis and disease monitoring.
Magnetic bead-based purification followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to profile human cerebrospinal fluids (CSF) proteins and peptides in a total of 82 samples from patients with definite MS (remission and relapse), NMO (remission and relapse) and primary progressive multiple sclerosis (PPMS). All study participants gave their written informed consent. We used a reagent set of chemically coated magnetic beads, reversed phase (C8) (ClinProtTM) and a-cyano-4hydroxycinnamic acid as the matrix solution. The eluted samples were then dropped onto a MALDI sample plate (AnchorchipTM), and spectra were obtained by an Autoflex II and a subsequent tandem MS analysis was performed by Ultraflex (Bruker Daltonics). The criteria for peak detection were: signal-to-noise ratio N5, 2-Da peak-width filter, and maximum peak number of 200. The pretreated data were graphed as spectra and evaluated by statistical analysis using the ClinProToolsTM software (Bruker Daltonics). Reproducible profiles were obtained as clear signals and approximately fifty peaks were detected from each of CSF samples. A differential distribution of samples from MS and NMO both in remission was noticeable, while samples from PPMS were not separated effectively using the same platform. One of the key variables contributing to the separation with an m/z of 3511 was defined as c-terminal fragment (182-212) of neuroendocrine peptide 7B2 by the tandem MS analysis.
The application of magnetic bead-based separation combined with MALDI-TOF-MS technique for CSF samples holds the potential to advance our understanding of the biochemical basis of MS and NMO. Further studies are required to validate the clinical effectiveness and disease specificity of the identified biomarkers.
6 -Role of granzyme B in T cells-induced neurotoxicity and neuroprotective strategies using the new serine-protease inhibitor Serpina3n 1 Osaka University, Osaka, Japan 2 Boehringer-ingelheim, Osaka, Japan 3 Toyonaka municipal hospital, Osaka, Japan Significant roles of Semaphorin in immune system were reported recently, while it has been investigated mainly as an axonal guidance molecule in the developing central nervous system (CNS). Class 4 semaphorin Sema4A is expressed in antigen presenting cells and T cells. It is suggested to activate T cells via T cell immunoglobulin domain and mucin domain (Tim)-2, and is a critical molecule for the Th1 differentiation.
Therefore, we generated monoclonal anti-Sema4A antibodies by immunizing Sema4A-deficient mice, and established ELISA system. We have measured serum levels of Sema4A in 41 multiple sclerosis (MS) patients, 10 patients with spinocerebellar degeneration (SCD) and 15 patients with encephalitis. Statistical analysis was performed by Welch's t-test. Serum Sema4A level was significantly higher in MS patients than patients with SCD and encephalitis (p b 0.001 and p b 0.02 respectively). Especially one-third of patients with MS exhibited marked high level of expression, which suggest the involvement of Sema4A in the pathogenesis of MS and may also suggest heterogeneity of MS pathogenesis. We next investigated the expression of Sema4A in experimental autoimmune encephalomyelitis (EAE) mice. Immunohistochemical examination revealed that infiltrating cells into the spinal cord of EAE mice exhibited marked expression of Sema4A.
Overall, these results suggest that Sema4A may be a potential biomarker for MS, and that it may be an important molecule for the pathogenesis of MS. Oligoclonal IgG bands in cerebrospinal fluid (CSF) are found in up to 95% of patients with multiple sclerosis (MS), a chronic inflammatory demyelinating disorder affecting the central nervous system (CNS). An important issue is whether these Igs recognize (1.) a disease-relevant structure [neural targets, e.g. GalCer]; or (2.) a cross-reactive infectious agent [e.g. Mycoplasma, E. coli], or whether they (3.) reflect intrathecal activation of B-lineage cells secreting Igs directed at undetermined specific antigens derived in the inflammatory process. We have postulated a lipid mimicry pathogenesis for the induction of MS autoimmunity, and examined CSF IgGs for cross-reactivity between myelin and microbial lipids focusing respectively upon: (a.) acetyl-cerebrosides designated as FMCs or 'fast migrating cerebrosides' of higher TLC Rf than GalCer and other myelin lipids, (b.) MfGL-II, a glyco-glycero-cholinolipid from M. fermentans, (c.) LPS of E. coli J5 (UK+) strain, and other bacterial lipids.
The analysis of 38 patients included 29 CSF samples from MS patients who were further classified into relapsing-remitting (RRMS) (16), secondary progressive (SPMS) (10), and primary progressive (PPMS) (3). Comparison was to nine other neurological disease (OND) controls divided into inflammatory (I-OND) (5) and non-inflammatory (NI-OND) (4). The elevated anti-FMC-7 antibodies were significantly higher in the PPMS group compared to SPMS group (66.67% vs. 0%, p = 0.0385) and in the RRMS in remission group compared to SPMS group (63.64% vs. 0%, p = 0.0039) while lesser changes were found for anti-GalCer and anti-sulfatide antibodies. It is striking and of considerable interest that highly elevated anti-microbial (M. fermentans, E. coli) antibodies were found in meningitis (3 patients), one patient with subacute sclerosing panenecephalitis (SSPE) and one patient with meningoencephalitis (I-OND group) compared to NI-OND control (p b 0.05). It is noteworthy that the anti-lipid antigen elevations occurred in tandem for both microbial and self antigens.
The findings are consistent with cross-reactivity of immunoglobulins in human MS CSF between microbial and myelin glycolipids in some MS patients, and suggest a role for microbial surface and myelin antigens, and particularly glycolipid antigens, in the pathogenesis of MS. Further examination of a larger number of MS and control Istituto Tecnologie Biomediche, CNR, Bari, Italy
We studied six monozygotic (MZ) twin pairs (5 female and 1 male) discordant for multiple sclerosis (MS) and two disease concordant MZ pairs (1 female and 1 male). Unrelated couples of patient-healthy control subjects were age-and sex-matched to each twin pair and analyzed as well.
All patients were free of immunomodulatory treatment. MZ twins and unrelated subjects were analysed independently to avoid that the expected high biological variation between these two groups might prevent the discovery of differentially expressed genes and exons. When array data from MZ twins were loaded and normalized by RMA in oneChannelGUI environment, the subsequent principal component (PCA) analysis confirmed the strong clustering of each twin pair (already observed with other platformsunpublished). To overcome this bias (the observed biological variability among couples of MZ twins was higher than the variability observed into each couple obscuring the identification of differently expressed genes), we imported array data into Partek GS software where an ad hoc ANOVA model could be implemented to handle this effect. After the batch removal a new ANOVA model was built in Partek GS in order to take into account the remaining sources of variation (patient status, gender, the age and all potential interactions). These ANOVA factors were then used to select lists of genes differently expressed and significant to a BH-corrected p-value b 0.05. We focused also on the detection of specific alternative splicing events by means of the identification of differently expressed exons. This analysis was performed in Partek GS environment using an ANOVA model in which the alternative splicing factor was set to the status of the patient.
The above parameters identified 10 differentially expressed genes and 1 exon in disease discordant MZ twins, 3 genes and 1383 exons in unrelated patients and healthy controls, 0 genes and 422 exons in healthy co-twins compared to unrelated healthy controls. 121 of these 422 exons were also in the list of 1383 exons differentially expressed between unrelated patients and controls.
The gradient in the number of differentially expressed exons among twins (1), among healthy twins and unrelated healthy subjects (422), and among unrelated patients and controls (1383) supports the hypothesis of an "MS trait" in the healthy co-twins. The relevance of the alternative splicing data is being verified by means of specific RT-PCR approaches.
Darlington Peter⁎ 1 , Johnson Trina 1 , Bar-or Amit 1 , Antel Jack 1 1
Neurons are susceptible to cytotoxic immune-mediated injury in neuro-inflammatory disorders including multiple sclerosis and acute and chronic encephalitides. The restricted expression of MHC molecules on neurons implicates injury mediated by MHC/antigen-independent immune effector mechanisms. Previously, we reported that innate immune cells (NK and gamma-delta-T cells) induced loss of human neurons in vitro consequent to initial destruction of supporting astrocytes (Darlington et al. J. Neuropath. Exp. Neurol. 2008, in press). Short-term anti-CD3-activated T cells did not kill neurons or astrocytes but caused caspase-dependent fragmentation of the cytoskeletal protein GFAP. Our goal was to further characterize the mechanism of such astrocyte injury and determine the overall consequence for neuronal survival.
Short-term anti-CD3-activated T cells induced sub-lethal caspase-3 activity, leading to caspase-dependent fragmentation of GFAP in primary human astrocytes. This occurred in a contact dependent manner. Further cytotoxicity experiments indicate that these effects could be attributed to CD8 T cells rather than CD4 T cells. Incubation of anti-CD3 activated CD8 T cells with the exogenous lectin phytohemagglutinin (PHA), which promotes adhesion between cell surface glycosylated receptors, resulted in actual astrocyte cytotoxicity leading to neuron loss. This lectindependent effect was not observed with short term anti-CD3 activated CD4 T cells. However, both polyclonally expanded (short term anti-CD3) and MBP-reactive Th1 CD4 T cells induced lectin-dependent astrocyte cytotoxicity. Th2 polarized CD4 T cells were less cytotoxic than their Th1 polarized counterparts.
This data indicates that the extent of T cell-mediated target cell injury is determined by both the intrinsic cytotoxic potential of these cells and the extent of cell-cell contact interaction with their targets. T cells with cytotoxic potential may induce sub-lethal injury of astrocytes when cell-cell interaction is limited; the long term consequence of such astrocyte injury on neuronal survival remains to be defined. Whether there is sufficient up-regulation of cell adhesion molecules in the inflamed CNS to support T cell mediated cytotoxicity and/or whether there are endogenous lectins capable of supporting immune-neural interactions in the CNS also remains to be established. from conventional multiple sclerosis (CMS) for their similarities in clinical manifestation. To investigate the feasibility of diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) assessment of microscopic fiber tract injury in the corpus callosum (CC) and other normal-appearing white matter (NAWM) in patients with NMO and multiple sclerosis (MS) .
In this study, we proposed a novel approach, using apparent diffusion coefficient (ADC) and fractional anisotropy (FA) of the brain derived from DTI and MRS as classification feature, to discriminate patients with RNMO/OSMS from CMS in Japan. Twenty patients with RNMO/OSMS (median expanded disability status score [EDSS] 4.6) and 23 CMS patients (median EDSS, range 2.5) were studied with T2 weighted images, DTI, MRS (chemical shift imaging and single voxel). Brain MRS allows in vivo examination of axonal integrity by quantifying the neuronal marker N-acetylaspartate (NAA), often expressed as a ratio to creatine (Cr). We have tested directly the relationship between changes in these DTI parameters and NAA concentrations in the MS lesion, NAWM, and normal appearing corpus callosum (NACC).
This study indicates that 1) FA in MS patients was significantly decreased in NACC and NAWM, possibly due to a reduction of smallcaliber axons; 2) the data showed significantly lower FA for posterior limb of internal capsule in RNMO/OSMS patients than in those in CMS patients and healthy volunteers after Bonferroni adjustment, 3) NAA/Cr in RNMO/ OSMS group was significantly decreased in the posterior lobe subdivisions of the WM. The decrease in FA in any of the CC and WM and the decrease of NAA in WM did not correlate with disease duration or EDSS score.
In conclusion, the proposed method on the basis of combined features is more effective for classification than those merely using the features separately, and it may be helpful in differentiating RNMO/ OSMS from CMS patient. controls than in MS (p = 0.016). No differences between groups in concentrations of IL-17, IL-13 and IL-10 were observed.
Our results indicate that no apparent deviation of immune regulatory T cell phenotypes can be seen at a systemic level with regard to transcriptional deviation and in vivo cytokine secretory patterns. Although both T-bet and RORC display higher relative expression in MS, representing a transcriptional deviation towards the Th17 and Th1 phenotypes, this has to be evaluated together with the finding that IFN-g was decreased in plasma of MS patients. Subsequent analysis of subgroups of MS, as well as cytokine levels and transcription factor expression in cerebrospinal fluid will ensue.
16 -The persistency of high levels of PSTAT3 expression in circulating CD4+ T cells from CIS patients favours the early conversion to clinically defined multiple sclerosis
Not all patients with clinically isolated syndrome (CIS) suggestive of multiple sclerosis develop clinically defined MS (CDMS). In this study we evaluated the expression of T-bet, pSTAT1, pSTAT3 in CD4+ and CD8+ T cell and monocytes from peripheral blood of CIS patients during and 3-6 months after the first clinical event. Furthermore we correlated the expression of transcription factors with patient clinical outcome and with IFNg production, IL6, IL17 and IL10 by PBMC.
In our study we included 18 CIS patients and 20 sex and age matched controls. Detection of pSTAT-1, pSTAT-3 and T-bet was performed by intracellular flow cytometry. The production of IFNg, IL17, IL6 and IL10 by PBMC was measured by ELISA. Higher levels of pSTAT1 and pSTAT3 were observed in CD4+T cells and in monocytes from CIS patients in acute phase than from controls. T-bet expression was higher only in monocytes from CIS patients in acute phase than in ones from controls. pSTAT1 and T-bet expression were higher in CD14+ cells from patients in acute phase than from patients in remission phase. IFNgamma, IL17 and IL10 levels were significantly higher in CIS patients in acute phase than from controls. IFNg levels decreased in remission phase while IL17 and IL10 levels remained high. During the follow-up period, 8/18 patients converted to CDMS. In acute phase both groups of patients who did or did not converted to CDMS showed higher expression of pSTAT1 and pSTAT3 in CD4+ T cells and higher expression of T-bet and pSTAT3 in CD14+ cells than controls while only non-converted patients showed higher pSTAT1 expression in CD14+ cells than controls. Both groups of patients showed higher production of IFNg and IL10 than controls. In remission CIS patients who converted to CDMS showed higher pSTAT1 and pSTAT3 expression in CD4+T cells than controls. In addition pSTAT3 expression was higher in CD4+ cells from converted than from nonconverted patients. Both groups of patients showed higher production of IFNg and IL10 than controls while only IL10 was higher in patients who did than in patients who did not converted to CDMS.
IL17 seems to play a pathogenetic role in CIS and the persistency of high levels of pSTAT3 in circulating CD4+T cells from CIS patients after the first clinical event may favor the early conversion to CDMS.
17 -The relapsing dynamics of multiple sclerosis depends on control properties of peripheral immune tolerance
Autoimmunity is characterized by the presence of a relapsingremitting dynamics such as in Multiple Sclerosis. The goal of our study was to demonstrate that this relapsing-remitting dynamic can be generated by a failure in the feed-back control of two competing populations regulating peripheral tolerance: effector and regulatory T cells.
We developed a computational model of the human immune system, using the Systems Dynamics framework and stochastic differential equations based in biological knowledge. Our analysis shows that by fine-tuning the parameters regulating the negative feedback between effector and regulatory T-cells, the immune system is able to generate a relapsing-remitting dynamics. Such autoimmune dynamic was stable and obtained in a wide range of parameters close to the normal functioning of the immune system. In addition, we found that stochastic stimulation of the immune system strongly influences the timing at which relapses appear, preventing their prediction. We also found some unexpected predictions because the increase in the activity of effector T-cells in some circumstances generates a strong control response that prevents the presence of relapses. Finally, we introduced perturbations into the model mimicking immunotherapies aimed to increase the activity of either effector or regulatory T-cells. We demonstrate that the effect of such therapies was highly dependent on the timing and the underlying dynamic of the immune system, implying that immunotherapies must be developed in a personalized manner and focused in obtaining a healthier dynamic in the control module of the peripheral tolerance, more than in keeping the absolute number of T-cells in certain ranges.
In summary, our study provides a view of autoimmunity as a dynamic process and provides new clues for developing immunotherapies. Helminth infections concurrent with MS significantly reduce disease severity, as previously shown by these authors. Growing evidence indicates that B cells play a key role in restraining unwanted self-reactive T cell responses in different autoimmune models in mice, and that helminth infections in MS patients give rise to a B cell population producing high levels of IL-10. Toll-like receptors (TLR) function as sentinels recognizing different microbial molecules. To elucidate the role of TLR in helminth infections, we examined the expression of TLR-2, TLR-4, TLR-5, and TL-9 on B cells in helminthinfected and in uninfected MS patients.
Peripheral blood CD19+ B cells from 12 helminth-infected MS subjects, 12 uninfected MS patients and 12 healthy controls were purified using magnetic cell sorting. Surface expression of TLR-2 on B cells was significantly higher in helminth-infected MS patients compared to uninfected MS patients and healthy controls. No differences in TLR-4, TLR-5 or TLR-9 expression were observed between groups. Moreover, in helminth-infected MS patients TLR-2 engagement induced preferential expansion of IL-10 secreting B cells in a MyD88-dependent manner. To examine whether this differential activation of regulatory B cells had an impact on T cell immune responses, B cells previously stimulated with TLR-2 ligands were cocultured with allogenic CD4+ FoxP3-T cells, and examined by flow cytometry for FoxP3 expression and IL-17 and IFN-gamma production. TLR-2 stimulated B cells were highly efficient in inducing FoxP3 expression on alloreactive T cells, converting over 50% to FoxP3 positivity, whereas TLR-4, TLR-5 and TLR9 engagements did not alter FoxP3 expression. Likewise, following TLR-2 stimulation, B cells induced the generation of IL-10 producing FoxP3-Tr1 cells. Moreover, the percentage of CD4+ T cells expressing IFN-gamma and IL-17 was significantly lower in cultures containing B cells stimulated with TLR-2 agonists, compared to those activated via TLR-4, TLR-5 or TLR-9. In this experimental setting, suppression of both IFN-gamma and IL-17 was abrogated when MyD88 expression was silenced by siRNA.
Our data indicate that IL-10 producing B cells link recognition of helminth products via TLR-2, providing a novel target for the parasites to suppress T cell-mediated autoimmune diseases.
19 -CD4+CD25+FOXP3+PD1− naïve regulatory T cells in acute and stable relapsing-remitting multiple sclerosis and their modulation by therapy Regulatory T cells (Treg) play a fundamental role in preventing immune reactions against self antigens. Recent data suggest that the intracellular expression of the programmed death receptor 1 (PD1) identifies a subset of naïve Treg with enhanced suppressive ability; antigen stimulation results in the surface expression of PD1. Because the role of Treg impairments in MS is still contradictory, we analyzed naïve (CD4+CD25++FoxP3+PD1−) and activated (CD4+CD25++FoxP3+ PD1+) Treg in peripheral blood of 50 relapsing-remitting Multiple Sclerosis (RR-MS) patients and of healthy controls (HC). CSF of 5 MS patients and 5 individuals with non-inflammatory conditions was also examined.
Results showed that, whereas CSF naïve Treg were significantly augmented in MS patients, these cells were comparable in the peripheral blood of patients and controls. Clinical sub analyses nevertheless demonstrated that peripheral naïve Treg were significantly increased in patients with stable (SMS) compared to those with relapsing (AMS) disease, and in patients responding to Glatimer Acetate (Copaxone; COPA) compared to AMS and COPA-unresponsive patient. In contrast, activated Treg were similar in CSF and peripheral blood of all groups analyzed. Naïve Treg were not increased in the peripheral blood of interferon b (IFNb)-responsive patients, but the suppressive ability of Treg was significantly higher in SMS and in COPA-or IFNb-responsive compared to AMS and COPA-unresponsiveindividuals.
The data herein suggest that naïve Treg play a pivotal role in the pathogenesis of MS and offer a biological explanation for disease relapse and for the mechanism associated with response to COPA and IFNb. Clinically isolated syndrome (CIS) represents first neurological symptoms suggestive of demyelinating lesion in the central nervous system (CNS) according to magnetic resonance imaging (MRI) with the risk of multiple sclerosis (MS) development. So far there are no sufficient immunological markers predicting even disease activity or efficiency of registered CIS treatmentinterferon beta.
Aim of the study: To compare immunological status before and after 6 months of interferon beta 1-a, 30 μg intramuscularly once weekly (IFNb) treatment in two groups of CIS patients: responders (R) and non-responders (NR) to IFNb therapy according to clinical and MRI disease activity within the first year of the treatment.
Patients and methods: Peripheral blood was taken before any treatment and after 6 months of IFNb therapy in 17 CIS patients (8 Raverage age 30 years, F/M = 5/3, 9 NRaverage age 32 years, F/M = 6/3). CIS was defined as a first demyelinating event with at least 2 lesions on T2W or FLAIR MRI images and positivity of oligoclonal bands in cerebrospinal fluid. NR was defined by occurrence of at least one relapse and one new T2W lesion on MRI during first year of the treatment. Flow cytometer FACSCalibur and monoclonal antibodies BD Bioscience were used for mononuclear cells immunophenotyping (CD3, CD4, CD8, CD19, CD3-CD16, 56+, and CD45RA subpopulation). Student t test and Pearson correlation were used for statistical analysis, intergroup paired comparisons were performed using MANOVA.
The only statistically significant correlation was found between amount of CD19+ cells in the peripheral blood before any treatment and relapse frequency during the first year of IFNb treatment (p = 0.004, r = − 0.9759) in five NR patients. There was no other statistically significant correlation or intergroup difference between any of the tested immunological markers and IFNb treatment responsivity.
Based on our preliminary results number of CD19+ cells in the peripheral blood may be one of the immunological markers in MS prognosis and determination of treatment efficacy. Multiple Sclerosis (MS) is an autoimmune central nervous system (CNS) disease characterized by inflammatory infiltrates in alongside demyelinated axons, axonal transections and degenerating neurons; the exact pathological process though is still unclear. We have been exploring the possibility that innate immune responses are involved in disease immunopathogenesis, in particular gammadelta T cells which are concentrated in MS lesions. We have previously reported on gammadelta T cell direct cytotoxicity and cytokine release, but here we examine the possibility that injury can also be mediated via antibody dependent cell cytotoxicity (ADCC). Autoantibodies to myelin components are found in MS patients. Activated gammadelta T cells up-regulate the expression of Fcg receptor (CD16) raising the possibility that they can bind antibody which facilitates ADCC-type interactions.
An in vitro experimental system was established using a humanized anti-CD20 antibody rituximab as the bridging antibody and CD20+ B lymphomas as targets. Cytotoxicity was measured with in-house developed multiple parameter flow cytometry technique. Our results show that, with E:T ratio at 4:1, the ADCC cytotoxicity of gammadelta T cells against the target cell lines was significantly increased by the presence of rituximab (p b 0.02), and this effect could be abrogated by removal of Fc portion of the antibody (p b 0.05 vs. integral rituximab) or by using CD16-effector cells (p b 0.05 vs. CD16+ population), suggesting a CD16-specific antibody-mediated cytolytic contribution. Furthermore, by using intracytoplasmic flow cytometry, we found that antibodycoated target cells could induce the production of both Th cytokines from gammadelta T cells. Interestingly, the capability of producing Th2 subtype cytokines is exclusively resided in the CD16+ population (p b 0.05).
Our findings tie together the innate and adaptive immune responses through an interaction that involves the stimulation of antibody production, possibly against CNS molecules such as myelin, via CD16 expressing gammadelta T cells that could use these antibodies to enact a guided attack on CNS targets via the mechanism of ADCC. This study offers further novel insights into the possible role of gammadelta T cells in MS that can involve both potentially pathogenic and immunoregulatory functions. Natural Killer (NK) cells are a major effector cell of the innate immune response. Their relevance to several autoimmune diseases is under investigation. Valleys in cytotoxicity been implicated in multiple sclerosis (MS) relapse. Cytotoxicity may be one way NK cells regulate the nature and scale of the autoimmune response. Our objective was to examine NK cytotoxicity in newly diagnosed treatment naive MS subjects, not in relapse, and compare it to age and sex matched healthy controls and controls from another static neurological condition: stable chronic spinal cord injury.
Methods: A four-hour chromium 51 release assay using a K562 target cell line was used, with the laboratory technician blind to the clinical status of the subject. Three different effector:target (E:T) cell ratios were studied, these were analyzed with paired t test.
Results: Eighteen MS subjects naïve to any immunotherapy, 23 spinal cord injured (SCI) subjects with static neurologic deficits, as well as their individually matched by age and sex healthy laboratory controls. There is a statistically significant reduction in NK percent kill in both the MS subjects as compared to their age and sex matched controls (at 25:1 mean 5.7 SEM 1.8 vs. 17.9 SEM 2.6; at 50:1 mean 14.7 SEM 2.7 vs 32.0 SEM 3.9; at 100:1 mean 25.0 SEM 3.9 vs 42.1 SEM 4.5). This difference was statistically significant at all three E:T cell ratios (t = 3.9, df = 17 p = 0.001; at 50:1 t = 4.50 df = 17, p = 0.003; at 100:1, t = 3.65, df = 17; p = 0.002). For the 23 SCI subjects there was a statistically significant reduction in NK percent kill as compared to their age and sex matched controls at only the 25:1 (mean 10.6 SEM 2.9 vs. 27.8 SEM 8.0; t = 2.34, df = 22, p = 0.03). Although there were trends for the other two E:T ratios, they did not reach statistical significance.
The MS subjects naïve to treatment with minimal neurologic deficits have a significant reduction in NK cytotoxic ability, as compared to health controls and as compared to a group with severe but static neurologic impairment without autoimmune disease. This supports the concept of intrinsic NK cytotoxicity dysfunction early in the MS disease process, possibly contributing to autoimmune dysregulation. Since there is evidence that their cytotoxic ability may be one way NK cells orchestrate their neuroregulation, these data would implicate them as potential targets for future therapy.
Vereyken Elly 1 , Dijkstra Christine 1 , Teunissen Charlotte 1 1
It is widely recognized that macrophages play an important role in the pathogenesis of multiple sclerosis (MS), having both beneficial and detrimental effects. Depletion of infiltrating macrophages has a suppres-sive effect on clinical signs of experimental allergic encephalomyelitis. Conversely, elimination of macrophages leads to impaired remyelination after lysolecithin induced demyelination. Different subtypes of macrophages exist with different functions in immune response and tissue repair. Two extremes are classically activated (CA) macrophages, which can be cytotoxic, and alternatively activated (AA) macrophages, which can be growth promoting. Both types of macrophages have been found in MS lesions, with CA being located mostly at the active border and AA more in the center of the lesion and perivascular. Due to these differences in localization our question became whether differently activated macrophages migrated differently and could be attracted by different cell types in the central nervous system (CNS).
We studied the migration of differently activated macrophages in organotypic CNS cultures and the effect of either demyelination or neuronal damage on migration. Finally, the intrinsic migratory capacity of macrophages was studied by directed migration over a filter under the influence of chemokines.
A higher number of AA macrophages migrated into untreated spheroids compared to CA macrophages. This difference in migration was also seen after the induction of demyelination or neuronal damage. Comparing demyelination and neuronal damage no difference in the number of migrating macrophages was observed. Both CA and AA macrophages could migrate deep into the spheroids and reach neuronal cell bodies and axons, however this was observed more frequently for AA macrophages.
To study whether the differences in migration were due to intrinsic migratory capacity of the macrophages, we studied the migration across a filter in a blind well chamber. A higher number of AA macrophages migrated in response to CXCL12 and MCP-1 compared to CA macrophages. Macrophages migrated very well towards neuronally conditioned medium, indicating the presence of a soluble chemokine in the medium.
The intrinsic migratory capacity of AA macrophages is greater compared to CA macrophages, even in the CNS environment. In spheroids AA macrophages migrate deeper, possibly due to neuronal attraction, which might also play a role in the localization of AA in MS lesions.
24 -Interferon-beta signal transduction, multiple sclerosis and auto-antibodies antibodies to phenotype specific cell subsets. The cells were then analyzed by multicolor FACS. In a second step we simplified the method for antibody detection by using a cell line. We have been able to evaluate signaling of interferon-beta in autologous sera and serum free media of treated and non-treated RRMS. By using a cell line and sera of RRMS patients we could rapidly detect auto-antibodies to interferon beta.
Multi colour flow cytometry in conjunction with phosphoantibodies is a powerful reproducible tool to detect immediate effects of agents and molecules such as auto-antibodies or inhibitors on signaling pathways in primary cells. Defects in immune regulation could result in a breakdown of immune tolerance leading to development of Multiple Sclerosis (MS). It was postulated that because of molecular mimicry with cross-reacting and yet undefined epitopes, autoreactive T cells are activated, peripheral tolerance is broken, and disease is initiated or reactivated. The immune impairment of MS is extremely complex. Because the PD-1/PD-L1 pathway was shown to be associated with the production of the immunoregulatory cytokine IL-10, an impairment in these molecules could be associated with the development of MS. The aims of this study were to analyse the possible role of PD-L1/PD1 costimulatory pathway in the onset and the progression of MS and to verify whether immunotherapy has an effect on the expression of these molecules.
We enrolled in the study 50 relapsing-remitting Multiple Sclerosis (RR-MS) patients subdivided in two groups: 26 with acute, relapsing disease (AMS) and 24 with stable disease (SMS). The expression of the costimulatory molecules PD-L1 and PD-1 as well as IL-10 production by myelin basic protein (MBP)-stimulated peripheral blood mononuclear cells was analysed by flowcytometry. The effects of different immunotherapies (Glatimer Acetate, Copaxone; COPA and interferon beta IFNb) were also evaluated.
Results showed that the percentage of CD14+ and CD19+ cells expressing PD-L1 was reduced in AMS compared to SMS individuals (p = 0.038); this was associated with an important decrease in IL-10 production by MBP-stimulated CD14+ and CD19+ cells in the same patients (p = 0.025) COPA and IFN-beta treatment did not influence PD-L1/PD-1 costimulatory molecules.
In conclusion our data suggest that impairments of thePD-L1/PD-1pathway resulting in a reduction of IL-10 production could play an important role in the pathogenesis of Multiple Sclerosis. Monitorage of the expression of these proteins could be a novel diagnostic tool. remitting multiple sclerosis (MS) for over a decade. Its mechanism of action remains elusive, although recent studies have suggested that multiple cellular and/or soluble factors might be involved in mediating GA's effects in MS and in experimental autoimmune encephalomyelitis (EAE). NK cells are potentially regulatory cells in MS and EAE. The fractalkine receptor, CX3CR1, is expressed on glial cells in CNS. Soluble fractalkine and CX3CR1 dictate the migration of NK cells to the CNS under pathological circumstances. Consequently, NK cells homing to the CNS is significantly impaired in CX3CR1 deficient mice. To investigate whether the impaired homing of NK cells to CNS influence the therapeutic effects of GA during EAE.
EAE was induced in CXCR1−/− mice by immunization of MOG35-55 peptide with adjuvant CFA. Groups of wild type mice and CXCR1−/− mice were treated with GA or PBS upon EAE induction. Clinical parameters were monitored and compared among the groups. GA conferred moderate protection against EAE in wild type mice (mean clinical score 2.43 ± 0.24 vs. 1.56 ± 0.19, p b 0.05; maximum clinical score 4.0 ± 0.50 vs. 3.12 ± 0.21, p b 0.05, respectively). In contrast, GA treatment did not alter clinical course of EA E in CXCR1−/− mice (mean clinical score 2.78 ± 0.28 vs. 2.84 ± 0.29, p b 0.05; maximum clinical score 4.13 ± 0.51 vs. 4.12 ± 0.62, p N 0.05, respectively).
Here we demonstrated that the therapeutic effects of GA in EAE are completely lost in CX3CR1−/− mice. Our current works focus on dissecting how NK cells might contribute to the therapeutic effects of GA using the combination of cell transfer and monoclonal antibodies.
27 -Induction of IL-12/IL-23 by Substance P in human PBMC and expression of Substance P receptor in peripheral blood of MS patients Substance P (SP) is a neuropeptide stored in the termini of sensory neurons. SP induces pro-inflammatory cytokines and mediates its effects by binding to specific Neurokinin (NK) receptors. Previous studies have shown that NK-1 receptor antagonist reduces clinical and histological signs of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). IL-23 is a member of the IL-12 cytokine family that drives a pathogenic T cell population involved in the initiation of EAE and possibly MS. The expression and function of SP and NK-1 receptor in MS is not clear.
Objectives: (1) To determine the effects of Substance P on IL-12p35, IL-12p40 and IL-12p19 expression in normal PBMC. (2) To determine the expression of NK-1 receptor in peripheral blood in MS.
Method: (1) Normal PBMC were treated with SP (10^-6 M), SP (10^-6 M) + SP NK-1 receptor antagonist, SP (10^-6 M) + NK-1 pseudoantagonist, 1 μg LPS and control for 24 h. Quantitative real time PCR (qPCR) was performed with IL-12p35, IL-12p40 and IL-23p19 primers. Values were normalized with beta2 microglobulin (B2MG). (2) RNA was extracted from 15 whole blood MS samples and 9 controls, qPCR was performed with NK-1 primers, data normalised via B2MG.
Results: (1) Treatment with SP increased the relative quantity of IL-12p40, IL-12p35 and IL-23p19 in PBMC 2) The relative quantity of NK-1 receptor RNA in MS patients was significantly lower than
Natalizumab therapy reduces the number of CD4+ T cells within the CSF ten-fold more than the number of CD8+ T lymphocytes. In addition, the effect on cell numbers persists at least 6 months after cessation of treatment.
To extend our studies on the prolonged, and differential, effect of natalizumab on T lymphocyte numbers in the CSF, we investigated the number and phenotypes of leukocytes, and the expression of major histocompatibility complex (MHC) I and II in cerebral perivascular spaces (CPVS). We hypothesized that natalizumab reduces the number of antigen presenting cells (APCs) in CPVS.
Inflammatory cell numbers in the CPVS of cerebral tissue were assessed by immunohistochemical staining from a patient with MS who developed progressive multifocal leukoencephalopathy (PML) during natalizumab therapy. Controls included location-matched cerebral autopsy material of patients without CNS disease, MS patients not treated with natalizumab, and patients with PML not associated with natalizumab therapy.
The absolute number of CPVS in the patient with MS treated with natalizumab was significantly lower than in all control groups due to extensive destruction of the tissue architecture. The expression of MHC class II molecules, and the numbers of CD209+ dendritic cells (DC) were significantly decreased in CPVS in the patient with MS treated with natalizumab. No CD4+ T cells were detectable.
Our observations may explain the differential and prolonged effects of natalizumab therapy on leukocyte numbers in CSF. Multiple sclerosis (MS) is an inflammatory disease of the central nervous system in which axonal damage and degeneration contribute significantly to the progressive irreversible neurological disability. Similar to pathogenic myelin autoimmunity, autoimmune responses to neuronal antigens may contribute to axonal damage and irreversible disability in MS. Autoantibodies to the axonal cytoskeletal protein neurofilament light (NF-L) are associated with cerebral atrophy in MS and we have recently reported that NF-L autoimmunity is pathogenic in mice. However the T-cell response to NF-L in MS patients has not been examined.
Here we identify and characterize T-cell proliferative responses to NF-L as compared to myelin oligodendrocyte glycoprotein (MOG) in MS patients and healthy controls. Using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay we show that while responses to MOG are dominated by CD3+CD4+ T cells, responses to NF-L were observed in both CD3+CD4+ and CD3+CD8+ T-cell populations. Both MOG and NF-Lreactive cells expressed CD45RO+, indicative of a memory phenotype. Moreover, in contrast to MOG-stimulation which predominantly induced interferon-gamma, both Th1 and Th2-type T-cell responses to NF-L were observed as indicated by the induction of interferon-gamma, tumor necrosis factor-alpha as well as interleukin-4.
The finding of T-cell responses to NF-L in MS patients may reflect transient activation of pathogenic potential but their presence also in healthy controls indicates that these cells are part of the normal immune repertoire. School of Biochemistry and Immunology, Dublin, Ireland MS patients treated with IFN beta have been reported to have reduced levels of pro-inflammatory cytokines such as IFN gamma and increased levels of anti-inflammatory cytokines, such as IL-10. However, the precise mechanism of action of IFN beta is unclear. Antigen presenting cells, particularly dendritic cells (DC), play a crucial role in directing T cell responses via the production of regulatory cytokines including the IL-12 family members and IL-10. IL-12 promotes the induction of IFN gamma-secreting T helper (Th) 1 cells, whereas IL-10 promotes the development of IL-10-secreting regulatory T cells. Recently, the IL-12 family member IL-23, together with IL-6 and IL-1, has been shown to promote the development of Th17 cells, which have been demonstrated to play a pathogenic role in autoimmune disorders. A protective role for IL-27, another IL-12 family member, has been demonstrated in the experimental autoimmune encephalitis (EAE) model, where it antagonises the induction of Th17 cells by up-regulating IL-10. Therefore, in this study we investigated the effect of IFN beta treatment on production of IL-27 by human DC.
DC were stimulated with recombinant IFN beta. After 24 h, cytokine concentrations were assayed by ELISA. Cells were stained for expression of ILT3 and analysed by flow cytometry. Alternatively, DC were cultured in the presence of IFN beta, activated with TLR ligands for 24 h, and used to stimulate allogenic CD4+ T cells. After 5 days, supernatants were assayed by ELISA for cytokines.
We demonstrated that IFN beta enhanced production of IL-27 and IL-10 by DC. Furthermore, stimulation of allogenic CD4+ T cells with DC grown in the presence of IFN beta increased T cell IL-10 and decreased IL-17 production. Treatment of DC with IFN beta also upregulated the expression of ILT3, a cell surface inhibitory molecule and marker of tolerogenic DC.
These data suggest that IFN beta may exert some of its protective effects by modulating the cytokine profile of both DC and T cells and by expanding or activating tolerogenic DC. Naïve CD4 T cells have a central role in initiating the autoimmune response in MS. Naïve T cells occupy an ecological niche in the periphery that is regulated by homeostatic mechanisms. Some investigators claim a naïve T cell homeostatic abnormality occurs in MS but the basis of this abnormality could not be clearly established.
Our objective was to test the hypothesis that RRMS patients have premature thymic involution leading to reduced generation of recent thymic emigrants (RTEs) and followed by compensatory homeostatic proliferation of naïve CD4 T cells.
To test this hypothesis, we used a FACSAria cell sorter to isolate subsets of naïve CD4 T cells (10 patients and 10 age-matched healthy controls), based on expression levels of CD31. Specifically, we isolated subsets of RTEs (CD31hi and CD31lo) and non-RTEs (CD31neg). Using real-time PCR, we quantified signal joint T cell receptor excision circles (sjTRECs) and beta TRECs in DNA from these subsets as markers of thymic output. Additional homeostatic markers (Bcl-2, CD127 and Ki-67) were analyzed by flow cytometry within the different subsets.
Results: In controls, true RTEs (CD31hi) showed a significant decline in TREC levels with increasing age, whereas in RRMS comparable cells had consistently low TREC levels irrespective of age. Second, in RRMS, RTEs and non-RTEs showed significantly increased expression of the IL-7 receptor (CD127), whereas non-RTEs had increased Bcl-2 levels.
We report first definitive evidence of a thymic abnormality in RRMS that leads to reduced thymic output and compensatory homeostatic proliferation affecting both RTEs and non-RTEs. These naïve CD4 T cell changes may increase autoreactivity and predispose to RRMS. Multiple Sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS). Cytokines and chemokines are soluble mediators of the immune system that are involved in the pathogenesis of MS by regulating transmigration of immune cells to CNS and mediating subsequent development of tissue damage. Altered cytokine and chemokines profiles in the cerebrospinal fluid and blood have been shown. The aim of this study was to examine whether these molecules could be used as markers of disease activity or markers of different subtypes of MS.
Methods: The study included 113 subjects: 74 patients with different subtype of MS, 17 patients with clinically isolated syndrome (CIS) and 22 healthy controls. Among MS patients 22 patients were treated with interferon-beta. The sera levels of 14 cytokines, including TRAIL, Fas, FasL, MIF, IL-10, TNF-alpha, IL-6, IL-12p70, IFN-gamma, IL-2, CXCL10, CCL2, CCL3 and CCL4 were determined using ELISA and Bio-Plex suspension array system.
The median levels of macrophage migration inhibitory factor (MIF) in sera were lower in untreated patients with MS than healthy controls (199.9 vs. 340.8 pg/ml; p = 0.008), especially in relapsing-remitting MS (RRMS) patients. There was also significant difference between RRMS and primary progressive MS patients (53.6 vs. 284.4 pg/m; p = 0.01). The levels of MIF were also lower in CIS patients when compared to healthy controls (186.2 vs 340.8 pg/ml, p = 0.004).
Also the levels of TNF-alpha were upregulated in MS patients when compared to healthy controls, but there were no differences between MS subtypes (4.8 vs. 3.7 pg/ml). In IFN-treated patients the levels of CXCL10 were upregulated when compared untreated patients (122.9 vs. 76.4 pg/ml; p = 0.01).
These results showed that MIF could be used as a potential MS subtype marker, but this observation needs to be validated in substantial patients groups.
Kondo Takayuki⁎ 1 , Komori Mika 2 , Tomimoto Hidekazu 2 , Tanaka Masami 3 , Tanaka Keiko 4 , Takahashi Ryousuke 2 , Matsuo Hidenori 1 , Saida Takahiko 5 1 Nagasaki Medical Center of Neurology, Nagasaki, Japan Multiple sclerosis (MS) in Japan is classified into two groups, conventional MS (CMS) and opticospinal MS (OSMS). It has been reported that while the former is associated with HLA-DRB1⁎1501, DPB1⁎0501 confers susceptibility to the latter. Following the finding of the positive association of neuromyelitis optica (NMO) and Japanese OSMS with antiaquaporin-4 (AQP4) antibody, the relationship among CMS, seropositive and seronegative OSMS has become a main focus of interests.
Because longitudinally extensive myelitis is the most prominent feature related to Anti-AQP4 antibody positivity, we grouped our patients into the groups of CMS, seropositive and seronegative long spinal (LS) MS with a contiguous spinal cord lesion 3 or more segments in length, and studied HLA class II association within each group. 41 patients with CMS, 53 with seropositive LSMS and 40 with seronegative LSMS were enrolled for this study. After PCR-amplification of HLA-DR genes, the DR alleles were determined by sequence-specific oligonucleotide probes. HLA-DP alleles were amplified by allele-specific primers to determine DP alleles. The data published by Nalajima et al.
(http://jshi.umin.ac.jp/hla_data/data.html) was used for control subjects.
Compared to control subjects, HLA-DRB1⁎1501 alleles tended to be increased in CMS patients (DRB1⁎1501: 18.3% vs. 11.6%, P = 0.09). Determination of HLA-DRB1⁎04 alleles remained ambiguous. Allele frequencies of DR4 carried by CMS were higher than the controls (42.7% vs. 24.4%, P = 0.0024) and LSMS (42.7% vs. 16.7%, P = 0.0023). The frequencies of DR4 in seropositive and seronegative LSMS also differed significantly (11.3% vs. 23.8%, P = 0.024). The frequency of DRB1⁎1502 carried by CMS was lower than in LSMS (3.7% vs. 11.8%, P = 0.03). DRB1⁎0101 was increased in LSMS, compared with controls (seropositive LSMS 8.5%, seronegative LSMS 11.3%, total LSMS 9.7% vs. controls 3.9%, P = 0.003) and CMS (9.7% vs. 1.2%, P = 0.01). Allele frequencies of HLA-DPB1⁎0501 in CMS, seropositive and seronegative LSMS and controls were 51.2%, 43.8%, 50.0% and 40.7%, respectively. No significant difference was observed. If we consider the linkage disequilibrium of DRB1 with DPB1, we failed to find a difference in the number of subjects with HLA-DPB1⁎0501.
Our study revealed that CMS and LSMS have different immunogenesis. However, we failed to show that particular DP alleles confer the susceptibility to any of the three groups, in contrast to previous reports by others.
34 -Soluble trail and BAFF levels in multiple sclerosis during interferon-β therapy 3 Hacettepe University, Oncology Institute, Departement of Basic Oncology Ankara, Turkey 4 Primer Medical Imaging Center, Ankara, Turkey
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family with immunomodulatory capacity having a protective role against autoimmunity TRAIL inhibits T cell activation, cell cycle progression, and IFN-gamma and IL-4 production and may be important in modulating disease expression by downregulation of mechanisms in T-cell-driven responses.
Being critical for B cell survival and maturation BAFF, affects both humoral and cell-mediated responses. Increased soluble BAFF (sBAFF) protein levels have been reported in several autoimmune disorders.
This study focuses on the expression of TRAIL levels on lymphocytes of multiple sclerosis (MS) patients and healthy individuals, and further evaluates the effect of β-interferon therapy on soluble TRAIL and BAFF levels in MS patients.
Age and gender matched 35 relapsing remitting MS patients and 18 healthy controls were included in the study. All patients were evaluated for prognosis, age of disease onset, disease duration and expanded disease severity score (EDSS). Also cranial magnetic resonance details as number of infra and supratentorial plaques, number of contrast enhancing plaques, corpus callosum atrophy and enhancement and brain atrophy have been analysed.
Karni Arnon⁎ 1 , Urshansky Natali 2 , Mausner Karin 1 , Fahoum Firas 2 1 Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel 2 Tel Aviv Sourasky Medical Center, Tel Aviv, Israel
Neurotrophins have been implicated to play a neuroprotective role in MS lesions. We have reported that in patients with RR-MS PBMCs secreted low levels of BDNF and its up-regulation after CD40 stimulation is defective. Here we studied the production and regulation of neurotrophins mRNA (BDNF, NGF and NT3) in immune cells of patients with RR-MS.
PBMCs from 30 patients with RR-MS (13 untreated patients and 17 patients treated with interferon-β) and 17 matched healthy controls (HC) were incubated for 1 h with anti-CD3 or anti-CD40 monoclonal antibodies (mAb) or their isotype controls (IC). mRNA quantity of BDNF, NGF and NT3 in PBMCs was studied by real-time qRT-PCR reactions on a LightCycler® 1.5 Instrument while GAPDH gene was run in parallel as internal control for each reaction set. Results are expressed by the production ratio of each neurotrophin mRNA vs. GAPDH mRNA. For the basal expression of neurotrophins mRNA: BDNF mRNA was similar in the patients (3.6 ± 1.0) and the HC (3.9 ± 0.9, p = N.S), NGF mRNA tend to be lower in the patients (1.4 ± 0.4) vs. HC (2.9 ± 0.8, p = 0.11) and NT3 mRNA was lower in the patients (2.2 ± 0.8) vs. HC (4.6 ± 1.2, p = 0.03). Stimulation of PBMCs with anti-CD40 mAb up-regulated mRNA expression of BDNF, NGF and NT3 as compared to the effect of IC in HC (BDNF mRNA: 9.5 ± 3.1 vs 3.0 ± 0.7, respectively, p = 0.04; NGF mRNA: 6.0 ± 0.8 vs. 2.8 ± 0.8, respectively, p b 0.05; NT3 mRNA: 10.2 ± 2.7 vs. 4.4 ± 1.2, respectively, p b 0.05). In the comparison of the up-regulatory capacity of CD40 stimulation using the ratio of mRNA production after CD40 stimulation vs. IC between HC and the patients: BDNF mRNA ratio: 2.7 ± 0.5 vs. 1.1 ± 0.1, respectively, p b 0.001, NGF mRNA ratio: 4.1 ± 1.3 vs. 1.4 ± 0.4, respectively, p = 0.008, NT3 mRNA ratio: 3.9 ± 0.9 vs. 1.1 ± 0.4, respectively, p = 0.006). No significant differences of neurotrophins mRNA expression were found between PBMCs of untreated and interferon-β treated patients.
The patients were followed-up under interferon-β treatment and their initial, 3rd, 6th, 9th and 12th month blood for TRAIL expression on lymphocytes and soluble TRAIL and BAFF levels were analysed by flow cytometry and ELISA respectively.
All patients were analysed for gender, age of disease onset, disease duration, initial symptoms, prognostic factors, and EDSS. MS patients' mean basal flow-TRAIL expression rates was 11.11 ± 15 while in control group it was detected as 2.16 ±1.64 (0-6). However, of MS patients, 17 (48.6%) had basal f-TRAIL levels as N3.8 (valuesN control mean basal trail+ 2SD) whereas others have b3.8. There was no significant difference in aspect of gender, age of disease onset, disease duration, prognosis and EDSS. Two groups were also similar for detailed cranial MRI features.
In the patient group with high f-TRAIL levels, after treatment with interferon-β for 3 months, mean f-TRAIL level was found to be 3.14 ± 5.30 (p b 0.025) whereas after 6 months it was 2.00 ± 3.21 (p b 0.025).
MS patients' mean soluble basal TRAIL levels were 634.75 ± 195.44 and it was 693.6 ± 379.36 (180-742) in the control group having no statistical difference (p N 0,05). Mean soluble TRAIL level was 634.75 ± 195.44 and this value significantly increased on the sixth month of therapy (1278.66 ± 868.42).
Mean basal BAFF levels of controls were 1.98 ± 0.14 while in MS patients it was 2.11 ± 0.2 (1.8-2.6) with statistical significance (p b 0.0.5). No statistical significance of sBAFF levels has been found during treatment for 9 months.
Our study evaluates the effect of β-interferon therapy on serial TRAIL and BAFF levels longitudinally. During the pre-treatment period we did not detect any significant difference in serum sTRAIL levels compared with healthy controls. Serum levels of sTRAIL were significantly higher on the 6th month than the pre-treatment values.
MS patients' mean basal flow-TRAIL expression rates were higher than control group, however, we also observed that there are two subgroups of patients expressing different TRAIL rates as similar or higher levels when compared with controls. TRAIL expression levels were significantly lower on the 6th month than the pre-treatment values. Detected decrease of TRAIL levels after therapy may depend on the cleavage of membranous TRAIL by specific metalloproteases into soluble TRAIL.
Supporting the literature of other autoimmune disease, in pretreatment period BAFF levels have been found significantly higher in MS group, however, interferon β treatment had no significant effect longitudinally.
Our data suggests that β-interferon can lead the modulation of soluble TRAIL levels leading to a secondary compensatory mechanism that downregulates the inflammatory response.
Haile Yohannes⁎ 1 , Pasichnyk Dion 1 , Giuliani Fabrizio 1 1
Infiltration of T cells and macrophages characterizes the lesions of inflammatory diseases of the central nervous system, such as Multiple Sclerosis (MS). Activated cytotoxic T cells release Granzyme B (GrB), a serine protease, and induce neurotoxicity by degrading the myelin and/ or injuring neurons. However, the separate effect of the subsets of T cells in terms of GrB expression and neurotoxicity has not been described. The objective of this study is to explore and compare the level of GrB expression on lymphocytes and its subsets (CD4 and CD8) as well as to evaluate the separate effect of CD4 or CD8 on human neuronal injury.
Human lymphocytes were activated for 3 days with anti CD3antibody before or after negative selection into CD4 and CD8 using magnetic separation. Expression of Granzyme B in the T cells subsets was measured by real-time PCR. Human cortical foetal neurons (HFNs) (15-20 wks age) were cultured alone or co-cultured with unactivated, activated whole T cells, CD4 or CD8 subsets on poly-ornithine coated culture plates. After 24 h, neuronal viability was assessed using immunocytochemistry for microtubule associated protein-2 (MAP-2) and neurons were counted manually. RT-PCR revealed that activated T cells express GrB above 40 fold in comparison to unactivated cells. Activated CD8 cells also expressed about 3 fold more GrB compared to CD4 cells. In contrast, when CD8 cells were activated in the absence of CD4 cells, the levels of GrB were comparable between CD4 and CD8 cells. The viability of HFNs was significantly low in the co-cultures with activated Tcells (39.1 ± 15%) and activated CD8 cells (38.2± 13.7%) whereas about 70% HFNs survived in the co-culture with activated CD4 cells.
Our findings demonstrate that anti-CD3-activation on lymphocytes triggers high expression of GrB by T cells. GrB mRNA expression correlates with neuronal killing. CD8 cells are the major effectors of neuronal killing. However, activation of CD8 T cells is dependent on CD4 cells for their GrB expression. Further studies are going on to evaluate the cytotoxic effect of post-selection activated CD8 cells.
Aranami Toshimasa⁎ 1 , Sato Wakiro 1 , Yamamura Takashi 1 1 Department of Immunology, National Institute of Neuroscience, NCNP, Kodaira, Japan
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system, in which activation of autoreactive CD4+ T cells targeting myelin antigens and differentiation into Th1 or Th17 effector cells are presumed to play a pathogenic role. It was reported that CD4+ T cells lacking costimulatory molecule CD28 expression (CD28null T cells) are expanded in MS patients and T cell clones derived from these T cells exhibited high ability to produce IFN-g. However, the specific antigen and the pathogenic role for these T cells in MS have been unclear. Here we report that CD28null T cells in MS are abnormally differentiated Th1 cells and are enriched in those specific for alphaBcrystallin (CRYAB), which is highly expressed in oligodendrocytes and astrocytes in active MS lesions but not in normal brains.
Age and sex-matched forty untreated MS patients in the remission state and nine healthy subjects (HS) were enrolled in this study. We examined cytokine production by these T cells upon polyclonal stimulation with PMA and ionomycin and found that they predominantly produce IFN-g but not IL-17 nor IL-4. We analyzed expression of T-bet, an essential transcription factor for Th1 cells, with intracellular Tbet staining and found that CD28null T cells are the only T-bet(high) population among CD4+ T cells. Albeit the higher expressions of T-bet in CD28null T cells than those in CD28+ T cells, CD28null T cells contain fewer cells expressing TIM-3, which has been reported to be induced on Th1 but not Th2 cells and regulate IFN-g production. When we analyzed the frequencies of T cells responding to ovalbumin (OVA), myelin basic protein (MBP) or CRYAB, we have found that the frequencies of T cells responding to CRYAB but not to OVA nor MBP in CD28null T cells from MS patients are significantly higher than those from HS. CD28+ T cells from both MS and HS specifically responded to neither antigen.
These results suggest that CD28null T cells are enriched in abnormally differentiated Th1 cells autoreactive to CRYAB and might contribute to the autoimmune inflammation in MS. Multiple sclerosis (MS) pathology was studied in vivo through evaluation of kinetics of lesion volume variations over time using frequent MRI scans. For this purpose patients with Relapsing Remitting MS course (n = 13) were followed and 29 new lesions showing persistent Blood Brain Barrier (BBB) damage, visualized by contrast enhancement for at least three observations were selected. Each lesion was analyzed using six consecutive observations. Each lesion volume was calculated using a semi-automated contouring method in FLAIR (mainly sensitive to water changes) and T1w sequences after administration of two different contrast doses of Gadolinium (Gd).
In the acute phase, lesions visualized by FLAIR sequences consistently resulted larger than T1 Gd enhancing volumes, but followed over time a similar kinetic profile. However once Gd enhancement disappeared, visualization by FLAIR sequences persisted, reaching a stable volume consistently smaller than the maximum volume of the Gd enhancing phase. Interpolation of the volume kinetic of each lesion with a multiparametric fitting suitable for describing diffusive expansion and contraction phenomena in biological environment, generated random walk type profiles describing in more detail the FLAIR volume kinetics. These profiles resulted from two different phenomena, each following two different kinetics: the first one, mainly associated to the earliest part of the curve, probably depending by the BBB disruption and by the inflammation; the second one, following the first but partly overlapping, probably reflecting the cellular infiltration cause of the irreversible tissue abnormalities.
These results indicate that tissue damage involves only part of the MS lesion visualized during the acute phase and that, a unifying hypothesis on the underlying pathogenic mechanism can be generated. This hypothesis supports a model of acute MS lesions with a "target" morphology organized as follows:in the center mainly the inflammatory infiltrate and tissue damage;in the inner periphery the BBB damage, caused by soluble inflammatory factors diffused from the infiltrate;in the external periphery the edema, made by fluids diffused from the BBB damaged area. In addition, when the acute phase is over and each lesion shrinks usually leaving a stabilized hyperintensity, the model implies that this stable abnormality is made by the center of the acute lesion: tissue damage and cell infiltrates persisting behind a healed BBB. Renewed interest in the role of B cells as antigen presenting cells (APCs) has occurred, following recent results demonstrating the beneficial effects of B cell depletion therapy on relapsing remitting multiple sclerosis (MS). Since this effect is fairly immediate, it is likely that peripheral B cells have a central role in the pathogenesis of the disease.
We have demonstrated that CD40L activated peripheral B cells are capable of efficiently presenting myelin antigen to autologous peripheral T cells. In contrast, CpG oligodeoxynucleotide activated peripheral B cells from both MS patients and healthy controls were unable to stimulate autologous T cells to proliferate in response to any antigen tested. A small population of B cells from MS patients but not healthy control B cells efficiently bound myelin basic protein by flow cytometry, but this did not correlate with APC function. In addition, we have begun to dissect the capacity of ex vivo peripheral naïve and memory B cells in antigen presentation. Thus far we have observed that both naïve and memory B cells are capable of presenting a recall antigen to autologous T cells from both healthy controls and MS patients. As predicted, memory B cells, but not naïve B cells from MS patients were able to present myelin oligodendrocyte glycoprotein to autologous T cells.
These results suggest that memory/activated B cells may be an important population of APCs in the context of MS.
Methyltioadenosine (MTA) have immunomodulatory activity by inhibiting the activation of NFkB and preventing methylation after Tcell activation, showing beneficial effects in animal models of Multiple Sclerosis (MS). The goal of our study was to compare MTA therapy against MS disease modifying drugs (DMD) and to assess tissue injury protection mediated by MTA.
MTA therapy was evaluated in the C57B6 mice immunized with MOG35-55 peptide developing chronic-progressive experimental autoimmune encephalomyelitis (EAE). Murine interferon beta (IFNbeta) and Glatiramer acetate, as well as MTA were administered i.p. every day from the day of immunization. Clinical and histological scores were recorded blinded. Axonal protection was quantified through confocal microscopy.
MTA therapy demonstrated a dose-response effect in ameliorating EAE in the C57 mice, with complete suppression of the disease at the higher dose (192 mcM/kg). At the intermediate dosage, MTA showed better efficacy than murine IFN-beta (5000 U/day) or GA (0.1 mg/day) in preventing EAE. Indeed, we found synergy between MTA and Glatiramer acetate, since suboptimal dosage combination of both drugs efficiently suppressed the disease. Tissue damage was prevented by decreasing the amount of axonal damage as quantified by confocal microscopy of labelled axons. Finally, we found no toxicity at any of the doses tested.
MTA have a potent immunomodulatory effect and display synergy with DMD, preventing axonal damage in models of MS.
The effects of Glatiramer Acetate (GA) in combination with Minocycline (MIN), a second-generation tetracycline, have been investigated on the course of EAE in mice, resulting in a significant reduction in disease severity and burden with attenuation of the inflammation, axonal loss and demyelination.
Here we investigate the effects of combination therapy with GA and MIN on the induction, maturation and phenotyping of blood monocyte-derived dendritic cells (DCs) in Multiple Sclerosis (MS) patients.
The expression of HLA-DR, CD11c, CD83 and CD1a was studied by flow cytometric analysis on immature (iDCs) and mature DCs (mDCs) from untreated and GA treated MS patients. Thirteen relapsingremitting MS patients and 13 healthy controls (HCs) were included in the study. Ten of the MS patient group were re-tested after a 2 month period of GA treatment. The marker expression on DC from untreated MS and HCs was studied in vitro in the absence or presence of GA and GA+MIN; and on DCs from GA treated MS patients without and with the in vitro addition of MIN.
We found that in vitro GA alone (p b 0.05) or in combination with MIN (p b 0.05) downregulated DCs antigen presentation capability (HLA-DR), whereas only the combination treatment affected (p = 0.05) also myeloid DCs activation (CD83) in both MS and HCs. Prolonged GA treatment (in vivo for two months) affected (p = 0.05) antigen presentation capability by DCs, whereas when treated in vitro with MIN these cells also reduced (p b 0.05) activation marker expression and myeloid phenotype acquisition (CD11c).
The present data demonstrate possible combination effects of GA and MIN on peripheral blood monocyte derived DCs in MS patients. Cytokine-neuroantigen (NAg) fusion proteins have promise as a new class of antigen-specific therapies for treatment of CNS autoimmune disease. The primary objective of this study was to assess therapeutic efficacy of selected cytokine-neuroantigen fusion proteins in the Lewis rat model of EAE.
Expression systems for several cytokine-NAg fusion proteins were derived in which the cytokine domain was comprised of IL-1RA, IL-2, IL-4, IL-10, IL-13, IL-16, or IFN-beta. The antigenic domain of each fusion protein was comprised of the major encephalitogenic determinant of myelin basic protein (referred to as the neuroantigen or NAg). The prediction was that the cytokine domain would bind the respective cytokine receptors on antigen presenting cells (APC) and simultaneously promote anti-inflammatory or tolerogenic activity by those APC while targeting the NAg domain to the class II MHC antigen processing pathway for enhanced presentation. The targeted presentation of NAg by these cytokine-conditioned APC would predictably inhibit NAg-specific T cells that interacted with those APC. These fusion proteins were expressed, purified, and subcutaneously administered in saline to test for inhibition of EAE. Three different cytokine-NAg fusion proteins were potent inhibitors of EAE. These fusion proteins incorporated the IL-2 cytokine as the N-terminal domain (the IL2NAg fusion protein), the secreted IL-16 cytokine as the C-terminal domain (the NAgIL16 fusion protein), or the IFN-beta cytokine as the N-terminal domain (IFNbetaNAg fusion protein). These fusion proteins were administered to rats once per week for a total of three injections over 3 weeks. Then at least one week later, rats were challenged with NAg in CFA to induce EAE. Compared to NAg alone, these fusion proteins strongly attenuated the subsequent active induction of EAE. These fusion proteins also inhibited EAE when administered after encephalitogenic challenge during onset of EAE. In NAgIL16 pretreated rats, the mechanism of tolerance was active as shown by adoptive transfer studies.
In conclusion, cytokine-NAg fusion proteins that incorporate IL-2, IL-16, or IFN-beta cytokine domains may comprise an effective tolerogenic therapy for CNS autoimmune disease. The objective of this study was to elucidate statins' therapeutic potential in suppressing chronic autoimmune response in multiple sclerosis (MS). Specifically, we studied: 1) statin-induced changes in dendritic cell (DCs) maturation and antigen presenting capacity; and 2) statin-induced changes in DCs' cytokine production, which may both modify Th17 cell differentiation and the development of autoimmune response in MS.
In order to assess the statin-induced changes in DCs' maturation, we measured surface expression of HLA class I and class II, CD80, CD86, CD40, and CD83 on the surface of DCs matured in the absence or presence of simvastatin. Our results on 10 patients with relapsing remitting (RR) MS revealed that simvastatin significantly decreased the percentage of HLA Class I (p = 0.017), CD40 (p = 0.023) and CD80 (p = 0.040) expressing cells, consistent with inhibition of DCs' maturation. Functionally, the effect of simvastatin-induced inhibition of DCs' antigen presenting capacity was confirmed in experiments with myelin basic protein (MBP) 83-99-reactive T-cell clones.
In RT-PCR studies that measured simvastatin-induced changes in DCs' cytokine production, we detected a significant up-regulation of IL-27 (p = 0.032) and IL-4 (p = 0.01), and inhibition of IL-6 (p = 0.042) and IL-23 (p = 0.043) gene expression. Measurement of secreted cytokines from the same DC cultures revealed up-regulated IL-27 (p = 0.01) and down-regulated IL-1b (p = 0.005), TGFb1 (p = 0.000008), and IL-23 (p = 0.01) protein level in statin-treated in comparison to the un-treated cultures.
Finally, we evaluated to what extent statin-induced inhibition of DCs' maturation and changes in DCs' cytokine secretion inhibit Th17 cell differentiation. We identified that both simvastatin-treated DCs cell-to cell contact and supernatant of simvastatin-treated DCs inhibited the Th17 cells differentiation.
Our results revealed that simvastatin suppresses DCs' antigen presenting capacity and changes DCs' cytokine production, which collectively inhibit Th17-cell differentiation.
5 -Case-report: Cladribine and alemtuzumab haematological treatment of chronic lymphatic leukemia and stabilizing effect on secondary-progressive multiple sclerosis Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS). Several MS forms are known: relapsing-remitting form (RRMS) at the beginning of MS with typical autoimmune inflammatory activity usually proceeds after 6-10 years into secondary-progressive form (SPMS) with prevailing neurodegeneration and variable residual inflammation. All current MS drugs are directed against inflammation with no significant neuroregenerative potential, therefore their effect is greatest at RRMS and is decreasing in SPMS. Several new drugs are now studied in MS including cytostatic agent cladribine and monoclonal antibody against CD52 cell surface marker of T and B cellsalemtuzumab, already used in haematooncological (HO) practice.
Objectives: To report stabilizing effect of cladribine and alemtuzumab HO treatment on SPMS.
Case-report: 51-year-old Caucasian man with MS history since 1974, first symptom optic neuritis of the left eye and secondary progressive course since 1985. Treatment history involved corticosteroids during MS relapses, combined immunosuppression with low-dose oral corticosteroid and azathioprine 50 mg daily (1996) (1997) (1998) , and later methotrexate 7.5 mg weekly (1999) (2000) (2001) (2002) (2003) (2004) (2005) Inflammation is a central process in the pathophysiology of Multiple Sclerosis (MS) and all available treatments for MS patients target the inflammatory process. Accordingly, in a gene expression study performed in the three main clinical forms of MS and in experimental autoimmune encephalomyelitis (EAE), an animal model for MS, we found an overall modulation of the expression of inflammatory genes and, in particular, we found an up regulation of CCR2, a chemokine receptor already described as associated with pathological inflammation in MS and EAE.
We therefore tested CCR2 functional activity in vivo in 150 SJL mice affected by relapsing-remitting (RR)-EAE, modulating its activity using the engineered monomeric MCP-1 variant named [8A]-MCP-11-2. We administered [8A]-MCP-1 both preventively and therapeutically. We compared the efficacy of different doses of [8A]-MCP-1 with methylprednisolone (reference compound) and we compared subcutaneous (sc) and intraperitoneal (ip) administration. Preventive treatment significantly delayed disease onset in a dose dependent way, but displayed no effect on other clinical parameters. We did not observe significant clinical effects when treatment was started after disease onset. Neuropathological analysis, on the other hand, showed efficacy of the therapeutic treatment with [8A]-MCP-1. We found statistically significant and dose-dependent decrease of demyelination, axonal loss and number of infiltrating cells in the mice spinal cord (CD3+ T cells and macrophages) in [8A]-MCP-1-treated mice as compared to controls. Immunological analysis revealed, as expected, that [8A]-MCP-1 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNγ-releasing effectors T cells. However, we observed an inhibition of IFNγ release upon antigen stimulation.
Taken together, these results suggest that the therapeutic mechanism of [8A]-MCP-1 may rely on interference with immune cell recruitment: it includes a mild clinical efficacy in a preventive setting and a neural protection from tissue damage evaluated when administration starts after the first clinical attack.
were comprised of an N-terminal cytokine domain (rat GM-CSF, M-CSF, or IL-6) and a C-terminal antigenic domain (NAg; the encephalitogenic determinant of myelin basic protein). The biological activities of the cytokine-NAg fusion proteins were examined through in vitro assays utilizing irradiated splenic APCs and myelin basic protein (MBP)-specific T cell clones. The GMCSF-NAg fusion protein was approximately 1000 fold more potent than NAg alone in stimulating antigenic proliferation of MBP-specific T cell clones, and the MCSF-NAg fusion protein was 10-100 fold more potent than NAg. The IL6-NAg fusion protein was only slightly more potent than NAg. The ability of the fusions proteins to potentiate antigenic proliferation was dependent on the covalent linkage between the cytokine and NAg domains, because the addition of GM-CSF or M-CSF plus NAg, as separate molecules, did not potentiate MBP-specific T cell proliferation. Additional data reinforced the concept that cytokine-NAg fusion proteins enhanced antigenic proliferation by targeting antigen to APCs. For instance, NAg targeting by GMCSF-NAg or MCSF-NAg was specifically inhibited by pre-treatment of the assays with the respective cytokine, GMCSF or MCSF, but not vice versa. In addition, cytokine-NAg fusion proteins specifically potentiated antigenic proliferation in the presence of particular APC subsets. GMCSF-NAg and MCSF-NAg were 100-1000 fold more potent than NAg alone in the presence of DC or macrophages, but these fusion proteins did not stimulate potentiated antigen recognition in the presence of B cells or T cell APCs.
In conclusion, the GMCSF and MCSF cytokine domains targeted the NAg domain to DCs and macrophages, but not to other APC subsets. These data indicate that cytokine-antigen fusion proteins may provide an important tool to study APC subsets that promote inflammatory or tolerogenic responses in experimental autoimmune encephalomyelitis.
Natalizumab is a recombinant humanized antibody directed against the integrin alpha-4-beta-1. This novel agent prevents the migration of immune cells into the brain and reduces the number of relapses by 68% in relapsing-remitting multiple sclerosis (RRMS). A significant increase of tumor necrosis factor (TNF) expression and production by peripheral blood mononuclear cells (PBMCs) as well as a trend towards worsened fatigue has previously been reported in 19 patients with RRMS treated with natalizumab for a period of 6 months. Here we report the relative expression of cytokines in both cerebrospinal fluid (CSF) and PBMCs in 22 patients with RRMS before and after 12 months of natalizumab treatment, and its long-term effect on the fatigue severity scale (FSS), expanded disability status scale (EDSS), multiple sclerosis impact scale (MSIS-29) and symbol digit modalities test (SDMT).
The relative mRNA expression of cytokines was measured by realtime RT-PCR methodology. Administration of natalizumab (300 mg iv infusion every 4 weeks) dramatically reduced the CSF mononuclear cell count (p b 0.0001). Expression of TNF and interferon-gamma (IFN-g) mRNAs was significantly increased in PBMCs (p b 0.001; p b 0.001) but interleukin (IL)-4, IL-10, IL-13, IL-17, IL-23 and transforming growth factor-beta remained unchanged. In contrast, in the CSF cells, expression levels of IFN-g and IL-23 were decreased (p b 0.01; p b 0.02) and IL-10 was increased (p b 0.01). Although EDSS improved significantly (p b 0.01), the FSS, MSIS-29 and SDMT remained unchanged compared to baseline scores in the entire cohort, however, these scores improved in patients with severe disease (EDSS N 4.5; n = 13).
Attenuation of immune cell trafficking to the brain and reduction of proinflammatory cytokines in the central nervous system compart-ment correlate to improvement in neurological functions measured with EDSS. Relative differences in other functional measures, such as fatigue, were only detected in the group with severe neurological disability. We speculate that increased production of proinflammatory cytokines in the periphery may impede the improvement of fatigue in natalizumab treated patients with RRMS. Azathioprine (Aza), 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) are thiopurine drugs widely used as immunosuppressants/ anti-inflammatory agents in organ transplantation and chemotherapy. As shown by several clinical trials, Aza is effective in modifying the course of MS and administered at lymphocyte-suppressing doses is well tolerated and effective in reducing MS new brain inflammatory lesions. In our study we investigated the action of 6-MP on human dendritic cells (DCs). We described for the first time that 6-MP impairs in vitro differentiation of DCs, has an inhibitory effect during DC activation processes, may inhibit fully activation process on DCs inducing a functionally less immunogenic phenotype and that 6-MP reduces DC CCR7 expression of the 50% and more, comparing to controls. All these findings add a novel action mechanism in Aza immune modulation.
DCs differentiated from CD14+ were cultured in the presence and absence of two doses of 6-MP (10-6 and 10-5M). Immature DCs were activated for 24 h with 1 μ/ml of LPS. Maturation state was evaluated as surface marker expression HLA-DR, CD14, CD80, CD86 and CD83 by flow cytometry. Dendritic cell macropinocytosis capability was examined by Dextran-FITC assay. DC function was evaluated as allostimulatory ability, antigen specific stimulation and CK production. CCR7 expression was evaluated by real-time PCR.
Our results show that 6-MP inhibits DC differentiation from CD14 precursors and reduce CD83 expression after LPS activation. We found that down regulation of CD83 results in less potent induction of allogeneic T cell proliferation and in reduced antigen specific proliferation, thus to treat with 6MP during DC LPS activation lead to a decreased stimulation of naïve and memory T cells. Moreover, our finding that DCs treated with 6MP retain endocytic activity after LPS stimulation indicates that reduced expression of CD83 not only determines a less efficient costimulation but goes together with a less mature, and therefore more tolerogenic, DC phenotype. When mature DCs were incubated with 6-MP the CCR7 expression was reduced, indicating that 6-MP down-modulate this molecule from the cell surface. Our data suggest that immunomodulatory effects of Aza involve both side of immune synapse: DCs and T cells, revealing a specific way of action. In the future it will be of interest to investigate ex vivo isolated DCs from treated patients together with the Treg subpopulation.
Celecoxib is a cyclooxygenase-2 (COX-2)-specific non-steroidal anti-inflammatory drug (NSAID) indicated for treatment of arthritis and pain. Recent studies have shown benefits in treatment of colorectal cancer, though associated risk of cardiovascular events may impede implementation. We have recently shown that a trifluoromethyl analogue of celecoxib (abbreviated as TFM-celecoxib), that lacks COX-2-inhibitory activity, induces cellular retention of IL-12 through a post-translational mechanism involving perturbation of Ca2+ in the endoplasmic reticulum and chaperones of the ER (Mol. Pharmacol. 69: [1579] [1580] [1581] [1582] [1583] [1584] [1585] [1586] [1587] 2006) . We have also demonstrated that celecoxib inhibits EAE by a COX-2-independent mechanism (Brain 129: 1984 (Brain 129: -1992 (Brain 129: , 2006 ) that is mediated by downregulation of cytokines, adhesion factors and chemokines. In the present study, we have analysed the mode of action of TMF-celecoxib in more detail and have, as well, assessed its effect in the EAE model.
Using recombinant cell lines established in our lab, we found that TMF-celecoxib blocks secretion of IL-12 (p40/p35 heterodimers), p80 (p40/p40 homodimers) and IL-23 (p40/p19 heterodimers) with same potency as wild-type celecoxib. A comprehensive analysis of 25 ERresident chaperones/foldases and associated co-factors showed that TMF-celecoxib induces significant transcriptional upregulation of GRP78, DNAJB9, ERO1LB and HERPUD1. HERPUD1 is part of a molecular mass protein complex involved in retrotranslocation of misfolded proteins prior to degradation. Further experiments using siRNA "knockdown" as well as immunoprecipitation of selected ER chaperones reveal a picture of multiple chaperone interactions involved in ER retention of these cytokines by TMF-celecoxib. We have also for the first time assessed the efficacy of TMF-celecoxib in EAE. TMF-celecoxib appeared to decrease the clinical score, and its effect was indistinguishable from celecoxib. Further analysis is being performed to assess its effect on IL-12, IL-23, IFN-gamma and IL-17 secretion in the EAE model. TMF-celecoxib may constitute the first drug for treatment of neuroinflammatory disorders, such as multiple sclerosis, that works through a mechanism disrupting cytokine folding in the ER. Its total lack of COX-2 inhibitory activity is beneficial, since inhibition of prostaglandin synthesis has been associated with deleterious alterations in endothelial cells, potentially contributing to development of cardiac events. Multiple sclerosis is the most common demyelinating disease of the central nervous system (CNS). Though the etiologic agent of MS is still unknown, epidemiologic data suggest that an environmental agent, such as a virus, may initiate the disease. Theiler's murine encephalomyelitis virus (TMEV) is a murine picornavirus that induces a demyelinating disease in susceptible strains of mice when injected intracranially. Theiler's virusinduced demyelination (TVID) lesions are similar to those seen in MS patients. Thus, TVID is a valuable animal model for the study of MS. Currently, interferon-beta (IFNB) is a major treatment option for MS patients. Unfortunately, IFNB treatment is not successful in all MS patients and often produces undesirable side effects. Thus, the search for alternative therapies is an area of intense investigation within the field of MS. Interferon tau (IFNT) is a Type I IFN produced in ruminants during pregnancy that is less cytotoxic than IFNB, but equally effective for immunomodulation. Cross-species reactivity of IFNT is key to its use as a treatment for human immune-mediated diseases, such as MS. The therapeutic potential of oral IFNT in the treatment of MS is now being assessed in Stage III clinical trials. The objectives of this study were to test the ability of IFNT to ameliorate TVID and to elucidate mechanisms of immunomodulation.
TMEV-infected mice were injected ip with encapsulated IFNT once every 14 days for eight weeks. Treatment began at 11 weeks post-infection. We hypothesized that IFNT would stabilize the clinical score, decrease inflammation and demyelination, and decrease Type 1 cytokines. Initial results revealed that IFNTtreated mice had improved clinical scores starting at 2 weeks posttreatment. Likewise, a test of coordination revealed that mice treated with IFNT regained function while non-treated mice exhibited increased disability. Histopathology revealed a decrease in inflammation and demyelination in treated mice. Lastly, splenicderived T cells stimulated ex vivo with TMEV, increased expression of both Type 1 and Type 2 cytokines [IFNG, TNFA, IL2, IL12, and IL10].
These results indicate the possibility of remyelination within IFNTtreated mice. Studies using electron microscopy are underway to test this hypothesis as are flow cytometric studies to elucidate phenotype of cells and expression of chemokine/cytokine receptors in IFNTtreated versus non-treated mice.
POSTER SESSION: Blood-brain barrier and chemokines 1 -Blood-brain barrier breakdown and repair following gliotoxic drug injection in the brainstem of streptozotocin-diabetic rats Bondan Eduardo⁎ 1 , Lallo Maria Anete 1 1 University Paulista, University Cruzeiro do Sul, São Paulo, Brazil Ethidium bromide (EB) causes local oligodendroglial and astrocytic disappearance, with primary demyelination, glia limitans disruption, supposed blood-brain barrier (BBB) breakdown and Schwann cell invasion. Hyperglycemia observed in Diabetes mellitus is known to cause some changes in the behaviour of glial cells. The aim of this study was to investigate the BBB integrity after the injection of EB into the brainstem of diabetic and non-diabetic rats.
Adult male Wistar rats were used and some received a single intravenous injection of streptozotocin (50 mg/kg), being submitted 10 days later to a single injection of 10 μL of 0.1% EB (group I) or 0.9% saline solution (II) into the cisterna pontis. Ten microlitres of 0.1% EB or 0.9% saline solution was also injected in non-diabetic rats (groups III and IV, respectively). The rats were anaesthetized, perfused through the heart from 24 h to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy studies and immunohistochemical staining using glial fibrillary acidic protein (GFAP) as an astrocyte marker. From each group some animals received colloidal carbon ink by intravenous route, 20 min before being submitted to euthanasia. In rats from groups II and IV, there was no sign of astrocyte loss and no leakage of ink from blood vessels in the injection site. In groups I and III, astrocyte disappearance began at 48 h and some areas were still devoid of astrocytic processes 31 days after. Leakage of carbon particles was seen from 48 h to 15 days in group I and from 48 h to 7 days in group III in the EBinduced lesions. Tight junctions did not show any detectable ultrastructural change due to the lack of perivascular astrocytes in groups injected with EB. Diabetic rats from group I presented delayed macrophagic activity and lesser remyelination in comparison to rats from group III. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, firstly appearing at 11 days in nondiabetic rats and by 15 days in diabetic rats.
Results indicate that short-term streptozotocin-induced diabetes hindered BBB reconstruction and both oligodendrocyte and Schwann cell remyelination in comparison to non-diabetic rats after EB injection. Interleukin-15 (IL15) is a proinflammatory cytokine with elevated concentrations in autoimmune diseases involving the periphery (e.g., rheumatoid arthritis) and CNS (e.g., multiple sclerosis). How circulating IL15 affects neuroinflammation has not been fully characterized. In this report, we will show the regulatory changes of the IL15 system in the endothelial cells composing the blood-brain barrier (BBB). A mouse model of lipopolysaccharide (LPS) treatment will be used to illustrate the mechanisms of BBB regulation.
There are four aspects of the study: pharmacokinetic assays of BBB permeability to blood-borne IL15; transcriptional regulation of IL15 receptor subunits at the BBB level; post-translational regulation of endothelial IL15 and its receptors which differed greatly from their respective mRNA transcripts; and evidence of CNS inflammation induced by IL15 and LPS. IL15 remained intact for at least 10 min after intravenous injection and reached CNS parenchyma with regional differences between brain and spinal cord. Both in-vivo and in-situ brain perfusion of IL15 showed that its permeation of the BBB was nonsaturable. LPS induced a significant increase of IL15 uptake by the brain and spinal cord, partly related to a higher general permeability of the BBB. There was a striking activation of the IL15 system in both cerebral endothelia and CNS parenchymal cells that mediated neuroinflammation.
The results suggest that the BBB is an interface for blood-borne IL15 to interact with the CNS in the basal state and during inflammation.
3 -Blood-brain barrier characteristics in multiple sclerosis Kooij Gijs 1 , van Horssen Jack 1 , van der Pol Susanne 1 , Dijkstra Christine 1 , de Vries Elga⁎ 1 1
The blood-brain barrier (BBB) is a selective barrier between the central nervous system (CNS) and the systemic circulation and is essential for maintenance and regulation of the neuroparenchymal environment and optimal neuronal functioning. It acts as a physical barrier due to the presence of well-developed tight junctions between brain endothelial cells that impede the entrance of circulating molecules and immune cells into the CNS. Specific transporter systems on the luminal and abluminal membranes regulate the transcellular traffic of small hydrophilic molecules and exclude potentially harmful compounds through multi-drug resistance efflux pumps.
Previously, we demonstrated that loss of BBB integrity is apparent in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis. Magnetic resonance imaging using the newly developed contrast agent, ultrasmall particles of iron oxide (USPIO) specifically imaged inflammatory events and BBB leakage in animal models and MS patients. Post-mortem analysis of well-characterized MS patient material also revealed alterations at the vascular level and the surrounding perivascular space. However, it remains unknown whether the molecular function of the BBB is affected in the course of MS and whether this contributes to the disease.
Immunohistochemical analysis in active and inactive lesions of post-mortem MS patient material reveals that the expression of multiple multi-drug resistance (MDR) proteins are decreased predominantly in chronic active lesions at the level of the brain endothelium. In contrast, no alterations in the expression of nutrient transporters could be detected. In our in vitro model for the bloodbrain barrier we demonstrated that both gene and protein expression of the glucose transporter (GLUT-1) and the MDR proteins were decreased by inflammatory agents. Interestingly, the efflux capacity of brain endothelial cells to pump out ligands of these multi-drug resistance pumps was diminished under inflammatory conditions.
Our data suggest that alteration of BBB function at both the physical and molecular level is a critical determinant in MS lesion formation. As such BBB dysfunction contributes to tissue damage due to accumulation of unwanted compounds and cells in the brain parenchyma.
4 -Interleukin-25 improves tumor necrosis factor-alpha-induced disruption of blood brain barrier properties in mouse brain capillary endothelial cells Sonobe Yoshifumi⁎ 1 , Takeuchi Hideyuki 1 , Kataoka Kunio 1 , Kawanokuchi Jun 1 , Mizuno Tetsuya 1 , Suzumura Akio 1 1 Nagoya University, Nagoya, Japan
Interleukin (IL)-25 is a member of IL-17 family cytokines including IL-17A and IL-17F. IL-25 mRNA is reportedly expressed by the activated T cells, lung macrophages, and lung endothelial cells. IL-25 promotes T helper type 2 (Th2) immune responses via producing IL-4 and IL-13. In addition, it is reported that IL-25-deficient mice are highly susceptible to experimental autoimmune encephalomyelitis (EAE) compared with wild type mice, suggesting that IL-25 regulates Th17 function via inducing Th2 cytokines. Interestingly, IL-25 mRNA is also reportedly detected in the brain tissue. However, the cellular source of IL-25 and its function in the brain remains unclear. Thus, we tested which cells express IL-25 and how IL-25 functions in the brain.
In this study, we used C57BL/6J mice. To assess the expression of IL-25 mRNA, we extracted total mRNA from the whole brain tissue, mixed glial cell culture, primary microglia, primary astrocytes, primary fibroblasts, primary neurons, and mouse primary endothelial cells (BCECs), and performed RT-PCR. To explore the protein expression, we performed immunohistochemistry and Western blotting. To evaluate the permeability of blood brain barrier (BBB), we used MBEC4, a BCEC line and an established BBB model, and performed permeability assays using Evan's blue albumin and counting transmigrated CD4+ T cells.
RT-PCR and immunohistochemical analysis showed that brain capillary endothelial cells (BCECs) produced IL-25, while microglia, astrocytes, neurons, fibroblasts, and oligodendrocytes did not. Additionally, IL-25 increased TJ proteins, occludin and claudin-5 and recovered their expression levels downregulated by tumor necrosis factor (TNF)-alpha in BCECs Permeability assay and CD4+ T cell transmigration assay confirmed that IL-25 partially suppresses TNFalpha-increased permeability and T cell migration across the BCEC.
Collectively, these results suggest that IL-25 produced by the brain endothelial cells protects against TNF-alpha-associated collapse of the BBB and controls inflammatory T cell migration across the BBB in MS or EAE. The destruction of blood-brain barrier (BBB) is an integral feature of neuroinflammatory process in pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study, we utilize Triptolide (TRD, a purified effective ingredient of Tripterygium wilfordii of Chinese herb) to EAE rats to investigate the kinetic changes of BBB, as well as the expression of intercellular adhesion molecule-1 (ICAM-1).
EAE was induced in Wistar rats and treated with TRD (100 μg/kg). Clinical score was evaluated among EAE, TRD-treated, and complete Freund's adjuvant (FCA) groups according to five forked method. Albumin in serum (S) and cerebrospinal fluid (CSF) samples of all rats were examined on the days 6, 8, 10, 12, 14, 16 post-inoculation (p.i.). The permeability of BBB was evaluated by calculating CSF to S albumin quotient (QA). At the same time, the brains and spinal cords were removed for immunohistochemistry staining.
The EAE model in our study presented one-way course of disease. The disease did not appear in FCA group, but the clinical symptoms occurred on day 12 p.i. in both EAE group and TRD-treat group. On day 14 p.i. the clinical symptoms reached the peak to display hindlimb paralysis, irritability, the rapid loss of body weight in EAE group. The mean clinical score in EAE group was higher than that in TRD-treated group (2.19 ± 1.68 vs 0.14 ± 0.08, p b 0.01). On day 8 p.i., QA reached the peak in EAE group, which was higher than that in FCA group (0.30 ± 0.05 vs 0.13 ± 0.05, p b 0.01). Then QA gradually decreased. In TRDtreated group, QA increased slightly in the initial stage, gradually increased and reached the maximum (0.19 ± 0.02) on day 14 p.i., which was later than that in EAE group. Our result suggests that the difference of QA was statistically significant between EAE group and TRD-treated group(0.17 ±0.09 vs 0.08 ± 0.07, F = 28.340, p b 0.01). Image analysis shows that the mean staining photodensity of ICAM-1 in TRDtreated group was lower than that in EAE group (2345.04 ± 1053.24 vs 3203.77 ± 1858.24, F = 17.11, p b 0.01) on days 12-16 p.i.
In early stage of EAE, the injury of BBB is prior to appearance of clinical symptom. TRD was not only down-regulated ICAM-1, but also repaired the BBB and ameliorated EAE.
Semaphorins are a family of soluble or membrane-associated proteins that are characterized by a conserved Sema domain. Although semaphorins were originally identified as axonal guidance molecules, it is emerging that several semaphorins play significant roles in immune response. For example, Sema4D is expressed in T cells and functions as a ligand to enhance the humoral and cellular immune responses via CD72. Sema4D-deficient mice are resistant to experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Another semaphorin, Sema4A provides a costimulatory signals to T cells and has a non-redundant role in the regulation of Th1/Th2 responses. Treatment of mice with anti-Sema4A antibody inhibited the development of EAE. Thus semaphorins have significant roles in the peripheral immune systems in pathological conditions such as EAE. However, it is unclear whether semaphorins are related with immune responses in the other systems such as the blood-brain barrier (BBB) or the central nervous systems (CNS). Here we investigate the expression of a semaphorin in the CNS of EAE and its relevance to the BBB. EAE was induced in C57BL/6 mice by immunization with MOG35-55 peptide and spinal cords of the mice were subjected to immunohistochemical analysis. The semaphorin is expressed in the infiltrating cells around the meninges in the spinal cord of EAE. Some of the cells expressing semaphorin were lymphocytes and the others were presumably macrophages. The plexin, one of the candidate receptors for the semaphorin, was expressed in lectin-positive cells which were supposed to be microglia and endothelial cells (ECs). From this expression pattern, we considered the possibility that the semaphorin is associated with immune cell trafficking through BBB. In the analysis in vitro, the semaphorin enhanced the expression of adhesion molecules on ECs.
These results indicate that semaphorin is expressed in infiltrating cells in the spinal cord of EAE and may be associated with trafficking of immune cells into the CNS.
7 -Interferon-beta regulates CD73 and adenosine expression at the blood-brain barrier Interferon (IFN)-beta treatment reduces the relapse rate in multiple sclerosis (MS) but its mechanism of action remains incompletely understood. Our aim was to clarify further the beneficial effect of IFNbeta in the treatment of MS. Ecto-5′-nucleotidase (CD73) is an ectoenzyme producing adenosine from adenosine monophosphate (AMP). AMP is abundantly present at sites of inflammation and, more importantly, adenosine, the product of ecto-5′-nucleotidase activity, possesses both anti-inflammatory and neuroprotective activity.
In the current study we elected to assess the influence of IFN-beta treatment on i) CD73 expression on the surface of primary cultures of human blood-brain barrier endothelial cells (BBB-ECs) and human astrocytes using immunofluorescence staining and flow cytometry, ii) transmigration of CD4+ T lymphocytes using an in vitro model of BBB, iii) ecto-5′-nucleotidase activity in the serum of MS patients using a radiochemical assay and finally iv) CD73 expression in postmortem MS brain samples and skin biopsies of MS patients using immunohistochemical staining. Our results show that IFN-beta increases the expression of ecto-5′-nucleotidase both on BBB-ECs and on astrocytes. As a consequence, transmigration of lymphocytes through BBB-ECs is significantly reduced. CD73 is strongly expressed in microvasculature in samples of post-mortem MS brain and moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto-5′-nucleotidase activity and skin microvascular CD73 expression after IFN-beta treatment.
Upregulation of ecto-5′-nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFNbeta on MS via enhancing the endothelial barrier function. ADAM-17 (a disintegrin and metalloprotease) is the principal convertase of tumour necrosis factor (TNF), which is a major immunomodulatory cytokine. In addition, ADAM-17 also cleaves a number of other membrane-bound proteins, including the chemokine and adhesion molecule, fractalkine (CX3CL1), which has been implicated in multiple sclerosis (MS) pathogenesis. ADAM-17 expression in endothelial cells in MS white matter and in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) has been shown by our laboratory to be up-regulated. Here we studied the in vitro expression of ADAM-17, its inhibitor (TIMP-3), and fractalkine in a human brain endothelial cell line, hCMEC/D3, at the mRNA and protein level under pro-inflammatory conditions. ADAM-17, TIMP-3 and fractalkine were up-regulated at the mRNA level under TNF treatment, however, only fractalkine was significantly so. Immunocytochemical analysis revealed that this increase in fractalkine expression also occurred at the protein level. To determine the shedding activities of ADAM-17 we then performed an ELISA assay. Cells treated with TNF at 100 ng/ml released 775.7 ng fractalkine/mg protein, which is 10 times greater than the amount of fractalkine bound to the cell membrane under the same conditions. We then sought to determine the protein expression of ADAM-17 following TNF treatment. A significant increase in ADAM-17 protein expression could not be determined using either immunocytochemistry, or western blotting and densitometry. We thus hypothesize that the increased shedding of fractalkine is due to increased enzyme activity of ADAM-17 and not increased protein expression of the protease. Activity assays for ADAM-17 under pro-inflammatory conditions using an InnozymeTM activity kit are ongoing to test this hypothesis. We are also investigating the immunohistochemical co-localisation and expression levels of ADAM-17 and fractalkine in MS and control brain tissue. The extent of associated inflammatory, demyelinated and neurodegenerative processes within the tissue is assessed in parallel.
This study highlights shedding of fractalkine in inflammatory conditions is a critical event which requires further investigation. Shed fractalkine from endothelial cells could mediate leukocyte recruitment and extravasation via modulating the expression of certain adhesion molecules or it could act to prevent fractalkine receptor mediated migration of leukocytes by masking the receptor.
9 -S100B as a novel and accessible determinant for the development and severity of monocyte-driven encephalitis in aids MacLean Andrew⁎ 1 , Redmann Rachel 1 , Ivey Nathan 1 , Didier Peter 1 , Lackner Andrew 1 1 Tulane National Primate Research Center, New Orleans, United States Despite a reduced incidence of HIV dementia in patients on highly active antiretroviral therapy (HAART), the improved survival has resulted in increased cumulative prevalence of nervous system complications of AIDS. Fundamental gaps remain in knowledge relating to mechanisms and pathways involved in the development of HIV-associated neurologic disease. Lesions associated with encephalitis include marked astrocytic and microglial activation with frequent multinucleated giant cells, high viral load and lesions consisting of multiple disseminated foci of inflammatory cells throughout the brain. The pathogenesis of the lesions is unknown, but it is thought to be associated with alterations in the BBB. Heretofore, it has not been possible to determine if an individual has encephalitis before post mortem examination.
In order to determine if it is possible to predict encephalitis in macaques infected with SIV we have correlated the levels of S100β protein in serum/plasma not just with the presence of encephalitis, but with the degree of encephalitis of a particular animal as determined by blinded histopathological evaluation. These studies were then taken further to quantify tight junction proteins. zo-1 was found to be diminished most in those animals with the highest degree of encephalitis/levels of S100β protein. Although disruption of the BBB is present in terminal AIDS, it is not as yet known when it begins. BBB disruption has been demonstrated in vivo using S100β protein in serum. Using this technique, it would be possible to determine when a particular animal was developing encephalitis, and thus be in a position to schedule a necropsy during these earliest stages.
By determining levels of the glial protein S100β in serum, we show that it is possible not only to determine correlates post mortem, but also to predict which individuals will have encephalitis before death. This could have profound impact on elucidating the pathogenesis of neuroAIDS using the rhesus macaque model allowing one to euthanize an animal during the earliest stages of neurological disease or to predict HIVE in patients infected with HIV and thus, tailor treatment administered.
10 -Sonic hedgehog is secreted by human astrocytes and promotes optimal blood-brain barrier functioning The blood-brain barrier (BBB), composed of tightly bound endothelial cells (ECs), regulates the entry of blood-borne molecules and immune cells into the CNS. Perivascular astrocytes regulate BBB permeability by secreting essential factors, the identity of which remains unclear. Recent studies indicate that the Hedgehog (Hh) signaling pathway in adult tissues plays an important role in vascular proliferation, differentiation and tissue repair.
Our study shows that human astrocytes express and secrete Sonic Hh and conversely, that human BBB-ECs bear the Hh receptor Patched-1 (Ptch-1), the signal transducer Smoothened (Smo) as well as transcription factors of the Gli family. Furthermore, we show that activation of the Hh pathway in BBB-ECs restricts the passage of soluble tracers (FITC-BSA and C14-sucrose), decreases the surface expression of intercellular cell adhesion molecule 1 (ICAM-1), and decreases BBB-ECs secretion of pro-inflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1). In our modified Boyden chamber assay, the migration of CD4+ lymphocytes across human BBB-EC monolayers is also reduced after BBB-EC treatment of Sonic Hh and Hh pathway agonists. In vitro treatment of BBB-derived ECs with inflammatory cytokines, TNFalpha and IFN-gamma, down-regulates the mRNA expression of Ptch-1, Smo and Gli-1, deregulating the Hh signaling pathway and preventing the barrier stabilizing properties of Sonic Hh.
Our data provide strong evidence for an anti-inflammatory and BBB-promoting effect of astrocyte-secreted Sonic Hh and suggest that a pro-inflammatory environment disrupts the BBB by impacting, at least in part, on Hh signaling in brain ECs.
The blood-brain barrier (BBB) regulates entry of both cells and molecules to the brain. Entry of cells and macromolecules to the brain parenchyma (BBB breakdown) correlates with clinical symptoms in inflammatory pathological conditions such as multiple sclerosis.
The Renin-Angiotensin system (RAS) is a complex hormone system with multiple functions primarily regulating blood vessel tonus and permeability. The active compound, angiotensin II acts on two receptors: AT1 and AT2.
The objective of this study was to examine the role of RAS at the BBB. Two distinct models of brain inflammation were used. The entorhinal cortex lesion model allows the study of an inflammatory response to traumatic brain injury at the site of stab wound with direct bleeding into the parenchyma, simultaneous with a milder inflammatory response at the distal dentate gyrus, where synapses degenerate, leading to glial activation. In the second model, MBP/CCL2 transgenic mice overexpressing the chemokine CCL2 in the brain show perivascular accumulation of leukocytes without other symptoms. Treatment with pertussis toxin (PTx) results in cellular infiltration to the parenchyma (Toft-Hansen et al. 2006 J Immunol.;177:7242). The presence and location of RAS components were investigated by realtime RT-PCR and immunohistology in these two animal models.
The AT1 receptor was upregulated on astrocytes in the dentate gyrus of the ipsilateral hippocampus 3 days post entorhinal cortex lesion. Horseradish peroxidase (HRP) leakage across the BBB was not observed at this site. In PTx-treated MBP/CCL2 mice BBB breakdown and loss of glia limitans integrity were shown by HRP leakage and loss of betadystroglycan (b-DG) staining. In this model AT1 staining was not observed whereas the other receptor subtype, AT2 was detected. AT2 staining was closely associated to GFAP and b-DG staining consistent with expression by astrocytes at the glia limitans. AT2 was also seen at the glia limitans at the site of stab wound in the entorhinal cortex lesion model. These findings suggest differential roles of the two receptors in brain inflammation and raise interesting new questions as to their functions.
Basivireddy Jayasree⁎ 3 , Buttmann Mathias 2 , Rieckmann Peter 3 2 Dept of Neurology, University of Wuerzburg, Wuerzburg, Germany, Germany 3 Dept of Neurology, University of British Columbia, Vancouver, Canada, Dept of Neurology, University of Wuerzburg, Wuerzburg, Germany, Canada Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin super family. It is a major constituent of endothelial tight junctions where it is thought to regulate tight junction assembly and immune cell transmigration. JAM-A expressed in epithelial tight junctions has been shown to regulate paracellular permeability for solutes. In thrombocytes, the monoclonal antibody M.Ab.F11, a potent JAM-A agonist, induced platelet secretion and aggregation. Here, we used this antibody to investigate JAM-A-mediated changes in cell morphology and paracellular permeability as well as underlying intracellular signaling mechanisms in an in vitro-model of the human blood-brain barrier (BBB).
Immortalized as well as low-passage primary human cerebral endothelial cells were cultured in vitro and stimulated with M.Ab.F11R antibody. Cell morphology was determined by phase contrast microscopy and immunocytochemistry. Paracellular permeability for solutes was assessed using fluorescent-labeled dextran in a transwell system. The role of MAP kinases was elucidated by specific chemical inhibitors. The phosphorylation status of MAP kinases was determined by Western blotting. Activation of JAM-A induced changes in cell morphology from a typical confluent cobble-stone pattern to a widening of intracellular spaces and development of lammellipodial and filopodial cell extensions. Actin staining showed a reduction of stress fibres. Permeability assays revealed increased permeability to small molecular weight dextrans of 3000 and 10,000 but not of 70,000 kDa. JAM-A activation induced delayed and sustained ERK phosphorylation while it did not activate JNK or p38. These JAM-A effects were largely neutralized by parallel treatment with chemical inhibitors of ERK phosphorylation but not of other MAP kinases.
JAM-A-induced alteration of cell morphology and increase of paracellular permeability for solutes could be abolished by ERK inhibitors. Therefore, ERK signalling but not disruption of interendothelial JAM-A interactions by antibody binding mediated the observed effects in our in vitro model of the human BBB. JAM-A or intracellular signalling pathways activated by JAM-A may be a novel therapeutic target in neurological diseases where a breakdown of the BBB and edema formation play a pathogenic role, such as in stroke or multiple sclerosis. Our study also shows the possibility of circulating antibodies that cause breakdown of blood barrier integrity.
13 -Ninjurin-1 is an adhesion molecule of the blood-brain barrier involved in monocyte recruitment to the CNS In Multiple Sclerosis (MS), inflammatory demyelinating lesions are associated with a compromised blood-brain barrier (BBB) and with perivascular infiltration of immune cells into the central nervous system (CNS). The movement of leukocytes from the blood to the CNS is orchestrated by many factors including cell adhesion molecules that enable immune cells to adhere and cross the BBB. Nerve injury-induced protein (Ninjurin)-1 was described as an adhesion molecule that promotes nerve regeneration, in a homotypic Ninjurin-1-Ninjurin-1 fashion. We recently identified Ninjurin-1 in a proteomic screen for BBB-associated proteins using human BBB-derived endothelial cells (ECs). Our goal is to study the role of Ninjurin-1 in the trans-BBB migration of immune cells.
Expression, regulation and localization of Ninjurin-1 on human BBB-ECs was determined by Western blot (WB), flow cytometry and immunocyto-and immunohisto-fluorescence. The functional role of Ninjurin-1 has been assessed using an in vitro model of the human BBB. We confirmed expression of Ninjurin-1 in vitro on BBB-ECs by WB and flow cytometry, in situ by confocal microscopy and we show regulation of Ninjurin-1 upon treatment with pro-inflammatory cytokines TNF, IFN-gamma and IL-17. Furthermore, Ninjurin-1 immunoreactivity was detected in vitro on astrocytes and in situ in MS lesion and in CNS material obtained from its animal model, experimental autoimmune encephalomyelitis (EAE). We demonstrate that Ninjurin-1 is present on peripheral blood immune cells, with a preferential expression on CD14 monocytes, as compared to CD4, CD8 or CD19 lymphocytes. Finally, using a modified Boyden chamber assay, we show that migration of ex vivo CD14 monocytes across the human endothelium is significantly reduced following treatment of BBB-ECs with a competing Ninjurin-1 peptide (PARWGWRHGPIN) and that Ninjurin-1 blockade increases BBB permeability.
Our findings suggest that Ninjurin-1 is a novel adhesion molecule of the CNS endothelium that acts as a regulator of monocyte migration into the brain.
14 -Doxycycline treatment decreases morbidity and mortality of murine NCC: Evidence for reduction of apoptosis, oxidative stress and MMP activity Alvarez Jorge⁎ 1 , Krishnamurthy Janani 1 , Teale Judy 1 1
Murine neurocysticercosis (NCC) is characterized by differential disruption of the barriers protecting the central nervous system (CNS) microenvironment. Among the host factors involved in this response, Matrix Metalloproteinases (MMPs) have been recently described as important players. Doxycycline is a commonly prescribed antimicrobial drug that also acts as an anti-inflammatory agent with broad MMP inhibitory properties. In this study we examined the effect of doxycycline treatment in the murine model of NCC.
Animals treated with doxycycline exhibit lower numbers of parasites and reduced morbidity and mortality through the course of infection. Although similar levels of leukocyte infiltration were observed in both treatments, doxycycline seems to provide the appropriate conditions for host survival as reduced levels of apoptosis and oxidative stress were detected among infiltrates as well as CNS tissue. As an established MMP blocker, doxycycline appears to reduce degradation of junctional complex proteins in parenchymal vessels and also lowered the levels of cytokines known to increase the permeability of CNS barriers. In addition, doxycycline treatment was associated with an overall reduction in the expression and activity of MMPs, particularly in areas of leukocyte infiltration.
These results indicate that partial inhibition of a broad range of MMPs establishes an environment within the CNS conducive to restrain the infection and promote host survival, suggesting the potential of doxycycline as a therapeutic agent to control the exacerbated response often found in CNS infections. The pathogenesis of idiopathic intracranial hypertension (IIH) remains unclear. IIH is almost always associated with obesity, which is recognised as a proinflammatory state, with increased expression of a number of adipokines, cytokines and chemokines including CCL2 (MCP-1). Whether these mediators play a role in the pathogenesis of IIH remains uninvestigated. The aim of this study was to investigate any significant differences in the expression of CCL2, IL-1a and leptin in IIH patients when compared to an age and sex matched control group. These 3 mediators were selected from a preliminary screen of forty two different cytokines and chemokines on an immunoblot which showed an apparent difference between IIH and control patients.
CCL2, IL-1a and leptin levels were measured using enzyme linked immunosorbent assay (ELISA) in the cerebrospinal fluid (CSF) and serum of thirty-seven patients with a diagnosis of IIH and forty-two control subjects. Controls were subdivided into three groups: patients with inflammatory neurological disease (n = 14), patients with noninflammatory neurological disease (n = 7) and patients presenting with headache or other neurological symptoms whose investigations were all normal (n = 20). Statistical analysis utilised Kruskal Wallis test and analysis of covariance.
CSF and serum levels of CCL2, IL-1a and leptin levels were not significantly different between IIH patients and the three control groups. Serum leptin levels were significantly associated with body mass index in all groups (p = 0.002).
Our findings suggest that CSF and serum CCL2, IL-1a and leptin levels in IIH patients do not differ compared to those who do not have the condition, suggesting that these specific mediators do not a play a role in the pathogenesis of IIH. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. T cells in inflamed lesions express the chemokine receptors CXCR3 and CCR5. The role of chemokines binding to CXCR3 receptors like interferon gamma-inducible protein 10 (CXCL10) or interferon-inducible T-cell alpha-chemoattractant (CXCL11) as well as CCL5 binding to CCR5 in the pathogenesis of MS is unresolved. The aim of the study was to estimate the levels of CCL5, CXCL10 and CXCL11 in serum and cerebrospinal fluid(CSF) in relapsing-remitting MS (RRMS) patients during relapse and stable stage of the disease and to compare results with those in the control group. We also decided to evaluate the effect of methylprednisolone (MP) therapy on CCL5, CXCL10 and CXCL11 serum levels in MS patients with relapse.
The studied groups consisted of 17 RRMS patients during relapse, 30 RRMS patients in stable stage of the disease and 25 controls. In the group of relapsing MS patients the blood samples were obtained both before steroid therapy and after five days treatment with MP in a dose of 1.0 g i.v. once daily. The levels of the chemokines have been measured by ELISA method. The levels of CXCL10 were significantly higher in the CSF of RRMS patients both during relapse (298.2 ± 143.8 pg/ml) and stable phase of the disease (323.7 ± 183 pg/ml) in comparison with the control group (152.4 ± 97.7 pg/ml, p b 0.001). The CSF levels of CCL5 were significantly higher in patients with relapses in comparison with stable RRMS, p b 0.005. The CXCL11 levels did not differ significantly from control values. There was no effect of MP therapy on the serum levels of CCL5, CXCL10 and CXCL11.
The results suggest involvement of CCL5 and CXCL10 but not CXCL11 in the pathogenesis of MS.
17 -Cleavage of CCL2 by MMPs 2 and 9 reduces cell migration which may supplement the effects of immunomodulatory therapy in multiple sclerosis Chemokines direct the migration of peripheral blood mononuclear cells (PBMCs) across the blood brain barrier (BBB) into the central nervous system (CNS); a process thought to initiate the tissue damage that occurs in multiple sclerosis (MS). Expression of the chemokine CCL2 has been demonstrated within active lesions in MS, but not in control brain. A low percentage of T cells and the majority of peripheral blood and cerebrospinal fluid monocytes express CCR2, the receptor for CCL2. CCL2 is thus considered to play a key role in the recruitment of PBMCs at the BBB in MS. Matrix metalloproteinases (MMPs), particularly MMPs 2 and 9, are involved in MS pathogenesis, including BBB disruption, and are colocalised with CCL2 in CNS inflammatory lesions. MMPs can cleave chemokines, thereby altering their activity. We investigated processing of CCL2 by MMPs 2 and 9, and the effects on chemotactic activity. Effects of in vivo immunomodulatory treatment on PBMC migration in response to CCL2 are currently under investigation, and will demonstrate whether drug treatment has specific effects which could be further influenced by truncation of CCL2.
In vitro processing of CCL2 by MMPs 2 and 9 was analysed using matrix-assisted laser desorption/ionisation quadrupole time-of-flight mass spectrometry (MALDI Q-TOF MS). Cell chemotaxis to cleaved and intact CCL2 was assessed using migration assays utilising calcein AM to fluorescently label cells, prior to addition to inserts with a fluorescentblocking membrane with 3 μm pores. Measurement of fluorescence from the underside of the membrane enabled acquisition of real time migration data. Assessment of the effects of in vivo drug treatments on PBMC migration to CCL2 pre-and post-treatment (3 and 6 months) in up to 50 patients on either interferon beta (beta interferon-1b, and -1a), glatiramer acetate or natalizumab, is in process. MMPs 2 and 9 each cleaved four amino acids from the N-terminus of CCL2 within 3 h, resulting in a two-fold reduction in migration of THP-1 cells (a monocytic cell line), compared to intact CCL2. Increases (3-5 fold) in migration of PBMC from untreated MS patients occurred within 90-120 min in response to CCL2.
In vivo migration is likely to be reduced to MMP2/9-truncated CCL2. Moreover, expression of MMPs 2 or 9 in the CNS in MS is likely to result in truncation of CCL2, which may augment the effects of immunomodulatory therapy in reducing further migration of monocytes and T cells across the BBB.
18 -Skewed pro-inflammatory innate immunity in immune deficient mice impairs cognitive function through inhibition of synaptogenesis Recent evidence suggests that T cells can influence neuronal plasticity by supporting learning and memory. For example, severe combined immune-deficient (scid) mice were shown to have impaired cognitive performance, which was reversed following injection of exogenous Tcells from genetically matched donors. The mechanisms underlying the beneficial effects of T cells in the brain are not well understood.
Here we show that scid mice possess fewer hippocampal synapses along with a reduced hippocampal expression of the synaptogenic protein, thrombospondin1 (TSP1), as compared to WT mice. Moreover, myeloid cells occupying subarachnoid and ventricular spaces exhibits a skewed pro-inflammatory phenotype in scid mice as compared to wild type controls. Pro-inflammatory cytokines produced by meningeal myeloid cells are proposed to diffuse into brain parenchyma and affect astroglial expression of TSP1. We show that while antiinflammatory cytokine, interleukin (IL)-4 increased TSP1 expression in astrocytes, pro-inflammatory cytokines suppress, such as IL-6 and TNF-a inhibit its expression, which is, supposedly, responsible for reduced synapses and for cognitive impairment observed in scid mice.
Our results not only shed a light on the mechanism underlying cognitive dysfunctions associated with immune decline but might also lead to a development of new future therapies for cognitive and mental conditions that target immune cells for the benefit of central nervous system. Using image-guided pathological analysis, we previously demonstrated that multiple sclerosis (MS) lesions which were abnormal on T2weighted, T1-weighted images (WI) and magnetization transfer ratio (T2/ T1/MTR lesions) showed axonal swelling and reduced axonal Na+-K+ ATPase, as contrasted with lesions abnormal only on T2-WI (T2-only lesions). Here, we compare leukocyte accumulation in T2/T1/MTR and T2-only lesions. To address inflammatory mechanisms, we characterized expression of the chemokine receptor/ligand pair CXCR4/CXCL12.
The study comprised autopsy brain tissue from five secondary progressive MS (SPMS) and five non-neurological control cases. Based on imaging characteristics, thirty regions of interest (ROIs) were excised. Using immunohistochemistry, we evaluated myelin status, leukocyte accumulation and CXCR4/CXCL12 expression in the selected MS and control ROIs.
Eight of ten T2/T1/MTR regions were chronic-active or chronicinactive demyelinated lesions. T2-only regions were histopathologically diverse, ranging from normal or partial myelination to complete demyelination. Demyelinated T2-only regions corresponded to chronic active lesions. Mononuclear phagocyte distribution in T2-only chronicactive lesions resembled that in T2/T1/MTR chronic-active lesions.
Parenchymal T-cells were significantly increased in T2/T1/MTR ROIs as compared to T2-only lesions and MS NWM. CXCR4 and phospho-CXCR4 expression was associated with reactive microglia and macrophages in active or chronic active MS lesions. CXCL12 expression was increased in astrocytic cells and vascular elements.
Inflammatory cell distribution and numbers correlated more with stages of demyelination than with T2/T1/MTR versus T2-only imaging characteristics. Therefore, imaging correlations to axonal pathology were not affected by differential inflammatory leukocyte distribution. Robust expression of CXCR4/CXCL12 on inflammatory elements in MS lesions highlights a role of this chemokine/receptor pair in CNS inflammation.
POSTER SESSION: Neuroimmune pharmacology and crosstalk 1 -A pilot trial of low dose naltrexone in primary progressive multiple sclerosis Multiple Sclerosis Study Center, Hospital of Gallarate, Gallarate (Va), Italy Naltrexone is an orally semi synthetic opiate antagonist licensed for the treatment of heroin and alcohol addiction. However, its opiate antagonist activity is completely abrogated at lower doses (=5 mg) while triggering a prolonged up regulation of β-endorphins (BE), an endogenous opioid with neuroprotective and immunomodulatory functions. A symptomatic effect on spasticity, pain and fatigue of low dose naltrexone (LDN) in multiple sclerosis (MS) has been reported, but only anecdotic observations are available so far.
A sixth-month, phase II, multicentre, open-label, therapeutic trial with LDN (4 mg/die) has been carried out in 40 patients (pts) with a diagnosis of primary progressive (PP) MS (21:19 female:male; mean age 53.4; median EDSS 6.0). Thirty-five (87.5%) patients completed the 6 months of therapy, 5 drop-outs occurred. Two major adverse events (5% of pts) of grade III (severe) and IV (life-threatening or disabling) occurred during the trial. The remaining adverse events were of grade I (not interfering with function) or II (not interfering with activities of daily living). Transitory haematological abnormalities (35% of pts), urinary tract infections (20% of pts), and mild irritability (12.5% of pts) were the commonest. Neurological disability progressed in only 1 (2.5%) patient at the end of the study. Regarding efficacy measures, a significant reduction of spasticity along with some evidence of a positive effect on fatigue and depression were found. As regards quality of life, there was a general trend of improvement between final and baseline evaluation albeit not statistically significant. Physical functioning, role physical, energy, and social functioning were the most benefited items. PBMC levels of BE (but not of MOR) started increasing 3 months after beginning of the therapy and were still increased 1 month after therapy discontinuation (p b 0.05). No association was found between allelic variants of the OPRM1 gene and responsiveness to treatment.
LDN is safe and well tolerated in patients with PPMS. We were also able to show some positive influence on spasticity, fatigue and depression. A pure placebo-like effect is improbable because of the temporal profile of the observed changes. A longer time of drug administration and a longer follow-up could definitely address whether or not LDN can be considered a reliable therapeutic option in MS patients.
Bipolar disorder (BPD) and schizophrenia (SCZ) are severe disorders representing an enormous social, familiar and individual burden, being SCZ the most disabling psychiatric disorder characterized by psychosis and cognitive impairment. It is well known that SCZ and BPD are associated with abnormalities in dopamine signaling pathway. Recent data in the literature have demonstrated altered expression levels of some proteins involved in the modulation of this pathway in both brain and peripheral tissues. It was shown that protein and mRNA levels of dopamine and cAMP regulated phosphoprotein (DARPP-32) was downregulated in dorsolateral prefrontal cortex (DLPFC) of patients with SCZ or BPD when compared to controls. Due to the difficulty to access brain tissue and the absence of objective laboratory tests for bio-markers, we measured DARPP-32 expression in blood cell sub-populations (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) taking advantage of the close relation of nervous and immune systems.
Using flow cytometry as the analytical method, our results have shown that the DARPP-32 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and was also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. These results showed that DARPP-32 expression in immune cells agree with reports of reduced DARPP-32 protein in the DLPFC of BPD or SCZ patients.
Our data suggest that DARPP-32 expression in lymphocytes and monocytes could be used as a source of bio-markers to help in the diagnosis and/or treatment response of neuropsychiatry disorders.
Mohan Shekher⁎ 1 , Fernando Samodha C. 2 The mu opioid receptor (hMOR) plays a significant role in opioid tolerance. Expression of hMOR has been shown to be differentially regulated following cytokine stimulation. We aim to determine the dose and time-dependent effects of IL-1beta and morphine on hMOR expression in SK-N-SH cells using quantitative real-time-PCR (qRT-PCR) and immunocytochemistry (ICC).
A time-dependent IL-1beta-induced up-regulation of hMOR was identified. 10 and 100 ng/mL of IL-1beta for 6 h (p b 0.001) and 12 h (p b 0.05) induced hMOR up-regulation as measured using qRT-PCR (n = 5). 10 and 100-fold pretreatment with IL-1 receptor antagonist (IL-1ra) reduced IL-1beta (10 ng/mL) induced up-regulation of hMOR, concluding a plausible IL-1R1 dependent mechanism. The effect of morphine (1, 10 and 100 03BCM) on hMOR expression showed a timedependent (24 h) down-regulation of hMOR mRNA expression (n = 5). Using Confocal microscopy, ICC showed that IL-1beta and morphine induced changes in hMOR mRNA expression are reflected in hMOR protein expression changes.
In conclusion, evidence for the interactions between IL-1beta, morphine and hMOR expression in vitro may elucidate the mechanism behind opioid-induced opioid tolerance. Next we aim to measure these changes in hMOR protein expression using immunoblot analysis. Future studies include elucidating the signal transduction pathways that result in changes in hMOR expression in neurons treated with IL-1beta and morphine concurrently. With the use of signaling inhibitors, we aim to determine if IL-1beta effect on hMOR expression in SK-N-SH cells is dependent on specific intracellular signaling proteins (p38MAPK-ERK-MEK-NF-kappaB). Understanding the neuroimmune interactions between IL-1beta and hMOR expression, may identify potential molecular targets for the prevention of opioid receptor expression down-regulation and perhaps opioid tolerance. EAE was induced in male, age-matched C57Bl/6 mice with myelin oligodendrocyte glycoprotein (MOG) peptide in adjuvant and monitored for disease severity using a 5-point scale (1, limp tail; 2, ataxia + limp tail; 3, weak hind limb; 4, complete hind limb paralysis; 5, death). Inhibitor was administered daily at 25 mg/kg p.o. beginning at 21 days post-immunization. Lymphocyte activation and cytokine production were measured in splenocytes following ten days of inhibitor treatment. We found that mice treated with MIF inhibitor had less severe EAE within seven days of treatment (cumulative disease index of 25.4 ± 15.9 vs. 48.9 ± 23.4). Inhibitor-treated mice also had a larger population of CD4 + 25 + FoxP3+ regulatory T cells (5.1% vs. 3.8%), but proliferation and production of the cytokines IL-10, IL-17, and IFNgamma in response to antigen were unchanged. Following conclusion of treatment, mice remained protected compared to vehicle-treated controls (cumulative disease index 46.3 ± 29.0 vs. 85.0 ± 40.4).
These findings demonstrate that an inhibitor of MIF is protective in EAE, potentially through the expansion of regulatory T cells. However, inhibitor treatment does not dampen the peripheral immune response to antigen or the production of inflammatory cytokines. This suggests an alternate role for MIF, perhaps in the trafficking of inflammatory cells into the CNS. Because of their bioavailability and mechanism of protection, these inhibitors could be a novel therapeutic approach for the treatment of ongoing MS and other inflammatory diseases. (Supported by NIH grant AI 064320 and NMSS grant RG3272)
Multiple sclerosis (MS) is an idiopathic inflammatory demyelinating and neurodegenerative disease of the CNS. It is currently believed that environmental and genetic factors contribute to both the onset and progression of the disease which occurs during early adulthood. Interestingly, both infectious agents and psychological stress have been suspected to play a role in the acquisition and progression of this disease since its early characterization in the late 1800s. Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus which upon intracranial inoculation of susceptible mice (i.e. SJL) causes a persistent infection that is characterized by an acute polioencephalitis followed by chronic inflammatory demyelinating disease that resembles human MS. We have been interested in the effects of stress on the pathogenesis of this experimental model of MS. In the current study we were interested in both the effects of chronic stress on virus-specific adaptive immune responses to TMEV in SJL mice and the mechanisms by which these responses are modulated.
Using known immunodominant viral peptides specific for either CD4+ or CD8+ T cells we found that stress decreased virus-specific T cell responses in both the spleen and CNS. Intriguingly, stress increased virus-specific circulating antibody responses in the serum of stressed mice when compared to non-stressed mice, but did not result in a shift from type 1 toward type 2 immunity as indicated by serum cytokines and T cell stimulation assays. Moreover, this suppression of T cell responses did not appear to be fully attributable to alterations in T cell percentages as indicated by flow cytometry. Finally, glucocorticoids were found to be partially responsible for the decreased responses on T cell function as the effects of stress were in part reversed by concurrent RU486 administration but mimicked by dexamethasone administration.
As T cells are thought to mediate viral clearance in this model, our data indicate that stress induced immunosuppression may provide a means for enhanced viral persistence within the CNS.
6 -Active immunization with amyloid-beta 1-42 significantly impairs memory performance in healthy mice Active immunization with Amyloid-beta 1-42 (Abeta) was shown to prevent amyloid plaque deposition in different mouse models of Alzheimer's disease (AD). Whether the induction of an immune response to Abeta also improves memory function is still controversial. Here, we identify the cognitive and immunological phenotype of healthy C57BL/6 mice immunized with Abeta and complete Freund's adjuvant (CFA).
Immunization significantly altered the behavioral phenotype of mice compared to control mice receiving Abeta or CFA only or Myelin oligodendrocyte glycoprotein induced EAE mice. Abeta and CFA immunized mice revealed decreased locomotor activity, reduced habituational learning and highly deficient spatial-learning abilities in an object recognition task. Quantitative rtPCR data demonstrated the upregulation of a number of macrophage and T cell related genes in the CNS of Abeta mice, in particular C3, CD3, CD14, Cxcl10, IL-1b, IFN-g and S100A8. Furthermore, reactive gliosis was detected in Abeta mice in the chronic phase of disease as shown by GFAP immunohistochemistry and by quantitative rtPCR for GFAP. In peritoneal macrophages from immunized mice, CD14, CSF 3, IL-1b, IL-6, MMP9, Nos2, S100A8 and TNF-a gene transcripts were highly upregulated.
In summary, immunization of healthy mice with Abeta and CFA induced an inflammatory cascade significantly impairing cognitive functioning. These results illustrate the potential danger of active immunization with neuronal antigens and demonstrate the complexity of immune responses to Abeta in the pathogenesis of AD. Abeta immunization in plaque pathology free animals even provides a suitable model for autoimmune mediated cognitive impairment.
Lewitus Gil⁎ 1 , Wilf-Yarkoni Adi 1 , Ziv Yaniv 1 , Shabat-Simon Maytal 1 , Gersner Roman 1 , Zangen Abraham 1 , Schwartz Michal 1 1
Depression in an animal model is often associated with reduced BDNF levels, and impaired neurogenesis in the hippocampus. Our previous observations that T cells recognizing CNS-specific antigens can regulate adult brain neurogenesis and expression of BDNF, created the basis of the present study of boosting T-cell levels to cope with depression.
Here we show that vaccination with a weak agonist of a myelinderived peptide ameliorated depressive behavior induced by chronic mild stress in rats. The behavioral outcome was accompanied by restoration of hippocampal BDNF levels and neurogenesis.
The results of the present study introduce a novel approach of vaccination with CNS-related antigens as a therapeutic means for fighting depression. Vaccination, as an antidepressant therapy, encompasses several molecular and cellular mechanisms that are known to be regulated by antidepressant drugs. Therefore, we suggest that immunebased therapies should be considered for treatment of depression. myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS.
The striatum, which is particularly prone to degenerate in MS, was rather selectively involved in EAE, and striatal neurons exhibited a marked up-regulation of AMPA receptor-mediated transmission since the presymptomatic phase of the disease. Altered striatal glutamate transmission was secondary to abnormal expression and phosphorylation state of AMPA receptors triggered by microglia activation, TNF-a release, and modulation of Arc/Arg3.1 early gene. Accordingly, application of activated microglia or of TNF-a, but not of activated T lymphocytes, replicated in normal neurons the synaptic defects observed in EAE.
These data reveal an important role of synaptic dysfunction and soluble factors released from activated microglia in the pathological process of EAE. Macroautophagy delivers cytoplasmic constituents for lysosomal degradation in skeletal muscle tissue and thereby could affect adaptive immune responses via delivery of intracellular antigens for MHC class II presentation on these cells.
We investigated constitutive and immune-mediated macroautophagy regulation and MHC expression in primary human myoblasts and in a rhabdomyosarkoma cell line (CCL 136) by immunocytochemistry, immunoblotting and flow cytometry. We identified TNF-alpha from complex mixtures of inflammatory cytokines as the predominant regulatory factor for macroautophagy in the skeletal muscle. While loss of macroautophagy did not predispose muscle cells for TNFreceptor mediated apoptosis, TNF-alpha enhanced HLA-DR surface levels in interferon-gamma treated myoblasts. The synergistic effect of TNF-alpha and IFN-gamma did not result in higher intracellular HLA-DR expression, suggesting that TNF-alpha facilitates antigen processing via macroautophagy for more efficient MHC class II loading.
Our findings establish a mechanism through which TNF-alpha regulates both macroautophagy and MHC class II expression in skeletal myocytes and suggest that TNF-alpha is a key mediator and a potential therapeutic target in T cell-mediated inflammatory myopathies in which macroautophagy is involved. Endogenous opioids as well as exogenous opiates affect immune responses. This is achieved by their indirect action via binding to opioid receptors expressed on cells of the neuroendocrine system and by their direct action via binding to opioid receptors expressed on immunocytes. The effect of direct action of opioids on immunocytes is modulation of their proliferation, activation and death.
Expression of opioid receptors of the kappa class (KOR) on cells of monocyte-macrophage origin has been reported. However, precise role(s) of KOR and their regulation have not been fully understood.
In this study we have examined the expression of KOR on cells of a murine macrophage cell line, J774. That was done at transcriptional and protein level by using semi-quantitative RT-PCR and Western blot, respectively. Mouse or rat brain cells, and mouse T-cell line R1.1, selectively expressing KOR, were used as positive controls. The data obtained have shown that J774 cells express KOR at both levels. Further, we examined the ability of pro-inflammatory cytokine interferon-gamma to regulate KOR expression. IFN-gamma increased KOR mRNA in a concentration-and time-dependent manner. This finding was confirmed by increased level of KOR protein in IFNgamma-treated J774 cells.
Collectively, the data presented have shown that J774 cells basally express KOR and that KOR could be up-regulated by IFN-gamma. This observation is in line with our previous finding that KOR-selective ligand Dynorphin-A modulates NO secretion and proliferation of J774 cells ( Cerebral ischemia is accompanied by an acute inflammation, involving the activation of microglia and the infiltration of neutrophils (PMN) and monocytes into the brain. Whether and how microglia can protect neurons from ischemic death is still debated.
Using a model of application of innate immune cells onto hippocampal slices (OHC), we investigated their effects on neuronal death after ischemia. We show that applied PMN exacerbated neuronal damage, whereas microglia protected neurons from oxygen glucose deprivation (OGD) in two ways. Applying an approach of transgenic OHC from mice (eYFP expression in neurons) and fluorescently labeled microglia, we observed that microglia is engaged in close physical contact with neurons after OGD. Blockage of microglia migration to the neurons after OGD abolished the microglia mediated protection. Moreover, the application of microglia and PMN simultaneously indicated that microglia counteracted the PMN neurotoxicity. Time-lapse imaging revealed the phagocytosis of PMN by microglia. Interference with the phagocytosis using RGDS and N-Acetyl-Glucosamine abrogated the microglial ability to reduce the PMN neurotoxicity.
Taken both observations together: microglia might protect neurons after ischemia in two ways: (i) directly by physical interaction with neurons and (ii) indirectly by clearance the inflamed tissue from toxic PMN. Thus, non-selective anti-inflammatory treatment after ischemia would be counterintuitive.
Cerebral ischemia is among other immunological events characterised by an early response of microglia cells within the endangered region and microglia from surrounding parenchyma. Migration of surrounding microglia to the site of injury is a key event in exerting the response in the respective region. Whether microglia cells then exhibit beneficial or detrimental effects is still highly debated. It has been shown that the serine protease thrombin is an inflammatory mediator that acts via protease-activated receptors (PAR1, PAR3 and PAR4) and modulates microglia function (e.g. cytokine production). We have been focusing on the role of PAR4 on microglia after ischemia.
Using an in vitro co-culture of primary hippocampal neurons and the microglia cell line BV-2, we show that microglia migration activity is dramatically enhanced after ischemia. Similar migration enhancement of microglia in the co-culture has been seen after selective PAR4 activation by an activating peptide without ischemic exposure. Moreover, we investigated the PAR4 dependent regulation of ED-1 expression that shows the lysosomal activity of phagocytotic cells. We found a down regulation of ED-1 expression after selective activation of PAR-4 on microglia cells. To characterise the cytokine production and thereby the status of microglia-activation we analysed the level of pro-inflammatory TNF-alpha in PAR-4 stimulated primary microglia and BV2 cells.
Our results implicate that microglia activation by PAR4 might regulate microglia function after cerebral ischemia. We recently demonstrated that cyclooxygenase 2 (COX-2) was expressed in dying oligodendrocytes at the onset of demyelination in the Theiler's Murine Encephalomyelitis Virus (TMEV) model of multiple sclerosis (MS). This finding is consistent with the hypothesis that COX-2 expression in oligodendrocytes might render these cells more susceptible to glutamate-mediated excitotoxicity in much the same manner that COX-2 expression in neurons increases neuronal cell death.
Objectives: Our goal was to determine whether COX-2 inhibitors can decrease demyelination in TMEV induced demyelination (TMEV-IDD) and limit excitotoxic death of oligodendrocytes in vitro.
Mice were infected intracerebrally with the DA strain of TMEV and either given a control diet or chow containing the COX-2 inhibitor CAY10452 (a derivative of celecoxib). Spinal cords of control and CAY10452-treated mice were harvested 35 days after infection and scored for inflammation and demyelination following Luxol fast blue staining. For the in vitro experiments, organotypic cultures were prepared from post natal day 8 spinal cords and grown on permeable filters at 37°C with 5% CO 2 . Cultures were then exposed to the excitotoxin kainic acid (KA) (300 μM) in the presence or absence of the COX-2 inhibitor CAY10452 (30 μM). Cell death in white matter and gray matter was assessed 24 h later by immunofluorescence confocal microscopy using the cell death indicator activated caspase 3 and the oligodendrocyte marker cyclic nucleotide phosphodiesterase (CNPase).
In the TMEV-IDD model of MS, COX-2 inhibitors had no effect on the score for inflammation but significantly decreased the amount of demyelination (p b 0.05 by the Mann-Whitney nonparametric t-test). In vitro, COX-2 inhibitors decreased the amount of KA-induced cell death in white matter and gray matter (p b 0.01 by ANOVA, Tukey-Kramer).
The COX-2 inhibitor CAY10452 can reduce demyelination in the TMEV-IDD model. In vitro, our results indicate that COX-2 inhibitors may have a therapeutic effect by limiting glutamate-mediated excitotoxicity and oligodendrocyte death.
Voulgari-Kokota Anda⁎ 1 , Delorme Bruno 6 , Taoufik Era 7 , Tseveleki Vivian 8 , Charbord Pierre 9 , Probert Lesley 10 1 Anda Voulgari-Kokota, Athens, Greece Bone marrow mesenchymal stem cells (MSCs) are a nonhematopoetic, multipotent stem cell population which can differentiate into osteoblasts, adipocytes and chondrocytes in vivo and in vitro. In bone marrow, MSCs support hemopoiesis providing the proper environment for hematopoietic stem cell renewal, proliferation and differentiation while they substitute osteoblasts, osteocytes and non-hematopoietic cells in bone marrow stroma. According to their supportive role, MSCs secrete a plethora of different cytokines, trophic factors and anti-apoptotic factors. It has been shown that MSCs are recruited to the sites of neural injury and inflammation and that they prevent neuronal death secreting neurotrophic and antiapoptotic factors as well as promoting local neurogenesis.
The aim of this study was to evaluate MSCs neuroprotective capacity and investigate pathways that are involved.
Bone marrow mesenchymal stem cells were flushed from C57bl/6 mice and cultured to passage 3. FACS analysis confirmed the purity of MSCs cultures (N = 98% cells CD11b-negative, CD44-positive and Sca1positive). Mouse embryonic cortical neurons were isolated from 15days C57bl/6 embryos. On div.5 for neurons, the two cell populations were cultured together using coculture inserts. Cells were then exposed to NMDA excitotoxicity (30 μM) and neuronal death was assessed 24 h later using LDH assay. Alternatively, embryonic cortical neurons on day 5 were cultured in MSCs' conditioned medium for 24 h and then NMDA was added in the culture.
Gene expression arrays which were performed using neuronal RNA samples showed that MSCs induce the expression of many neuroprotective and neurotrophic factors by neural cells, suppressing at the same time a variety of apoptotic molecules.
Our results show that the presence of MSCs or MSCs-conditioned medium significantly protect neurons in the excitotoxic environment.
5 -Cytotoxic effects of myasthenia gravis sera on cardiomyocytes in vitro different antibody composition and varying disease severity. Cytotoxic effects were assessed by evaluating morphology, measurement of adenylate kinase (AK) release from the cells, and by using flow cytometry after propidium iodide and Annexin-V staining.
Incubation with MG sera did not induce morphological changes in the cells compared to cells treated with sera from healthy human donors. Perinuclear vesicles and inclusion bodies were present in a minority of cells treated with human sera, regardless of whether patient or control serum. The mean normalized AK release value from cells treated with MG sera in concentration 1:10 was 80.3% of controls (standard deviation = 28.8). One-way ANOVA analysis revealed no significant difference between the patient and control groups (p = 0.24). When comparing patients within the MG group, no significant differences were detectable (p = 0.09). Girardi cells treated with MG-sera in concentration 1:10 for 24 h prior to flow cytometric examination showed a cell death rate of 9.3%, compared to 8.4% for control serum. The standard error of the mean for the two groups was 1.26 and 1.45, respectively (p = 0.69, One-way ANOVA test). Less than 1% Annexin-V positive cells (indicative of apoptosis) could be detected in both the MG patient and the control group. There was no systematic variation according to antibody profile or concentration, MG severity or other clinical parameters, and the observations were not dose or time dependant.
We conclude that MG sera have no cytotoxic effect in our heart cell culture system. If there is a causal link between MG and heart disease, a direct affection of the cardiomyocytes is not the most likely mechanism. EBV latency phenotypes were characterised for the lines used here; these lines were then screened for the expression of neurotrophin ligands and their receptors: the selective high affinity Tropomyosin receptor kinases TrkA, TrkB and TrkC and the (common) low affinity, tumour necrosis factor receptor member, p75NTR. FDC-like cell lines were also analysed for neurotrophin component expression. This was to question FDCs as potential providers of paracrine neurotrophin signalling to Burkitt's lymphoma B cells.
Neurotrophin and neurotrophin receptor expression was detected by flow cytometry, reverse transcription polymerase chain reaction (PCR) and real time PCR methods.
Cell lines with the full complement of EBV latency genes expressed were positive for the neurotrophin, Brain Derived Neurotrophic Factor (BDNF) and all neurotrophin receptors in question. Burkitt's lymphoma cells expressing limited EBV latency genes revealed more restricted expression of neurotrophin components. FDC-like lines also express neurotrophin and neurotrophin receptors, thus paracrine signalling between Burkitt's lymphoma cells and FDCs may occur via this axis, perhaps to enhance B cell survival.
Multiple sclerosis (MS) is a common cause of severe neurological disability resulting from the interruption of myelinated tracts in the central nervous system. Familial aggregation of cases and differences in disease risk among ethnic groups residing in the same geographical regions are well documented, supporting the role of genetic influences in MS pathogenesis. However, its modest heritability reflects complex genetic effects and multifaceted geneenvironment interactions. The human leukocyte antigen (HLA) gene cluster in chromosome 6p21.3 represents the strongest susceptibility locus for MS genomewide. The primary signal arises from the HLA-DRB1 gene in the class II genomic segment of the locus, but complex hierarchical cis and trans allelic effects were detected, including a protective signal in the class I region near the HLA-C gene.
The past two years have seen substantial progress in the development of laboratory and analytical approaches to perform large-scale genome-wide association studies in MS and other complex disorders. A trio-based study using 500,000 SNPs arrays in 931 MS families was recently completed and reported. The classic HLA risk locus stood out with remarkably strong statistical significance (P b 1 × 10 − 81 ). The study also yielded several promising non-HLA leads, which were pursued in an independent dataset. A combined analysis of more than 12,000 samples identified a number of genes associated with disease susceptibility, including the interleukin 2 receptor alpha chain (IL2RA also known as CD25) on chromosome 10p15, which has been also implicated in the pathogenesis of type 1 diabetes and Graves' disease; CD58 on chromosome 1p13; and IL7R (CD127) on chromosome 5p13. Progress in high-throughput genotyping and sequencing technologies and a better understanding of the structural organization of the human genome, together with powerful brain imaging techniques that refine the phenotype suggest that the tools may finally exist to identify the full set of genes influencing the pathogenesis of MS.
Baylor College of Medicine, Houston, Texas, USA Whereas Watson-Crick DNA base pair changes have long been recognized as a mechanism for mutations, rearrangements of the human genome including deletions, duplications, and inversions have been appreciated only more recently as a significant source for genetic variation. Diseases that result from DNA rearrangements have been referred to as genomic disorders. Rearrangements of our genome can be responsible for inherited as well as sporadic traits. Rearrangements associated with genomic disorders can be recurrent, with breakpoint clusters resulting in a common sized deletion/duplication, or nonrecurrent and of different sizes.
The analyses of breakpoints in the proximal short arm of chromosome 17 (17p) reveal nonallelic homologous recombination (NAHR) as a major mechanism for recurrent rearrangements, whereas nonhomologous end-joining (NHEJ) can be responsible for many of the non-recurrent rearrangements. Genome architectural features consisting of low-copy repeats (LCRs), also called segmental duplications, can stimulate and mediate NAHR. There are positional hotspots for the crossovers within the LCRs. We recently elucidated a DNA replication mechanism for nonrecurrent rearrangements that we termed FoSTeS -Fork Stalling and Template Switching. Rearrangements introduce variation into our genome for selection to act upon and as such serve an evolutionary function analogous to base pair changes. Genomic rearrangements may cause Mendelian diseases, complex traits such as behavioral and psychiatric traits (e.g. autism, schizophrenia), or represent benign polymorphic variations. The latter copy number variations (CNVs) are ubiquitous in the human genome. In fact, any two humans contain more base-pair differences due to structural variation of the genome than resulting from single nucleotide polymorphism (SNPs). The mechanisms by which rearrangements convey phenotypes are diverse and include gene dosage, position effects, unmasking of coding region mutations (cSNPs) or other functional SNPs, creating gain-of-function fusion genes or loss of function gene mutations at the breakpoints, and perhaps through effects of transvection. De novo genomic rearrangements have been shown to cause both chromosomal and Mendelian disease, as well as sporadic traits, but our understanding of the extent to which genomic rearrangements, gene CNV, and/or gene dosage alterations are responsible for common and complex traits remains rudimentary. During cellular differentiation, both permissive and repressive epigenetic modifications must be negotiated to create cell-type specific gene expression patterns. At cell fate decision checkpoints, dynamic developmental decisions are made to establish specific epigenetic patterns, but the mechanisms by which these heritable states are created, especially the loss of repressive methyl marks, have been elusive. The Tbox transcription factor family is critically important in several developmental systems ranging from embryogenesis to the differentiation of adult tissues. In the immune system, the T-box factors T-bet and Eomes are responsible for cellular differentiation events in several cell types, most notably Th1 cell differentiation. We have explored the mechanisms by which developmentally important transcription factors contribute to the establishment of epigenetic patterns by utilizing T-bet as a model. A comprehensive mutagenesis study surprisingly suggested that in addition to DNA binding, the highly conserved T-box domain is responsible for recruiting chromatin-modifying activities to target gene promoters. By examining point mutations known to be responsible for several different human genetic diseases found within the T-box factors Tbx3, Tbx5, Tbx20, Tbx22 and T-pit, we have found that two distinct surfaces of the T-box domain recruit chromatin-modifying activities to promoters and are required for the functional activation of T-bet target genes. Our data suggest that T-bet is required and sufficient to alleviate repressive H3K27 methylation marks and that this is mediated through a functional interaction with the demethylase JMJD3. After repressive modifications are removed, a permissive chromatin environment is created by the T-box dependent recruitment of an H3K4 methyltransferase to the target genes. Collectively, the data suggest that the ability of the T-box DNA binding domain to functionally establish the epigenetic state of its target genes is required for its ability to initiate cell-type specific developmental gene expression patterns. Characterization of encephalitogenic T cells is critical for identifying therapeutic targets for multiple sclerosis (MS). To this end, we have used small interfering RNA (siRNA) to suppress molecules expressed by encephalitogenic T cells to determine if the molecule is essential for immune-mediated demyelinating disease. Using a siRNA specific for T-bet, a transcription factor, we found T-bet to be critical for the differentiation and effector function of encephalitogenic T cells. Systemic administration of T-bet specific siRNA inhibites the onset of experimental autoimmune encephalomyelitis (EAE) and treatment of established EAE with this siRNA ameliorates disease. We found that the interferon-γ, STAT1, and IL-23 receptor genes are all directly regulated by T-bet. In addition, the pathogenic capacity of both myelin-specific Th1 and Th17 cells is greatly diminished when T-bet is suppressed. Interestingly, adoptive transfer of myelin-specific Th1 cells results in the generation of host-derived Th17 cells found only in the CNS. Thus, it appears that the CNS is a conducive environment for the differentiation of Th17 cells. However, systemic administration of T-bet siRNA following adoptive transfer of Th1 cells prevents the generation of Th17 cells in the CNS, indicating that T-bet plays a role in the differentiation of Th17 cells. In addition, myelin-specific T cells from mice with spontaneous EAE fail to transfer EAE if T-bet is suppressed, confirming a critical role for T-bet in spontaneous autoimmune demyelinating disease. In an effort to determine the role of myelin-specific Th17 cells in pathogenesis, we have found Th17 cells differentiated in vitro with IL-6+TGFβ do not transfer EAE and are a distinct population from the pathogenic Th17 cells generated in vivo. This data suggest that T-bet may be a viable therapeutic target for MS, capable of suppressing both Th1 and Th17 cells.
The La Jolla Institute for Allergy and Immunology, La Jolla, CA, United States
The intestinal mucosa forms the largest body surface exposed to exogenous antigens, both innocuous and pathogenic non-self antigens. Consistent with chronic antigen exposure, the gut mucosa also lodges a large collection of activated lymphocytes that under physiological conditions remain in an immune quiescent state. This so-called "gut physiological inflammation" is controlled by distinct regulatory mechanisms, including different types of regulatory DCs and T cells. An important molecule in this context is TGF-β, abundantly produced in the gut and crucial for both systemic and mucosal immune-regulation. Among multiple roles, TGF-β has the capacity to block Th1 and Th2 differentiation and to convert naïve CD4 cells into Foxp3-expressing induced Treg cells. Paradoxically, TGF-β also displays pro-inflammatory roles and in the presence of pro-inflammatory cytokines such as IL-6, IL-21, IL-1β and TNF, TGF-β promotes the conversion of naïve T cells into effector Th17 cells. This contrasting deviation puts TGF-β as a principal controller of immune responses and underscores a central role of this cytokine in orchestrating the pro-and anti-inflammatory nature of adaptive immunity. Nowhere else is a critical regulation of this balance between productive and suppressive immunity, of more significance that at the mucosal surface of the gut where efficient immune protection against pathogens has to coincide with maintenance of the mucosal barrier integrity and selftolerance as well as tolerance towards the vast load of harmless nonself antigens derived from food and beneficial commensal bacteria. We identified a metabolite of the nutrient vitamin A, retinoic acid, as a key regulator of TGF-β-dependent immune responses, capable of inhibiting the IL-6-driven induction of pro-inflammatory Th17 cells and promoting the peripheral differentiation of anti-inflammatory Foxp3 + induced Tregs. This intestinal induced regulatory mechanism allows for environmental factors to control aberrant endogenous immune responses from the outside in. The induced immune tolerance is not irreversible however and in the presence of innate danger signals retinoic acid effects condense or synergize with innate responses to promote or enhance protective immunity. Dendritic cells (DCs) are antigen presenting cells that influence the activities of all types of lymphocytes, including B, NK and T cells. Two main pathways of DC development are recognized: 1) the myeloid DCs (mDCs) with two unique subsets: Langerhans cells and interstitial DCs; and 2) the plasmacytoid DCs (pDCs), a main source of the antiviral and immunomodulatory type 1 interferons. Subsets of DCs display unique combinations of surface markers, such as lectins, that endow them with distinct phenotypes and functions. DCs also are an unavoidable target in the prevention, treatment and development of disease. In the settings of infection and cancer, microbes and tumors can exploit DCs to evade immunity, but DCs also can generate resistance, a capacity that is readily enhanced with DC-targeted vaccines. In the context of patient care, DCs are providing new approaches to disease pathogenesis and therapy. In particular it is clear that DCs play a critical role in the process of vaccination. To address this, we are exploring new vaccine designs that harness DCs. In cancer, DCs are generated ex vivo by culturing either blood monocytes or bone marrow progenitor cells with cytokine combinations. These DCs are then loaded with tumor antigens in different forms and injected back into the patients. This has resulted in the induction of clear tumorspecific immune responses as well as clinical responses. We are expanding this strategy to other cancer types as well as to infectious diseases, such as HIV. Novel strategies have been proposed to directly target the antigens to DCs in vivo. Multiple DC surface molecules have been considered as targets, as they need to allow internalization of the antigen cargo and its processing for presentation on both MHC class I and class II molecules. We are characterizing DC subsets that may be effective targets for anti-DC antibodies. These antibodies can be fused to antigens and carry them directly to the desired DCs for maximum immune response against a variety of infectious diseases.
Neuroimmunology Division, Inst. Experimental Immunology, Department of Pathology, University Hospital of Zurich, Switzerland
During the formation of an immune synapse between APCs and Tcells, cytokines have the capacity to orchestrate the ensuing immune response. In the context of MS and its animal model EAE, the precise function and phenotype of the encephalitogenic disease-mediating T cell remains elusive. For more than 10 years, the cytokine secretion profile of effector TH cells divided them into TH1 and TH2 cells and "immune deviation" from pathogenic TH1 to regulatory TH2 cells was widely held to be an attractive therapeutic strategy for the treatment of MS. This paradigm was dismantled when it was discovered that IL-23 rather than its TH1 inducing sister cytokine IL-12 is essential for the development of autoimmune disease. IL-23 was demonstrated to drive the expression of IL-17 in TH cells and the term TH17 cells was coined. The clear association of this newly described T H 17 phenotype with autoimmune pathogenicity now implicates T H 17 cytokines as critical mediators of chronic autoimmune diseases. However, this notion relies almost exclusively on correlations and associations and a causative link between TH17 cytokines and pathogenicity has yet to be made. In order to study the actual function of TH17 cytokines in the context of autoimmunity in vivo, we generated a number of transgenic and gene-targeted mice and discovered that the current notion of the pathogenic TH17 cells is again far too simplistic and erroneous. I will discuss that TH17 cytokines, while clearly associated with pathogenicity, do not or only marginally contribute to the development of autoimmune CNS-disease and that the master cytokine IL-23 drives pathogenicity independent of TH17 polarization. B cell contribution to MS pathophysiology has generally been considered based on their potential to react to particular CNS antigens and produce CNS-directed autoantibodies. The presence of abnormal CSF immunoglobulin (Ig) with typical oligoclonal band (OCB) pattern, pathologic features of demyelinating lesions and molecular analyses of B cell lineagetogether have implicated contribution of CNSspecific antibodies in MS (e.g. complement-mediated injury, antibody-directed cytotoxicity (ADCC). While elegant approaches have confirmed that at least a portion of the CNS Ig bind myelin targets, it remains unclear whether all CNS antibodies directly contribute to pathogenesis, are generated as a consequence of tissue injury, or represent epiphenomenon of active CNS inflammation. Certain CNSdirected antibodies may actually be beneficial such as through the support of remyelination, or by helping to remove debris including growth inhibitory molecules such as Nogo, thereby providing a more permissive environment for axonal regeneration.
B cell contribution to MS has also been considered beyond their capacity to become antibody-producing cells. Distinct B cell subsets have been identified that function as effective APC to T cells and normal human B cells are now recognized to actively contribute to the regulation of immune responses in part through secretion of distinct effector cytokine profiles in a context-dependent fashion. In some patients with MS, chronically activated B cells have been described in structures reminiscent of germinal centers that may affect the CNS independent of T cells as well as promote ongoing T cell activation and propagation of the MS disease activity. A deficiency in the ability of MS patient B cells to produce the downregulatory cytokine IL-10 has been reported, implicating them directly in the dysregulated MS immune process. Promising results from early phase MS studies of B cell depletion with rituximab (anti-CD20 antibody) further support a role for B cells in disease pathogenesis, and the rapid onset of action of this strategy (without obvious effects on IgG levels) suggests that antibody-independent functions of B cells likely contribute to the observed therapeutic effect.
Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy Epstein-Barr virus (EBV) infection has been implicated in several major autoimmune diseases, including multiple sclerosis (MS). In MS, evidence supporting a pathogenic role for EBV comes from epidemiological, immunological and histopathological studies. The presence of abnormal deposits of EBV-infected B cells/plasma cells in post-mortem brain tissue from cases with acute, relapsing-remitting and progressive MS courses is compatible with abnormalities in humoral immunity, such as persistence of B-cell clones that expand and produce immunonoglobulins intrathecally and formation of intrameningeal B-cell follicles (as latent EBV infection promotes B-cell activation and growth), and with increased immune reactivity to EBV in the patients' blood and cerebrospinal fluid (as both the latent and lytic EBV proteins are potent immunogens and elicit strong B-and T-cell immune responses). EBV could promote chronic brain inflammation through mechanisms that involve autoimmunity (e.g., molecular mimicry or rescue of self-reactive B cells) and/or virus-induced immunopathology resulting from the inability of the immune system to control viral infection in the CNS properly. In this talk, I shall present findings that expand previous evidence of dysregulated EBV infection in the MS brain and discuss a scenario in which cytotoxic immune responses targeting intracerebral EBV deposits may play a major role in maintaining a chronic inflammatory condition in the MS brain. Accumulating data indicate that B lymphocytes and antibodies are involved in the pathogenesis of multiple sclerosis (MS). Rituximab is a monoclonal antibody that depletes circulating CD20+ B cells in humans. We report the results from an investigator-initiated trial of rituximab in relapsing MS patients.
30 relapsing-remitting MS (RRMS) patients with continued clinical and magnetic resonance imaging (MRI) activity despite treatment with an FDA-approved immunomodulatory drug (IMD) were enrolled. Subjects received 4 doses of 375 mg/m 2 rituximab over 4 weeks, and continued to take IMD. Twenty-three subjects underwent two spinal taps, at one week pre-treatment and at week 24 after treatment. Immunoglobulin (Ig)G concentration, IgG index, IgG synthesis rate and electrophoresis for oligoclonal bands were performed by a central lab. The concentrations of B and T lymphocytes in cerebrospinal fluid (CSF) were determined by flow cytometry. ELISAs were used to quantitate chemokine levels in CSF before and after rituximab.
Post-treatment, CSF B cells and T cells were decreased significantly compared to pre-treatment, as reported (Cross et al. J. Neuroimmunol. 2006 ). To address why T cells decreased following treatment with a monoclonal antibody targeting only B cells, an array of chemokines was compared in CSF before and after treatment. In 19 of 23 subjects, CSF CXCL13 levels decreased by N20% after treatment. In 4 subjects, CXCL13 remained unchanged post-treatment. CXCL13 did not increase in CSF in any subject post-rituximab treatment. The proportional decline in CSF CXCL13 correlated with the proportional decline in T cell numbers in CSF.
No significant reductions in CSF IgG index, IgG concentration or number of oligoclonal bands were observed. A trend toward decreased IgG synthesis rate post-treatment was noted.
B cells and T cells were significantly reduced in CSF at six months after rituximab. The decline in T cell numbers correlated with alterations in the CSF chemokine pattern, specifically with a decline in CSF CXCL13 levels.
National MS Society USA (RG3292), National Institutes of Health (K24RR017100; MO1RR00036), Genentech, Inc. and Biogen Idec. LP is a Fellow of the NMSS USA (FG1665-A-1). Although the presence of leukocytes within MS lesions is indisputable, the route and adhesion molecules by which these cells access the brain are still not fully understood. Using a lipid raft-based proteomic approach we identified melanoma cell adhesion molecule or MCAM/CD146 as an immunoglobulin superfamily member potentially involved in leukocyte migration across the blood-brain barrier (BBB).
Using an in vitro model of the human BBB and the animal model of MS, experimental autoimmune encephalomyelitis (EAE), we characterized the role and function of MCAM in immune cell migration to the brain. We demonstrate that MCAM is expressed on BBB endothelial cells, in vitro and in situ, and that MCAM expression can be upregulated with inflammatory cytokines in vitro. Since MCAM's ligand is MCAM itself, we analysed MCAM expression on peripheral blood leukocytes from human healthy donors and MS patients. MCAM is detected on a subset of activated memory CD4+ and CD8+ lymphocytes and the proportion of MCAM+ T cells appears to be higher in MS patients compared to controls. Furthermore, in vitro microscopy studies suggest that MCAM plays a role lymphocyte adhesion and diapedesis.
Our preliminary data provides evidence that MCAM is expressed by both the BBB and the immune cells and suggest that MCAM is potentially involved in MS lesion formation. The aim of this project was to identify novel targets of an autoimmune response in multiple sclerosis (MS) patients and to test the potential pathogenic relevance in an animal model.
To this end myelin glycoproteins were purified from human postmortem brain and separated by two-dimensional electrophoresis. Binding of IgG from MS sera was screened by Western Blot. Mass spectrometry of immunoreactive spots identified contactin-2 as a novel target of the disease associated autoantibody response. Contactin-2 and its rat homolog TAG-1 (transiently-expressed axonal glycoprotein 1) are sequestered in the juxtaparanodal domain of myelinated axons, but are also expressed by adult cortical neurons as we show by immunohistochemistry. Contactin-2/TAG-1-specific autoantibodies were detected in both the sera and cerebrospinal fluid (CSF) of some MS patients and were shown to recognize native antigen on the surface of transfected cells by flow cytometry. This autoantibody response was accompanied by contactin-2-specific Th1/Th17 T-cell responses in peripheral blood as demonstrated by ELISPOT and proliferation assays. The pathogenic significance of these autoimmune responses was then explored in adoptive T cell mediated experimental autoimmune encephalitis (EAE) models in the rat. Adoptive transfer of TAG-1 specific T-cells induced an encephalitis characterized by extensive inflammation of white and gray matter tracts of the spinal cord and the cortex. Co-transfer of TAG-1 specific T cells with a myelin oligodendrocyte glycoprotein (MOG)specific mAb generated focal perivascular demyelinating lesions in the cortex similar to those reported in MS.
This study identifies contactin-2 as a novel autoantigen targeted by T cells and autoantibodies in MS and suggests that these responses may contribute to the development of gray matter pathology in this disease. Recently, there has been increasing evidence that CD8 T cells can participate in Multiple Sclerosis (MS) pathophysiology. In vitro and in vivo studies suggest that CD8 T cells may be involved in both the loss of myelin and axonal damage, the latter being regarded as a cause of permanent neurological impairment observed in MS. However, a directly damaging interaction between antigen-specific CD8 T cells and the central nervous system (CNS) has not yet been unambiguously documented.
We have therefore established an organotypic CNS slice culture system derived from transgenic mice expressing GFP+ myelin. This "ex vivo" approach allowed us to directly monitor the interaction between OT1 derived CD8 T cells and myelinated axons by confocal microscopic live imaging. Strikingly, we observed direct myelin damage mediated by CD8 T cells only when (1) slices had been pulsed with SIINFEKL peptide, the cognate antigen for OT1 T cells, and (2) T cells were activated. Furthermore, we detected a decrease of neurofilament at sites of repeated T cell-myelin contact. In some instances, we could even document the transsection of myelinated axons.
We have thus shown for the first time that myelin loss can be the result of a direct immunologic attack by CD8 T cells. Importantly, this damage was confined to an antigen-specific interaction and could not be observed in an unspecific set-up, e.g. with wildtype CD8 T cells or after pulsing the slice with control peptide. Accordingly, we suggest that the observed transsection of myelinated axons is composed of at least a two-step mechanism: first, an antigen specific process causing myelin damage by CD8 T cells, presumably by release of perforin/granzyme, and second, axonal injury as the result of "collateral damage". The high prevalence of autoimmune diseases in industrialized countries may, in part, be due to reduced early life exposure to common microbial agents and a more hygienic environment. To investigate this concept, we modeled the effects of neonatal exposure to a common pathogen-associated molecular pattern, lipopolysaccharide (LPS) in experimental autoimmune encephalomyelitis (EAE) by assessing LPS-mediated effects on neuroimmune parameters and ensuing neurological outcomes.
A single intraperitoneal exposure to LPS as neonates delayed the onset and reduced severity of MOG-induced EAE compared to littermate PBS-exposed mice (p b 0.05). T lymphocyte proliferation in response to MOG-exposed antigen presenting cells (APCs) was suppressed among T cells derived from LPS-exposed animals although there was a selective expansion of CD4 + FoxP3+ T cells mediated by APCs from the LPS-exposed group (p b 0.05). Splenic CD11c+ dendritic cells from LPS-exposed animals exhibited reduced expression of MHC Class II together with induction of CD80 and CD86 prior to and during EAE (p b 0.05). Spinal cord transcript levels from animals with EAE revealed both CD3-epsilon and F4/80 were lower in LPS-compared to PBS-exposed animals, which were associated with increased IL-10 levels in the LPS-exposed group (p b 0.05). Neuropathological studies disclosed reduced axonal and myelin loss in spinal cords from the LPScompared to the PBS-exposed animals with EAE, which was associated with increased FoxP3 immunoreactivity. Indeed, there was a robust inverse correlation between EAE severity and the number of FoxP3+ T cells detected in the spinal cord (p b 0.05).
These findings support the concept of early-life pathogen exposure biasing the immune system towards a more tolerant state with improved disease outcomes and might provide new insights for neuroprotective immunotherapies.
Kawachi Izumi⁎ 1 , Arakawa Musashi 1 , Yanagawa Kaori 1 , Nishizawa Masatoyo 1 1 Department of Neurology, Brain Research Institute, Niigata University, Niigata, Japan IL-17 plays a critical role in autoimmune and allergic conditions: disease progression of experimental allergic encephalitis is severely attenuated in IL-17-deficient mice and IL-17-secreting lymphocytes have been detected in the cerebrospinal fluids of multiple sclerosis (MS) patients, but their identity and properties remain elusive. Our objective is to characterize and evaluate the function of IL-17-producing cells in MS.
Peripheral blood was obtained from patients with relapsingremitting MS, other neuro-inflammatory diseases (ONID), and healthy volunteers (HS). We analyzed the expression of several chemokine receptors on the cell surface of peripheral mononuclear cells (PBMC), and detected intracellular IL-17 and IFN-g by flow cytometry. Within the CD4 T cell population (CD3+CD4+CD8−), the frequency of IFN-gproducing cells (TH1), IL-17-producing cells (TH17), and Foxp3expressing Treg cells in PBMC did not differ between MS, ONID, and HS. However, within the double negative T (DN T) cell population (CD3 +CD4−CD8−), IFN-g-producing cells decreased in frequency in PBMC from MS as compared to HS. More strikingly, IL-17-producing cells were more frequent in DN T cells from MS as compared to ONID and HS. Furthermore, we found that the sorted DN T cell population in MS had higher expression of transcripts encoding RORgt (RORC) than that in HS by the real-time quantitative RT-PCR. These results suggest that the distinct pool of IL-17-producing DN T cells in MS might be larger than that in ONID and HS. Innate T cells, including gd T cells, Va24i and Va7.2iT cells constitute a large percentage of DN T cells. In mice, it has been shown that innate T cells such as NK1.1negVa14iT and gd T cells have the ability to produce IL-17 in airway neutrophilia and other disorders. But in this setting, it was impossible that we detected gd T cells and Va24iT cells expressing IL-17 in PBMC from MS. Therefore we suspect that the production of IL-17 derived from not only TH17 per se, but also distinct lineage of innate T cells, except gd T and Va24iT cells, might be critical elements contributing to autoimmune disorders such as MS.
We identified the distinct population of DN T cells expressing IL-17 in MS. Future study should be performed to investigate the identification of innate T cell lineage with production of IL-17, and pathogenesis of the IL-17/IL-23 axis in MS.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS. Studies on experimental autoimmune encephalomyelitis have shown a pathological function of Th17 cells. Th17 cells have been recently identified as a further T effector cell type, producing the proinflammatory cytokine IL-17 and expressing the transcription factor RORgammat. However, their role in MS remains to be elucidated.
We examined the frequency of IL-17A-producing CD4+ T cells in the blood and the cerebrospinal fluid (CSF) of MS patients at different stages of the disease, and during remission and exacerbation. Our data demonstrate that while the frequency of Th17 cells in peripheral blood remains constant during the disease course the number of Th17 cells dramatically increases in the CSF during relapses. At the same time, we observed a significant decrease of Th1 cells in the CSF of patients in relapse. Th17 and Th1 T cell clones were generated from peripheral blood and CSF of MS patients. Such clones have been phenotypically analyzed by flow cytometry, and gene expression profiles obtained from microarray analysis and RT-PCR.
Altogether, our data suggest that Th17 cells have a high pathogenic potential and thus they are likely to contribute to the pathology of MS. We developed T cell receptor (TCR)-transgenic mice (RR mice) carrying a TCR specific for myelin oligodendrocyte glycoprotein (MOG) peptide 92-106 presented by I-As. RR mice on the SJL/J background spontaneously develop relapsing-remitting and, in fewer cases, chronic progressive experimental autoimmune encephalomyelitis (EAE).
Development of spontaneous EAE involves recruitment of MOG autoreactive B cells from the endogenous repertoire by transgenic T cells. Autoantibody formation is dependent on the presence of the MOG autoantigen and occurs only in TCR transgenic substrains prone to develop EAE. MOG reactive autoantibodies are secreted from B cells within the CNS and bone marrow and are directed against conformational MOG epitopes. We hypothesize that MOG is released from subclinical lesions and that autoantibodies are formed subsequently either in the local lymph node or directly in the CNS. Lesions in the CNS are characterized by massive demyelination and deposition of immunoglobulins; in vitro anti MOG antibodies activate complement leading to lysis of MOG expressing transfectants. Therefore, we consider the formation of MOG autoantibodies as a pro-pathogenic step which enhances severity of spontaneous EAE.
RR mice represent the first spontaneous animal model for the most common form of Multiple Sclerosis (MS), relapsing remitting-MS and are especially appropriate to study the spontaneous autoimmune T-B cell cooperation. MuSK antibody positive myasthenia gravis (MuSK-MG) has more prominent bulbar and facial involvement than AChR antibody positive myasthenia gravis (AChR-MG). MuSK antibodies appear to be predominantly IgG4 (non-complement fixing) although IgG1 and IgG2 antibodies are also seen at lower levels. However it remains unclear how these antibodies impair neurotransmission.
We immunised C57BL/6 mice with 30 μg recombinant human MuSK or phosphate buffer solution (with Freund's adjuvant). Two further boosts were then given. Some mice also received pertussis toxin at days 0 and 2.
The mice immunised with recombinant MuSK had antibody titres equivalent to those seen in the human disease. These MuSK antibodies were predominantly IgG1 (non complement fixing) and IgG2c (complement fixing) determined using an immunoflourescence cell based assay and ELISA. Moreover these antibodies appear to be able to activate complement in vitro. The MuSK immunised mice showed minimal limb muscle weakness on an inverted screen/horizontal bar although there was a trend towards impaired swallowing ability.
Ex vivo we found a modest but consistently significant reduction in both miniature endplate potential (MEPP) and endplate potential (EPP) amplitudes with preservation of quantal content (QC), confirming postsynaptic dysfunction. The mean MEPP was 0.96 mv (±0.03) in MuSK immunised group compared with 1.27 mv (±0.05) in the control group (p b 0.0001; unpaired t-test). Furthermore we identified a modest but significant reduction in both endplate area and length of the neuromuscular junctions in diaphragm muscle using fluorescently labelled a bungarotoxin. The mean area was 222.7 square micrometres (±4.98) in the MuSK immunised group compared with 290.2 square micrometres (±4.90) in the control group (p b 0.0001; Mann Whitney test).
These studies demonstrate that MuSK antibodies are able to activate complement in vitro, and to impair neurotransmission and cause morphological changes at the neuromuscular junction in vivo. This provides further evidence to their potential pathogenic role in causing MG.
3 -Anti-CD20 B cell depletion in immune intervention of central nervous system autoimmune disease
To investigate the immunological mechanisms associated with anti-CD20 mediated B cell depletion in experimental autoimmune encephalomyelitis (EAE).
Background: B cells and myelin-specific antibodies (ab) both play a pathogenic role in CNS autoimmune disease. Recent studies indicate that naïve B cells may also have a regulatory function. Anti-CD20 mediated B cell depletion, approved for treatment of B cell lymphoma and rheumatoid arthritis, may also reduce inflammation in multiple sclerosis (MS). To understand its immunomodulatory properties, B cell depletion was tested in EAE induced either by recombinant mouse MOG 1-117 (rMOG), which activates B cells and directs secretion of ab against conformational MOG or by its encephalitogenic T cell determinant MOG p35-55, a model that does not require B cells in its pathogenesis.
EAE was induced in human CD20-transgenic C57BL/6 mice in which anti-hCD20-ab (m2h7) depletes B cells. For EAE prevention, mice received weekly injections of m2 h7 or IgG2a-isotype starting 21 days prior to immunization. In reversal-experiments, mice were randomized to treatment at onset of paralysis.
Anti-CD20 treatment reversed paralysis induced by rMOG, which was associated with reduction in CNS inflammation, frequency of CNS B cells and myelin-specific ab. Frequencies of myelin-specific Th1 and Th17 cells, as well as FoxP3+ regulatory T cells (Treg), were reduced. Remaining antigen-presenting cells (APC) from B cell-depleted mice did not promote Th1 and Th17 differentiation of myelin-specific T cells. In contrast, B cell depletion exacerbated MOG peptide-induced EAE. While Treg were also reduced, Th1 or Th17 polarization was not altered. When used as APC, B cells from rMOG-, but not from MOG peptide-immunized mice stimulated naive MOG-specific T cells to proliferate to rMOG.
Anti-CD20 B-cell depletion has differential effects on MOG peptide-and MOG-protein induced EAE. In peptide-induced EAE, depletion of exclusively naïve B cells appears to be solely associated with a reduction of Treg. In protein-induced EAE however, depletion of myelin-specific B cells results in reduced frequencies of Th1 and Th17 due to the lack of T cell activation by residual APC. An important contribution of B cells and autoantibodies has been demonstrated in the pathogenesis of multiple sclerosis (MS) leading to interest in the use of such autoantibodies as diagnostic or prognostic biomarkers. Using a cDNA phage display library derived from MS brain plaques, we applied 'Serological Antigen Selection' to identify antigenic targets specifically interacting with antibodies present in the cerebrospinal fluid (CSF) of MS patients. A panel of 8 antigenic targets was identified which, after a detailed serological analysis on a large panel of individual MS patient and control CSF showed 86% specificity and 45% sensitivity in discriminating MS patients and controls. The objective of this study was to further characterize these novel antigenic targets and associated antibody biomarkers for MS.
Analysis of the immunoreactive profiles revealed that autoantibody reactivity was demonstrated in different MS subtypes and was found at an early stage in a subset of MS patients. Moreover, an increasing reactivity towards the antigenic targets with clinical disability was demonstrated.
Detailed bio-informatic analyses was performed on the 8 CSF-reactive epitopes which ranged in size from 11 to 121 amino acids. This revealed a novel antigen SPAG16. The remaining antigens constitute novel epitopes encoded by transcript variants of known genes. Several clones represented epitopes from cerebral proteins, or epitopes from novel cDNA sequences originating from brain libraries. Of note, 3 of 8 antigenic targets represented epitopes similar to viral proteins such as EBV.
At present, we have successfully expressed recombinant proteins for 4 of the antigenic targets and obtained synthetic peptides for the remaining candidates. Immunoreactivity for each purified recombinant protein and synthetic peptide was confirmed. We are currently extending our screening on these purified proteins and synthetic peptides. In addition, we have used the synthetic peptides and expressed proteins for monoclonal antibody production and are performing immunohistochemical staining on experimental autoimmune encephalomyelitis tissue with these monoclonal antibodies.
In conclusion, autoantibody profiles against epitopes derived from MS brain tissue could serve as diagnostic markers or form the basis for the identification of a subgroup of MS patients who may benefit from specific therapies. Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) in which the autoimmune response is directed to yet unidentified white matter antigens. A distinctive feature of MS is the presence of a local immune response within the CNS which give raise to IgG oligoclonal bands (OB) in the cerebrospinal fluid (CSF). One of the still obscure pathogenic aspects of MS concerns the identification of the antigen(s) targeted by IgG OB.
In order to identify the MS autoantigen(s) recognized by OB IgG present in the CSF, we have applied a proteomic approach coupled with immunoblotting. Human white matter proteins have been separated by bi-dimensional electrophoresis and transferred to nitrocellulose membranes, which have been probed with CSF IgG from 12 MS patients all showing CSF OB; for each case, we have separated IgG OB from non-OB IgG on agarose gels after isoelectrofocusing; the reactivity of each IgG fraction has been analyzed in parallel with whole CSF IgG by 2D immunoblotting. The presence of IgG OB-specific autoantigens has been then assessed by the comparative analysis of immune-reactive spots obtained with the two IgG fractions. Preliminary results showed that CSF OB IgG in all cases recognized a restricted number of neural protein isoforms; among these, a specific isoform of transketolase (TK) was recognized by CSF OB IgG of most MS patients. A more detailed analysis will provide a complete picture of the fine reactivity of OB IgG.
The results obtained indicate that TK is a major autoantigen in MS, since a specific TK isoform is the target of the autoimmune response in the CSF of about 50% of MS patients and here we showed that such molecule is specifically recognized by OB IgG. The identification of additional autoantigens in those cases not targeting TK will be instrumental to define the complete scenario of the molecular targets of the autoimmune response in MS. . We now tested whether local BAFF production is linked to local Ig production in MS lesions.
Local Ig production and expression of 18 TNF family members were quantified by qPCR in active and inactive MS lesions. We observed elevated transcripts of BAFF and its relative APRIL (a proliferationinducing ligand) in both active and inactive MS lesions. Local IgG production within MS lesions correlated strongly with local BAFF and to a lesser extent with APRIL transcription.
These results underscore the relevance of the BAFF/APRIL system for maintenance of B lineage cells in the chronic inflammatory process in the CNS of MS patients. Thus BAFF and APRIL are promising therapeutic targets. Gray matter disease in multiple sclerosis is poorly understood in part due to a lack of mouse model. We examined the role of CXCR6 in entry of T cells into the white and gray matter of the CNS during experimental autoimmune encephalomyelitis (EAE) with and without a secondary injury to the cerebral cortex.
We used intravital two-photon laser scanning microscopy in intravital mice to image the role of CXCR6 in gray and white matter disease in the spinal cord and brain. CXCR6-deficient mice were susceptible to EAE induced by MOG (35-55) peptide in Complete Freund's Adjuvant. At the peak of EAE, GFP+ T cells were predominantly CD4+, exclusively present in the spinal cord, and absent in the brain in both CXCR6gfp/gfp and CXCR6gfp/+ mice. In the spinal cord, GFP+ T cells were mostly restricted to the white matter with preferential locomotion along the rostral-caudal axis in both genotypes. During remission (Nday 20 EAE), IL-17 and interferon-γ producing T cells were N65% GFP+, whereas Foxp3+ cells were 20% GFP+ with no difference in the ratio of Foxp3+ to IL-17 producing T cells in both genotypes. Recruitment of GFP+ T cells to the gray matter was further evaluated by inducing a small laser injury in layer 1 barrel cortex during day 16-20 post EAE induction. Laser injury recruited GFP+ T cells into the cortical gray matter of the CXCR6gfp/+, but not, CXCR6gfp/gfp mice. CXCL16, the ligand for CXCR6, was induced in the spinal cord as well as the brain of the active (day 16) and recovering (Nday 20) EAE mice.
CXCR6 is necessary for entry of T cells in gray matter of EAE mice. We propose a two hit model for T cell infiltration in the cerebral cortex requiring both neuro-inflammation induced CXCL16 expression and local tissue injury.
The therapeutic potential of adult neural stem cells (aNSCs) has been suggested in several models of central nervous system (CNS) insult. aNSCs injected intravenously can migrate and home to damaged areas of the CNS. Previously, we analyzed the expression of integrin and chemokine receptors before and after in vitro differentiation of aNSCs, in an effort to elucidate their potential contributions to adhesion, migration and homing to damaged areas of the CNS. Here, we show elevated expression of CXCL12 [Stromal cell derived factor 1a (SDF-1a)] in inflamed areas of the CNS in mice with experimental autoimmune encephalomyelitis (EAE). We also show upregulated expression of CXCR4, the receptor for CXCL12, in differentiated aNSCs.
Studies are now focused on searching the source of CXCL12 in insulted CNS, and on the involvement of other molecules potentially involved in migration and homing.
Immunostaining analysis has shown that reactive astrocytes found in large number in the inflamed regions of EAE mice are the major source of elevated levels of CXCL12 in the inflamed spinal cord. We also found oligodendrocyte progenitor cells immunoreactive for NG2 and CXCL12 as another potential source of increased CXCL12.
Interestingly, the transcript of the non-classical cytokine, receptor activator of NF-kB ligand (RANKL) was found in the inflamed CNS compared to CNS of healthy mice. Further analysis revealed that the expression of Osteoprotegerin (OPG), the RANKL decoy receptor, was significantly induced following differentiation of aNSCs. The activating receptor RANK was not expressed by undifferentiated or differentiated aNSCs.
Studies on the effect of pro or anti-inflammatory cytokines on the differential expression of OPG and the involvement of CXCR4/CXCL12, CXCR5/CXCL13 and OPG/RANKL in the migration of aNSCs towards insulted areas of the CNS are now under investigation.
3 -Aberrant CXCR3 signaling due to CXCL10 deficiency impairs the anti-viral response to herpes simplex virus type 1
Carr Dan⁎ 1 , Wuest Todd 1 1 The University of Oklahoma Health Sciences Center, Oklahoma City, United States CXCL9 and CXCL10, chemokines that are chemotactic for NK cells, activated T cells, and dendritic cells are highly expressed during viral infection. We hypothesized that the absence of these chemokines would increase the sensitivity to infection. To test this hypothesis, a neurotropic pathogen, herpes simplex virus type 1 (HSV-1) was evaluated in mice deficient in CXCL9, CXCL10, the receptor for these ligands, CXCR3, or a combination of CXCL10 and CXCR3.
In the present study, following ocular herpes simplex virus-1 (HSV-1) infection, viral titers were elevated in the central nervous system (CNS) of CXCL10 deficient (CXCL10 −/−) mice. Moreover, CXCL10 deficiency resulted in a more severe phenotype in comparison to mice deficient in CXCR3 or CXCL9. Deletion of CXCL10 resulted in impaired recruitment of NK, dendritic, and antigen-specific CD8+ T cells to the nervous system. Although NK and dendritic cell mobilization were similar between CXCL10 −/− and CXCR3 deficient (CXCR3 −/−) mice, CXCR3 −/− mice did not exhibit any modification in mobilization of antigen-specific CD8+ T cells. The defect in trafficking of HSV-1specific CD8+ T cells to the CNS of CXCL10 −/− mice is not due to the absence of CXCL10 signaling through a chemokine receptor other than CXCR3. CXCL10 and CXCR3 double deficient mice exhibit no significant change in recruitment of HSV-1 specific CD8+ T cells compared to wild type control mice indicating the defect observed in CXCL10 −/− mice is a result of defective CXCR3 signaling.
There is a selective loss in resistance to HSV-1 infection in CXCL10 −/− mice directly related to the aberrant recruitment of HSV-specific CD8+ T cells to the nervous system and is dependent on CXCR3 expression.
Toft-Hansen Henrik⁎ 1 , Løbner Morten 1 , Owens Trevor 1 1
Leukocytes accumulate spontaneously in the perivascular space of transgenic mice that express the chemokine CCL2 (CCL2 Tg) in the central nervous system (CNS) under control of a myelin basic protein promoter. This is not associated with clinical pathology. We have previously shown that systemic injection of pertussis toxin (PTx) to these mice induces parenchymal infiltration across the glia limitans, and that this correlates to the development of encephalopathy with stupor-like symptoms and weight loss (Toft-Hansen et al., 2006, J Immunol). This experimental model allows us to dissociate events involved in leukocyte migration across the endothelial barrier and across the glia limitans, as two separate components of the neurovascular unit.
We have used laser microdissection followed by real-time RT-PCR on amplified RNA to determine gene expression by cells in the perivascular area in CCL2 transgenic mice treated with PTx. Since PTx causes recruitment of both monocytes/macrophages as well as T cells to the CNS of CCL2 Tg mice, we investigated if expression of CD11c and class II transactivator (CIITA), molecules related to antigen presentation, was regulated. We found lower levels (normalized to GAPDH) of both CD11c and CIITA expressed in PTx treated CCL2 Tg mice compared to PBS treated CCL2 Tg control mice. The expression by perivascular cells of cytokines IL-6, TNF-alpha and IFN-gamma was not altered by PTx, whereas IL-17 went from being detected at a relatively low level in only 1 of 6 control CCL2 Tg mice to being detected at higher levels in 5 of 6 PTx treated CCL2 Tg mice.
These results may reflect a differential participation of IFN-gamma and IL-17 expressing cells in migration across the neurovascular unit. To further analyze involvement of antigen presentation and T cell receptor specificity, we have crossed CCL2 Tg mice with 2D2 TCR Tg mice whose T cells are specific only for myelin oligodendrocyte glycoprotein (MOG). The double-transgenic offspring shows no signs of spontaneous clinical disease, suggesting that trans-endothelial migration of MOG-specific T cells to the perivascular space is not sufficient to induce encephalopathy. Prior studies indicate that CNS expression of the chemokine CXCL12 normally occurs along basolateral surfaces of the microvasculature, thereby controlling leukocyte access to the parenchyma. In patients with multiple sclerosis (MS) and in mice with experimental autoimmune encephalomyelitis (EAE), however, CXCL12 is relocated to lumenal surfaces of CNS venules, allowing egress of autoreactive leukocytes from perivascular spaces. As the relocation of CXCL12 highly correlates with the extent of perivascular infiltrates, we hypothesized that leukocyte-derived inflammatory mediators relocate CXCL12 via altered expression of CXCL12 receptors.
We examined CXCL12 relocation after intravenous administration of cytokines to naïve mice and in cytokine receptor-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG). We also identified perivascular leukocyte subsets during induction of EAE and determined whether leukocyte-derived cytokines affect the endothelial cell expression of the CXCL12 receptors CXCR4 and CXCR7. Examination of CNS specimens from MOG-immunized mice at preclinical time-points revealed that venular CXCL12 relocation occurs prior to the onset of clinical disease and that 100% of these venules contain perivascular CD3+, but not CD11b+, cells, which expressed IL-1β and were CD4+, CD8+ and γδTCR+. Administration of IL-1β, but not TNF-a, induced CXCL12 relocation in 90.1% of vessels and mice with targeted deletion of IL-1R did not relocate CXCL12 at the microvasculature. In contrast, MOGimmunized, TNFR1 −/− mice exhibited both relocation of CXCL12 and a predominance of perivascular γδT cells, suggesting a role for these cells in relocating CXCL12 at the BBB. In addition, bone marrow chimeric experiments revealed that loss of CNS IL-1R determines the severity of EAE. Finally, in vitro treatment of brain endothelial cells with IL-1β induced the expression of CXCR7 mRNA.
These results suggest that the CNS trafficking of γδT cells during autoimmunity leads to altered expression of CXCL12 at the BBB via increased endothelial cell expression of CXCR7. We studied five patients with acute axonal motor neuropathy (AMAN) and 2 patients with acute inflammatory demyelinating polyneuritis (AIDP). All patients had an antecedent Campylobacter jejuni infection and 6 patients had serum antibodies against GM1 and/or GD1a.
We isolated monocytes from 4 healthy subjects and 7 GBS patients. Immature dendritic cells expressing CD1 molecules, cultured with autologous T cells, were stimulated with LPS obtained from two CJ strains isolated from GBS patients and with GM1 or GD1a. The T cell response to LPS and to gangliosides was determined by the release of the proinflammatory cytokines IFNg and TNFa. In all patients a tight correlation existed between T cell reactivity to the gangliosides and individual antibody responses to the same ganglioside.
In conclusion, our findings provide the evidence of molecular mimicry also at the level of T cell responses, and suggest an important contribution of cellular immunity in the pathogenesis of GBS.
Guillain-Barré syndrome (GBS) is an immune-mediated polyneuropathy which typically develops after infection with Campylobacter jejuni. The antibodies that are produced to clear the infection cross-react with gangliosides present in peripheral nerves causing nerve damage and neurological disease. The polyreactive character of these antibodies as well as the lack of a memory response suggests a T-cell independent mechanism for B-cell activation. Since dendritic cells (DC) play an important role in the presentation of bacterial antigens and can regulate B-cell activity, we investigated whether lipooligosaccharide (LOS) antigens isolated from Campylobacter jejuni strains can activate DCs and subsequently induce B-cell proliferation.
Monocyte-derived DCs from healthy donors were stimulated with control Escherichia coli lipopolysaccharide (LPS) and with LOS from six different C. jejuni strains isolated from GBS patients. We observed that C. jejuni LOS was more effective than control E. coli LPS in upregulating the costimulatory molecules CD80, CD86, CD40 and HLA-DR indicating full DC activation. In addition C. jejuni LOS induced a dose-dependent secretion of the cytokines IL-6, IL-8, IL-10, IL-12p40 and TNF-alpha. LOS from C. jejuni did not enhance anti-IgM induced proliferation of mucosal B-cells, however when culture supernatant of DCs stimulated with C. jejuni LOS was added, B-cell proliferation was significantly increased. The effect of C. jejuni LOS was partly dependent on the presence of sialic acid since LOS from cst-II mutant strains as well as sialidase-treated LOS was less effective in inducing DC activation and B-cell proliferation.
Our results indicate that LOS from GBS-associated C. jejuni can activate DCs to produce soluble factors that subsequently enhance Bcell proliferation. This effect is partially dependent on the sialydation of LOS. Further unraveling of the cellular mechanism behind the antibody response causing GBS is necessary to develop novel strategies to limit disease progression and improve clinical outcome. antecedent infection. Intravenous immunoglobulin and plasma exchange are efficacious treatments. Recently, nerve growth factor (NGF) as a new drug is commonly used in many peripheral nerve disorders, including GBS, in China. We aimed to determine whether NGF was safe in Guillain-Barré syndrome, and to make preliminary observations of its efficacy.
We reported this study at two university hospitals, located in the west and north of China, respectively. The study was designed as a parallel group randomized controlled trial. GBS demyelination group 58 patients were divided into treatment group A (20 patients, 14/6) and controlled group B (38 patients, 18/20); GBS mixed group 53 patients were divided into treatment group C (32 patients, 16/16) and controlled group D (21 patients, 10/11); GBS axonal degeneration group 44 patients were divided into treatment group E (21 patients, 10/11) and controlled group F (23 patients, 13/10). Group A, C, E were treated with intravenous immunoglobulin and intramuscular NGF 9000AU (purityN = 98%, specific activityN = 5.0 × 10^5 AU/mg protein) daily for up to 8 weeks. Group B, D, F were only treated with intravenous immunoglobulin. Patients' neurological function and electrophysiology test were evaluated at the beginning and end of the treatment. The rate and extent of recovery and motor nerve conduction velocity (NCV) in group A were more better than those in group B (P b 0.05). Recovery rate, full recovery rate, motor NCV and motor nerve wave range in group C were greatly better than those in group D (P b 0.05). Clinical recovery rates and full recovery rates showed no obvious difference between group E and F (P N 0.05), but motor nerve wave range in group E demonstrated obviously higher than that in group F (P N 0.05). In addition, no serious adverse events were found.
Nerve growth factor, as a kind of safe treatment, possesses significant effects on the recovery of demyelination and axonal degeneration in GBS. Circulating nucleic acids (CNAs) have been shown to have diagnostic utility in human diseases. The aim of this study was to sequence and organize an extensive catalog of CNAs and compare the normal range to CNAs from patients with Acute Multiple Sclerosis (AMS).
Serum DNA from 34 otherwise healthy humans and 13 AMS patients was extracted, amplified, sequenced using pyrosequencing (454/Roche) and categorized by: 1) origin (human versus xenogeneic), 2) functionality (repeats, coding or non-coding genes), and 3) chromosomal localization and genomic frequencies.
3.1e5/8.9e4 (normals/AMS) sequences were derived from a total of 5.1e7/1.6e7 nucleotides, of which 84%/79% was attributed to known database sequences. Approximately 99% of the known sequences were genomic and 1% were xenogeneic (bacterial or viral). Of the genomic sequences, 34% originated from repeats and 40% from annotated genes, which is within the expected range. Interestingly, coding sequences were significantly underrepresented (0.25% ± .07% vs 1.25%; p b .001). AMS patients comprised an overrepresentation of sequences from HS17 and ALU sequences (p b 0.001), reaching an odds ratio of 90 (p b 0.001; c-index: 0.94). Unexpectedly, one normal was found to have HBV genotype F sequences.
Whereas the distribution of CNA was mostly comparable to the genome, the under-representation of CDS argues strongly against a solely apoptotic origin of CNAs. The results for HS19 and ALU, already found to be associated with MS, could open a diagnostic horizon using this technology, as well as the capability for uncovering occult infections.
Vosslamber Saskia⁎ 1 , van der Voort Laura 1 , van Baarsen Lisa 1 , Heijmans Roel 1 , Crusius Bart 1 , Killestein Joep 1 , Polman Chris 1 , Verweij Cor 1 1
Multiple sclerosis (MS) is a heterogeneous disease, reflected by a differential response to therapy, in particular IFN-b. Previously, a heterogeneous pharmacological response to IFN-b therapy in RRMS patients was described. Evidence was provided that the baseline status of the IFN type I activity negatively correlates with the pharmacological effects of IFN-b treatment.
It is hypothesized that the inter-individual differences in the IFN type I response program at baseline, and concomitant variation in IFNb responsiveness may be the result of inter-individual variation in IFNb biology. In this study we investigated the role of genetic variation in IFN signaling components in relation to the heterogeneous pharmacological response to IFN-b.
To determine biological response, peripheral blood was collected from 30 RRMS patients before and during IFN-b therapy. From 20 untreated RRMS patients peripheral blood was collected at two time points over a three to twelve month time period to analyze the stability of baseline values over time. Baseline stability and biological response rate, were analyzed by Taqman Low Density Arrays (TLDA) using the mean gene expression level of a set of 10 IFN type I response genes. Genetic variation was determined in several components of the IFN signaling cascade using Taqman Genotyping Assays and conventional genomic PCR. The ratios of gene expression levels before and during therapy were compared with the occurrence of genetic variation in components of the IFN signaling pathway.
The extent of the biological response correlated negatively with the baseline expression of the type I IFN response gene set (R = −0.3891; P = 0.0336). The baseline stability over time was reflected by a correlation efficient of r = 0.54; P = 0.029 (n = 20). Next we determined the association of genetic variation in IFN signalling components with IFN type I response gene activity at baseline and after pharmacological intervention with IFN-b. This analysis revealed a significant association between genetic variation in one of the IFN signalling components and baseline IFN type I response gene expression (P = 0.0198). Accordingly, a significant reduced biological response was observed for patients who contained the genetic variant (P = 0.0057).
Variation in the IFN signaling may determine (part) of the heterogeneity in the pharmacological and clinical response to IFN-b in RRMS. Multiple sclerosis (MS), and its mouse model EAE, are autoimmune (AI) diseases that depend on both genetic and environmental factors. Shared immunopathology genes, e.g., tyrosine kinase-2 (Tyk2), may also be shared AI disease genes. B10.DI-tyk2 mice have a single 2538 G to A missense mutation in tyk2 making them AI arthritis resistant and T. gondii susceptible, unlike the wild-type B10.Q/Ai (B10.Q/Ai-Tyk2) substrain.
We induced EAE in B10.DI-tyk2 and B10.Q/Ai-Tyk2 mice with MOG79-96-CFA using a double injection protocol (2× MOG-CFA). B10. Q/Ai-Tyk2 mice were resistant (0/46) to EAE while B10.Q/Ai-Tyk2 (31/ 36) and reciprocal F1 hybrids (42/51) were susceptible (p b 0.0001). Antigen specific production of RANTES, IL-6 and IFN-gamma was specifically increased in B10.Q/Ai-Tyk2 and F1 mice. Thus, tyk2 is a shared AI susceptibility gene, and EAE can be complemented genetically by one wild-type Tyk2 allele. Next, we tested whether EAE in B10.DI-tyk2 mice could be complemented by environmental factors using MOG79-96-CFA + pertussis toxin (PTX) (1× MOG-CFA + PTX). B10.DI-tyk2 mice immunized with 1× MOG-CFA + PTX were significantly more susceptible to EAE than 2× MOG-CFA immunized mice (33/37 vs. 0/46; p b 0.0001). A large number of Th1 and Th17 cytokines and chemokines increased when PTX was included as an ancillary adjuvant. In contrast only IFN-gamma, IL-6 and RANTES changed significantly with both genetic and PTX complementation.
In summary, tyk2 is both a shared immunopathology and AI disease susceptibility allele that can be complemented both genetically and environmentally. Such SNPs are of considerable significance given the potential role of gene-by-environment interaction in MS susceptibility. The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of the peripheral nervous system which is characterised by proximal muscle weakness and autonomic dysfunction. Approximately 60% of patients have an associated small cell lung carcinoma (SCLC), whilst approximately 90% of patients have autoantibodies to voltage-gated calcium channels (VGCC). These transmembrane proteins are located at the presynaptic nerve terminal, in the cerebellum and on the surface of the SCLCs. It has been postulated that antibodies raised against determinants on the tumour may cross react with the VGCC at the nerve terminal resulting in the neurological dysfunction, and such antibodies may convey a survival advantage to these patients.
One hundred consecutive, unselected patients with biopsy-proven SCLC were recruited at time of tumour diagnosis from hospitals within the Trent region of the UK and assayed for autoantibodies to VGCC and onconeuroantigens. The survival rates of patients with paraneoplastic neurological syndromes or positive onconeural antibodies and SCLC, and patients with SCLC alone were compared.
Overall, 10 patients (10%) had positive VGCC antibodies. Eight patients had P-/Q-type VGCC antibodies (range 70-1101 pM), one had low levels of N-type VGCC (97 pM), whilst one had both P-/Q-type and N-type VGCC antibodies (795 and 69 pM respectively). Of these ten patients, only four had typical clinical and electrophysiological features of LEMS. A further 6 patients had onconeural antibodies (Hu antibodies in 2 patients and Hu and Ri, Ri, amphiphysin, and Ma2 in four others). The median survival of the four antibody positive LEMS patients (19.6 months, p = 0.038, unpaired two-way t-test) was considerably greater than that for the antibody negative (8.9 months) or all antibody positive patients (10.5 months).
Although preliminary, these results indicate that the presence of functionally active autoantibodies confers an increased survival advantage compared to antibody negative patients. Additionally, the VGCC antibody-positive LEMS survive longer than patients with similar VGCC autoantibodies but without neurological symptoms. In 50% of the patients with Lambert-Eaton myasthenic syndrome (LEMS) a small cell lung carcinoma (SCLC) can be found. We reported Sox-1 antibodies to be specific and rather sensitive (64%) for paraneoplastic LEMS by phage display screening. This study reports preliminary results of a more amenable assay system.
We cloned the Sox-1 gene and used recombinant Sox-1 protein in an ELISA to titrate serum samples. We tested 12 healthy controls, 11 patients with SCLC, 46 patients with LEMS without a SCLC and 45 patients with SCLC and LEMS. A sample was considered positive if the OD value was at least 0.1 and more than three SD over the average OD of healthy controls.
In SCLC-LEMS patients 35/45 (76%) tested positive. Of the twentynine samples tested in both assays, twenty-three tested positive in ELISA and seventeen (58%) by phage display. In our ELISA, positive values were found in 17% of the LEMS without SCLC and 9% in SCLC. Median and average OD values were 0.62 and 1.46 for SCLC-LEMS, 0.08 and 0.09 for LEMS without SCLC and 0.05 and 0.05 for HC, respectively. High titers above an OD of 0.4 were found only in the SCLC-LEMS group (26/45: 58%), while none tested strongly positive in the other two groups (p b 0.0001). To rule out false positives an immunoblot can be useful to confirm low OD titers.
In five patients Sox-1 and VGCC-titers were available before and after treatment; in all titers were strongly positive before treatment and all were diminished after treatment. VGCC became negative in two.
High titer Sox-1 antibodies are highly specific for LEMS patients with SCLC. An ELISA using recombinant protein is as sensitive and specific as a more elaborate phage display assay.
Biollaz Gregoire 1 , Bernasconi Luca 1 , Cretton Christine 1 , Püntener Ursula 1 , Frei Karl 1 , Fontana Adriano 1 , Suter Tobias⁎ 1 1
Glioma are among the most fatal tumors. This has been attributed to immunosuppressive features of both the tumor and the CNS. However, the relative contribution of either the glioma or its localization has not been investigated.
We report here that the syngeneic GL261 glioma triggers a protective immune response only when growing subcutaneously, despite the fact that also intracerebrally grown gliomas are infiltrated by DC and T cells. This failure to control intracerebral gliomas correlates with increased immunosuppressive conditions in intracerebral tumors: tumor infiltrating dendritic cells from intracerebral gliomas are not able to stimulate T cell proliferation in vitro; brainlocalized GL261 gliomas are characterized by significantly higher numbers of Foxp3+ regulatory T cells and higher expression of TGF-b1 and TGF-b2 mRNA when compared to GL261 gliomas in the skin. Moreover, we show that DCs from intracranial tumors induce in vitro higher numbers of regulatory T cells than subcutaneous tumor DCs.
Our data show that not the tumor but its location dictates the efficiency of the anti-tumor immune response.
Mesenchymal stem cells (MSC) are a promising therapeutic tool for autoimmune diseases because couple immune-suppressive properties with multi-differentiation potential. Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by widespread demyelination and axonal loss. The aim of this study was the assessment of the clinical efficacy and the molecular mechanisms of adipose-derived MSC (A-MSC) in chronic EAE.
We administered A-MSC to treat MOG35-55 induced EAE in C57BL/6J mice by two distinct experimental protocols. The intravenous administration of A-MSC before disease onset (preventive protocol) significantly ameliorated the clinical course and decreased spinal cord inflammation and demyelination. The effect was mostly due to peripheral impairment of T cell function, as GFP+ A-MSC migrated and persisted into lymphoid organs inhibited T cell proliferation and production of pro-inflammatory mediators. In addition, a significant number of A-MSC was also found within inflamed spinal cord, where a small proportion underwent glial differentiation. In addition to the preventive effect, A-MSC significantly reduced EAE clinical and pathological scores when administered after disease stabilization (25-28 days post-immunization; therapeutic protocol), with a significant reduction of demyelination and axonal loss. In this experimental condition, migration of A-MSC to lymph nodes as well as ex vivo inhibition of T cells was significantly reduced. The A-MSC administered by the therapeutic protocol efficiently migrated into the inflamed CNS; within demyelinated areas, A-MSC infiltration was accompanied by increased number of oligodendrocyte progenitors of donor origin. A significant subset of A-MSC expressed alpha4 integrins and anti-alpha4 antibody inhibited their penetration into inflamed CNS of mice with established disease.
Taken together our data suggest that A-MSC produce a beneficial effect in chronic EAE by a bimodal mechanism, similarly to neural precursor cells. Depending on the inflammatory background, these cells may inhibit the autoimmune response in lymphoid organs in early disease stages and/or promote local neuroregeneration when administered during chronic phase of disease. We previously demonstrated that systemic infusion of mesenchymal stem cells (MSC) induces peripheral tolerance against encephalitogenic myelin antigens. However it is still unknown whether systemic delivery of MSC could affect the immune response inside the central nervous system (CNS) and if local delivery (intra-cysternal, i.c.) may result in a significant clinical advantage.
Thus, we compared the effect of MSC injected systemically or i.c. in myelin oligodendrocyte glycoprotein (MOG) immunized experimental autoimmune encephalomyelitis (EAE) affected C57Bl/6J mice. We observed that both route of administration led to similar improvement of clinical and histology scores. No significant evidence of transdifferentiation was observed in both groups. Systemic infusion of MSC induced an increase of CD4+ FoxP3+, T regulatory cells and a decrease of Th17 effector cells isolated from the CNS of treated mice compared to controls.
Thus, the immunomodulatory activity of systemically infused MSC occurs not only through the induction of peripheral tolerance but also through the inhibition of migration of effector cells and the expansion of T regulatory cells in the target organ. We evaluated the MSC-mediated effects in MOG-induced experimental autoimmune encephalomyelitis by histology and molecular analysis of central nervous system specimen showing that they decreased axonal sufferance through the inhibition of astrogliosis and microglia activation and the upregulation of genes involved in the BMP and Notch pathways.
Finally, we demonstrated that MSC express arginase, an enzyme that plays important roles in several biological processes including nitric oxide (NO) production by limiting the pool of arginine available for NO synthase. As consequence, N9 microglial cells, cultured in the presence of MSC, decrease NO production.
These findings suggest that MSC therapeutic effect may be the result of an impaired arginine metabolism.
Thus, irrespective of the lack of neural differentiation, MSC exert a remarkable neuroprotective effect in EAE. The University of Texas Health Science Center at Houston, Houston, Texas, USA Magnetic resonance imaging (MRI) was first used to image living subjects with multiple sclerosis (MS) ∼25 years ago. Despite limitations of early relatively low field scanners and crude imaging sequences it was immediately evident that MRI provided an important portal to the pathology of this disease; pathology that previously could only be adequately sampled once in a patient's lifetime. Early studies based completely on the constraints placed on the relaxation properties of water molecules imposed by the structural characteristics of their microenvironment provided an early look at well established MS lesions or plaques. The introduction of paramagnetic contrast agents and their use in longitudinal serial scanning of patients unveiled the very dynamic nature of lesion formation and evolution over time. MRI has secured an important place in the diagnosis of the disease, in monitoring the evolution of the disease process and in the investigation of the potential benefit of new treatments for the disorder. MRI also provides important information for the categorization of disease activity that may be crucial for understanding the results of investigations into the aberrant immune responses of MS patients; phenomena that appear to be fundamental to our current understanding and approaches to manage this potentially devastating disorder. At least in the early clinical phases of MS, MRI not only aids in securing the diagnosis of the disease, but also enables short-term prognosis as to the disease's course. While the specificity of MRI findings for the pathologic changes that are present in affected tissues places some limitations on the correlation between quantitative MRI findings and clinical disability that currently constrains the use of MRI is a true surrogate, they have not undermined the ability of serial MRI to measure the biological efficacy of new therapeutic agents. Central issues that are actively being addressed include whether composite measures of conventional MRI parameters can provide better concomitant and predictive correlations with clinical disease status in the short or long term, if better pulse acquisitions or higher field imaging can better delineate cortical pathology and its evolution, and do the more global changes in brain pathology as seen with advanced MRI approaches reflect distributed change in neuronal pathways that are the consequences of classic focal lesions or do they bespeak an independent neurodegenerative process. Subtle changes in T cell effector function are clinically critical, e.g. in autoimmune disease, allergy, and insufficient immune responses to cancer. As the foundation for the design of molecular therapeutics it is thus important to understand, how modest changes in receptor engagement on the T cell surface are processed under varying physiological circumstances in the complex T cell signaling network to regulate T cell effector function. The ability of signaling intermediates to interact with each other in such networks is encoded by extensively studied biochemical properties. In addition and much less well understood, the concentrations of signaling intermediates inside live cells vary in time and space. We refer to these variations as 'spatiotemporal patterns'. They are critical for how the efficiency of signaling is regulated in live cells, as enrichment of two proteins in the same location at the same time increases their interaction probability. Spatiotemporal patterning needs to be studied in a systems approach. The spatiotemporal patterning of an individual signaling intermediate only becomes of interest when it can be compared to the patterns of multiple other components of the same network. First, we have therefore determined the spatiotemporal patterns of 26 elements of T cell activation in the activation of primary T cells by antigen presenting cells. The most striking finding was that every signaling intermediate had a unique spatiotemporal pattern. Such diversity of spatiotemporal patterning is of critical significance. Since spatiotemporal patterning differed dramatically between signaling intermediates, the likelihood of signaling intermediates to interact with each other varies substantially in time and space during T cell activation; spatiotemporal patterning has to be a significant regulator of T cell activation. Second, we have provided causal support for this hypothesis. Addressing a particular signaling intermediate, the central regulator of cell polarity Cdc42, we have shown that subcellular localization not overall cellular activity is most critical for Cdc42 function. Together, these data establish that the regulation of spatiotemporal pattering in T cell activation is a critical element in the control of the efficiency of T cell activation. Molecular therapeutics targeting such regulation may be of great utility in modulating T cell activation in autoimmune disease, allergy, and insufficient immune responses to cancer.
How MR-based technology is changing our views on the pathophysiology of multiple sclerosis
Neuroimaging Research Unit, Department of Neurology, Scientific Institute and University Ospedale San Raffaele, Milan, Italy A dramatic paradigm shift is taking place in our understanding of the pathophysiology of multiple sclerosis (MS). An important contribution to such a shift has been made possible by advances in magnetic resonance imaging (MRI) technology, which allows accurate and quantitative estimates of structural central nervous system (CNS) damage to be obtained from patients with MS and to follow its evolution over the course of the disease with high sensitivity.
In the past few years, the use of modern quantitative MR techniques has indeed reshaped the picture of MS leading to the formulation of the so called "axonal hypothesis" (i.e., changes in metabolism, morphology, or density of the axons are important determinants of functional impairment in MS). In this context, metrics derived from magnetization transfer and diffusion-weighted MRI enable us to quantify the extent of structural changes occurring within T2-visible lesions and normal-appearing tissues (including gray matter) with increased pathological specificity to irreversible tissue damage over conventional MRI. Proton MR spectroscopy adds valuable pieces of information on the biochemical nature of such changes. Finally, functional MRI is providing new insights into the role of cortical adaptive changes in limiting the clinical consequences of MS-related irreversible tissue damage. As a consequence of the extensive application of these MR techniques our current understanding of the pathophysiology of MS is that it is not only a disease of the white matter, characterized by focal inflammatory-demyelinating lesions, but is also associated to more subtle and diffuse damage throughout the white and gray matter of the entire CNS. The inflammatory and neurodegenerative components of the disease process are present from the earliest clinical phases of the disease, but appear to be, at least partially, dissociated. In addition, recovery and repair play an important role in the genesis of the clinical manifestations of the disease, involving both structural changes (e.g., clearance of inflammation and remyelination) and plastic reorganization of the cortex. This new picture of MS has important implications in the context of treatment options, since it suggests that agents that protect against neurodegeneration or promote tissue repair may have an important role to play alongside agents acting on the "more classical" inflammatory component of the disease. Anti-GQ1b ganglioside antibodies are found in the acute phase sera of more than 90% of the Miller Fisher Syndrome (MFS) patients. These antibodies are believed to be the pathogenic factors leading to neuromuscular paralysis. We have previously shown in an in vitro mouse model using diaphragm muscle-nerve preparations that anti-GQ1b antibody binds presynaptically at the neuromuscular junction and activates the complement system, culminating in the formation of the membrane attack complex (MAC; C5b-C9). Functional and electrophysiological analyses showed block of synaptic transmission at the neuromuscular junction, after an initial period of a dramatically high and asynchronous release of acetylcholine, and associated paralysis of the muscle. Morphology showed MAC deposition at the neuromuscular junction and neurofilament degradation. Since complement involvement with the formation of MAC is an important factor in membrane injury in general, it forms an attractive pharmacotherapeutic target.
We have tested two experimental drugs that are able to inhibit the complement cascade at the level of C5, without affecting the immunoprotective role of upstream complement components. Eculizumab (Alexion Pharmaceuticals) is a humanized mouse monoclonal IgG that specifically binds human C5 and blocks its cleavage into C5a and C5b, the latter being the first component of MAC. rEV576 is a recombinant form of a soft tick saliva protein, which also effectively inhibits C5 cleavage in humans. We used our mouse model for MFS to assess the efficacy of these drugs in an antibody-mediated neuropathy.
The results demonstrate that both drugs are effective blockers of MAC formation in the in vitro mouse model and thus completely prevent the development of neuronal damage and associated paralysis. Eculizumab was also tested in an in vivo mouse MFS model, generated through intraperitoneal injection of anti-GQ1b antibody, followed by normal human serum as a source of complement. While untreated mice developed respiratory paralysis due to transmission block at the diaphragm neuromuscular junctions and showed functional and morphological hallmarks of terminal motor neuropathy, intravenous injection of eculizumab was found to protect mice against complement-mediated damage in murine MFS.
Narcolepsy is a lifelong, debilitating disease caused by a deficiency in the orexin/hypocretin signalling pathway in the brain. The orexinexpressing neurons are located in the lateral hypothalamus and play an important role in sleep regulation and feeding. Orexin is absent from the CSF of N90% of patients with narcolepsy/cataplexy and postmortem studies have demonstrated that the neurons are absent in narcoleptics. The disease is very highly associated with the MHC class II allele, DQB1⁎0602, suggesting that the condition is immune-mediated.
Our hypothesis is that the destruction of the orexin neurons is caused by autoimmunity and more specifically by T-cells that cross the blood brain-barrier and target the orexin expressing cells. Our aim was to generate an animal model of narcolepsy by immunizing mice that express human MHC class II molecules with a peptide derived from preproorexin (Orx 1-14). This peptide had previously been shown to bind to DQB1⁎0602.
Mice (females 10-12 weeks old) were immunised subcutaneously with 100 μg of the peptide or PBS in Complete Freunds Adjuvant (CFA). 500 ng of Pertussis toxin (PTX) was injected intraperitoneally at day 0 and 48 h post-immunisation in all animals. 9 days after immunisation, lymph node derived T-cells were tested in proliferation assays.
Orx 1-14 peptide was very potent in activating the cells in vitro and the Orx 1-14 sensitive T cells were DQ6 restricted. Groups of immunised and control animals were tested for sleepiness and motivational status for up to 12 weeks after immunisation. No significant differences were observed between the experimental and control groups. Finally, brains from various stages after immunisation (day 9, day 21, and week 8) were analysed for numbers of orexin neurons in the hypothalamus, T-cell infiltration and for markers of immune and neuronal activation. No neuronal loss or activation was observed and practically no T-cells entered the brain.
We observed strong peripheral T-cell activation, restricted to DQ6. However, this immune activation had no effect on the behaviour of the animals or generated any brain pathology. This lack of symptoms may be explained by the fact that the T-cells in the periphery did not cross the blood-brain barrier (BBB) despite the use of PTX as an adjuvant. Further immunisation protocols are in progress. The pathogenesis of inflammatory autoimmune diseases of the peripheral nervous system leading to demyelination and/or axonal damage is still incompletely resolved. In particular, it is controversial to which extent (i) the autoimmune-mediated destruction of peripheral nerves results in secondary damage of the central nervous system and (ii) CD4 and CD8 T cells contribute to the disease.
To address these questions we applied the murine model of P0106-125 induced experimental autoimmune neuritis (EAN). Immunization of C57BL/6 mice with P0106-125 resulted in severe axonal damage and mild demyelination. Importantly, mice developed a dying back axonopathy with apoptosis of a large fraction of neurons in the anterior horn of the lumbar and thoracic spinal cord and a progressive neurogenic muscular atrophy. T cell depletion experiments identified CD4, but not CD8 T cells as decisive mediators of EAN. CD4 T cells were the major cellular source of antigen-specific Interferon-γ and Interleukin-17 production, regulated the number of tumor necrosis factor+ and inducible nitric oxide synthase+ macrophages in the diseased sciatic nerve, mediated axonal damage, and, subsequently, neuronal apoptosis and neurogenic muscular atrophy. In contrast, demyelination of peripheral nerves was only slightly ameliorated in CD4 T cell-depleted mice.
In conclusion, P0106-125-induced EAN is a CD4 T cell-mediated autoimmune disease affecting both the peripheral and central nervous system. These observations have implications for the pathogenesis of autoimmune T cell-mediated diseases of peripheral nerves, and clinically relevance for the development of specific therapeutic strategies aiming to inhibit the autoimmune attack against peripheral nerves and to prevent a dying back axonopathy, in particular, by conferring axonal protection, which is of upmost importance for the final clinical outcome.
Kiyozuka Tetsuhito⁎ 1 , Fujioka Toshiki 1 , Kudeken Tsukasa 1 1 Toho University, Tokyo, Japan
To investigate the effect of statin on experimental autoimmune neuritis (EAN) atorvastatin was given orally to EAN rats daily. EAN was induced in female Lewis rats by injecting synthetic peptide of bovine myelin P2 protein with CFA. EAN rats fed with vehicle only were used as control. Clinical signs were assessed daily using 8 grade-score system. Cauda equina (CE) were removed at 4, 10, 12, 14, 18, 21, and 24 days post-immunization (DPI). mRNA expression of IFN-gamma, IL-10, IL-12 p35, IL-17 and IL-23 p19 in the CE was studied using semiquantitative RT-PCR. Both groups developed EAN at 12 DPI simultaneously although atorvastatin-treated rats showed milder signs and earlier recovery. Mononuclear cell infiltration seen in the CE of atorvastatin-treated rats was also milder than that of control rats. RT-PCR revealed that IFN-gamma and IL-12 p35 mRNA expression at 10 DPI and IL-10 and IL-17 mRNA expression at 12 DPI in atorvastatintreated rats were decreased compared to the control rats. Conversely IL-23 p19 mRNA expression through 14 DPI and thereafter in atorvastatin-treated rats was increased compared to the control rats. In EAN atorvastatin suppressed the neurological deficit and Th1 cytokines although reciprocal up-regulation of Th2 cytokine was not seen. Moreover suppression of IL-17 was observed in atorvastatintreated rats with up-regulated IL-23 p19 mRNA.
Out data suggest that, in EAN, atorvastatin suppressed Th1 and Th17 activity irrelevant to Th2 cytokine up-regulation or IL-23 p19 suppression. Previous studies have shown that anti-ganglioside GD1a antibody causes neuromuscular presynaptic blocking on mouse phrenic nervediaphragm preparations. The aim of this study was to determine whether anti-GM1-or anti-GD1a-positive sera affect neuromuscular transmission in human limb muscles.
Axonal-stimulating single fiber electromyography was performed in the extensor digitorum communis muscle of four patients with acute motor axonal neuropathy (AMAN) with anti-GM1 and GD1a antibodies and four with acute inflammatory demyelinating polyneuropathy (AIDP). Anti-ganglioside antibodies were measured by ELISA. Anti-GM1 or GD1a antibodies were positive in all four AMAN patients and in none of AIDP patients. All AMAN patients showed normal jitter and no blockings in the acute phase of the disease (within 2 weeks after onset). In AIDP patients, jitter was normal in three and slightly increased in the remaining one.
Neuromuscular transmission does not appear to be impaired in AMAN patients with anti-GM1/GD1a antibodies, and this suggests that these antibodies do not affect neuromuscular transmission in human limb muscle. Our results failed to support the findings in experimental studies in the mouse phrenic nerve. Anti-ganglioside antibodies would involve the nodes of Ranvier of distal motor axons near the motor nerve terminals, but not the neuromuscular junction itself. Early studies evidenced the presence of inflammatory infiltrates in the muscles of some facioscapulohumeral muscular dystrophy (FSHD) patients. In order to evaluate the contribution of inflammation to FSHD pathogenesis and progression in this study we evaluated in peripheral blood mononuclear cells (PBMC) from FSHD patients and controls the percentages of pSTAT1, T-bet and pSTAT3-positive CD4+, CD8+ and CD14+ cells. Moreover we correlate the percentage of T-bet, pSTAT1 and pSTAT3 expressing cells with PBMC production of IFNg, IL6, IL10, TNFa, IL12, TGFb and disease severity, evaluated by Magnetic Resonance Imaging (MRI) and clinical examination.
In our study we included 25 FSHD patients and 15 sex and age matched controls. Detection of pSTAT-1, pSTAT-3 and T-bet was performed by intracellular flow cytometry. The production of IFNg, IL6, IL10, TNFa, IL12, TGFb by PBMC was measured by ELISA. No difference was found in the percentage of circulating CD4+pSTAT1+, CD8+pSTAT1+T cells and CD14+pSTAT1+ cells, CD4+pSTAT3+, CD8+pSTAT3+T cells and CD14+pSTAT3+ cells, CD4+T-bet+, CD8+T-bet+T cells between FSHD patients and controls while a higher percentage of CD14+T-bet+ cells was observed in FSHD patients than in controls. We found higher IFNg, IL6, TNFa, IL10 and IL12 levels in FSHD patients than in controls while no difference was observed in TGFa levels.
When we divided FSHD patients in two different groups according to MRI parameters, a higher percentage of CD8+pSTAT1+ and CD8+T-bet+T cells and CD14+pSTAT1+ cells, CD14+pSTAT3+ cells and CD14+T-bet+ cells was found in peripheral blood of FSHD patients with inflammatory pattern than in patients without inflammatory pattern and in controls. In addition the number of CD8+pSTAT1+ and CD8+T-bet+T cells correlated with MRI-T2 score. FSHD patients with MRI inflammatory pattern showed significant higher IFNg, TNFa and IL12 levels than both FSHD patients without inflammatory pattern and healthy subjects. IL6 and IL10 production was higher in FSHD patients with inflammatory pattern then in FSHD patients without inflammatory pattern and both groups of patients showed higher IL6 and IL10 levels than controls.
Our data suggest that inflammation, mainly mediated by CD8+T cells, may play an important role in FSHD promoting the muscle pathology and favouring the disability progression. The thymus has been implicated as a possible site of origin that triggers autoimmunity such as myasthenia gravis (MG). Several groups have previously reported the analysis of regulatory T cells (Treg) in MG thymus. However, certain pattern in the changes of T cell subsets has not been revealed.
To examine the pathogenesis of MG, we analyze what patterns of Treg and IL-17 producing T cells (Th17) are observed in the thymus between the specimens from MG (MG+) and non-MG (MG-) humans.
Thymic tissue consisted of MG-group (child thymus with no immunological diseases, n = 6; adult thymus with no immunological diseases, n = 5; and adult thymus with thymoma, n = 5) and MG+ group (adult thymus with no neoplasmas, n = 9; adult thymus with hyperplasia, n = 6; adult thymus with thymoma, n = 6). Child and adult thymi without immunological diseases were obtained at cardiac surgery. In the samples of thymoma, nonneoplastic thymic tissues adjacent to thymoma were analyzed. We performed immunohistochemical analysis of thymic sections. To visualize Treg and IL-17 producing cells, antibodies specific for Foxp3 and IL-17 were used in combination with anti-CD4 antibody. All data were obtained with Leica confocal microscopy. Quantification of Foxp3 positive cells and IL-17 signals was measured by using ImageJ software. This study was approved by the Ethics Committee of University of Tokushima.
1) The number of Foxp3 positive cells and the pixels of IL-17 signals in thymic medulla were higher than those in the cortex. 2) The number of Foxp3 positive cells in child thymi was higher than that in adult thymi. However, there was no significant difference in the number of Foxp3 positive cells among adult thymi with and without MG. 3) IL-17 signals in MG+ thymi seemed to be more prominent than those in MG-thymi.
IL-17 producing cells rather than Treg may be altered in the thymus of MG patients. To investigate the occurrence of AQP4-Ab in Japanese patients with MG.
To detect AQP4-Ab, we employed immunofluorescence using HEK 293 cells transfected with human AQP4. Sera from 41 patients with MG being followed up in our department were collected after obtaining written informed consent.
Two patients with MG had symptoms related to CNS disturbance. MRI investigation of the brain and spinal cord revealed a long spinal cord lesion in one patient with recurrent optic neuritis and paraplegia consistent with NMO, and demyelination in the midbrain tegmentum and cerebral white matter in the other. Only these patients were positive for AQP4-Ab. Development of MG and sub-sequent thymectomy preceded the development of multiple sclerosis in both cases.
Patients with MG frequently exhibit AQP4-Ab. The occurrence of this antibody is strongly related to the development of CNS demyelinating disorder in such patients. Myasthenia Gravis (MG) is an autoimmune disorder affecting the neuromuscular junction. Muscle specific kinase (MuSK) antibodies are found in a variable proportion of patients with generalised disease who are acetylcholine receptor (AChR) antibody negative. Typically these patients have more prominent ocular, facial, bulbar, neck and respiratory weakness and often require long term immunosuppressive therapy.
We document the clinical course of two such patients who have been under long term follow up at our centre. We correlated this with their MuSK antibody titres using the standard radio-immunoprecipitation assay. We examined the IgG subclasses and ability to activate complement (C3/Membrane Attack Complex) in vitro at several time points during their disease using a cell based assay. Finally we analysed the functional effects of the IgG antibodies on cultured C2C12 muscle cells.
The MuSK antibodies in both patients were mainly of the IgG4 subclass although IgG2 and IgG1 antibodies were also seen later in the disease course. We found that the MuSK antibodies have the ability to activate complement in vitro, although the role of complement in vivo remains unclear. We also demonstrate that MuSK antibodies from these patients have variable ability to cause dispersal of acetylcholine receptor clusters in fully differentiated C2C12 muscle cells.
These studies show that MuSK antibodies cause instability of preformed AChR clusters which has potential to disrupt neuromuscular transmission in vivo. Furthermore, IgG subclass switching and increased ability to activate complement in vitro may be responsible for fluctuations during the disease course. Long-term corticosteroid (CS) therapy is effective in treating myasthenia gravis (MG), but the problem of the cumulative side effects and toxicity still needs to be resolved. Some studies showed that the therapeutic efficacy of cyclosporine (CyA), a potent immunosuppressive agent, in MG, and CyA is expected to reduce CS dosage. However, CyA has side effects including dose-dependent nephrotoxicity and hypertension.
In 2000, a new oral microemulsion form CyA (meCyA) was developed. meCyA is equally biologically active with much lower dosage than the old formulation. The present study was to evaluate the CS-dosage reduction effect and safety of meCyA therapy in generalized MG.
Twenty three patients with generalized MG [14 women and 9 men; age median (60 y.o.) range 30-74 y.o.] were enrolled in this study. The diagnosis of MG was based on typical clinical features, positive response to edrophonium or neostigmine, decremental response at low-frequency repetitive nerve stimulation, and a positive anti-acetylcholine receptor antibody assay. All the patients underwent thymectomy and were receiving CS daily prior to meCyA therapy.
The initial meCyA dosage of 3-5 mg/kg was divided into two doses per day and then gradually reduced during treatment to a minimal efficacious dosage by monitoring meCyA blood levels(adjusted to maintain a trough level less than 200 ng/mL), renal function (serum creatinine and BUN) and on the basis of clinical response (MG-ADL score).
After meCyA therapy, the daily CS dosage was significantly reduced from 18.5 ± 14.2 mg to 5.8 ± 4.9 mg, while the MG-ADL scores significantly improved from 7.4 ± 4.4 points to 3.8 ± 2.8 points at the 6-month point. There was also a significant decrease in the HbA1c levels following the therapy. The side effects of meCyA encountered (an increase of serum creatinine, trichosis and hypertension) were all mild and tolerable. meCyA therapy is effective and safe in reducing CS dosage in patients with generalized MG. Patients with early-onset Myasthenia Gravis (EOMG; onset b age 40) have autoantibodies to the acetylcholine receptor (AChR); also lymph node-like infiltrates in the thymic medulla, including T cell areas and germinal centers, which are implicated in MG pathogenesis. Most of those with muscle-specific kinase (MuSK) antibodies have normal thymi, whereas N60% of the remaining patients have low affinity antibodies to AChR and thymi similar to those in EOMG (though some from either subgroup are atrophic). To investigate the potential for lymphocyte trafficking, we compared the thymic lymphatic vessels (LV) and high endothelial venules (HEV) in MG patients with those in infant and aged-matched non-myasthenic controls.
We stained paraffin sections of thymi from 73 MG patients and 22 controls for lymphatic vessels (anti-LYVE-1 mAb), HEV (MECA-79 mAb) and blood vessels (anti-Von Willebrand factor pAb). Frequency, disposition and cellular content of both HEV and LV were analyzed over entire sections using a semi-quantitative scoring system. We also double-stained by immuno-fluorescence for CD20, CD1a, CD3, Ki67 or FOXP3 on lymphocytes.
We found LV at similar overall frequencies in all adult samples. Most were in the fat/septa around the residual thymic tissue, and appeared empty/collapsed. However, in control infants, and even in most adult MG samples with infiltrates (90% in EOMG and 55% in those with low affinity AChR abs), we also found some LV within the thymic tissue at cortico-medullary junctions or near infiltrates. Notably, these 'internal LV' were usually dilated/packed with CD1alymphocytes, a few of which were CD20+; of the many CD3+ cells, a few were FOXP3+, consistent with emigrant status. Neither lymphatic endothelial cells nor lymphocytes showed signs of cell division (they were negative for Ki67). As expected, HEV were restricted to the infiltrates in MG thymi and appeared empty. Finally, a small proportion of atrophic MG samples showed more LV (containing lymphocytes) and HEV in the remaining parenchymal areas than in adult controls; they may be relics of 'burnt-out' infiltrates.
Overall, the results suggest increased accessibility of MG thymi to incoming lymphocytes (via HEV) and increased export via lymphatics into the general circulation. That agrees with the involvement of the thymus in pathogenesis of EOMG and, notably, also in MG patients with low affinity antibodies to AChR. T-bet, a tissue-specific transcription factor, controls Th1 differentiation and IFN-gamma production, affects IgG class switching and pathogenic autoantibody production. New evidence suggests that Tbet also regulates the Th17 cells. In this study we examined the influence of T-bet deficiency (T-bet−/−) on the development of experimental autoimmune myasthenia gravis (EAMG), an animal model of human myasthenia gravis (MG). MG and EAMG are T celldriven, antibody-mediated autoimmune disorder of neuromuscular junctions.
EAMG was induced in T-bet−/− and C57BL/6 mice by immunization of acetylocholine receptor (AChR) combined with adjuvant. Onset, incidence and severity of EAMG were compared between T-bet deficient mice and control mice. The responses of T helper cells (Th1/ Th2/Th17) and B cells as well as the phenotypes and functions of regulatory cells were investigated. We show that T-bet −/− mice are less susceptible to EAMG, which was associated with a defect of Th1 T cell, B cell and dendritic cell (DC) responses. This was correlated with an expansion of CD4+CD25+ regulatory (Treg) cells as well as an enhanced response of Th2 and Th17 cells.
These results suggest that T-bet might provide a potential therapeutic target for myasthenia gravis.
The severity of symptoms shows an intraindividual correlation with titers of IgG autoantibodies in myasthenia gravis (MG) with antibodies to the acetylcholinereceptor (AChR) or to muscle specific kinase (MuSK), and the Lambert Eaton myasthenic syndrome (LEMS). However, the interindividual correlation between titers and symptoms is weak. Evidence exists that the inflammatory properties of IgG depend on the type of glycosylation of the Fc moiety affecting the interaction with Fc-gamma receptors. A high degree of galactosylation of IgG antibodies has been associated with clinical remissions in rheumatoid arthritis. Differences in glycosylation might also play a role in the autoimmune myasthenic syndromes. Autoantibodies are mainly IgG1 and IgG3 in AChR MG and LEMS, but IgG4 in MuSK MG. Both AChR MG and LEMS occur as a paraneoplastic syndrome or as an idiopathic autoimmune disease in association with the HLA B8-DR3 haplotype. In contrast, MuSK MG is not known as a paraneoplastic disorder, and has a different HLA association. To explore the immunopathogenesis in more detail, we studied the glycosylation profile of IgG in these three myasthenic syndromes.
Fc-part tryptic glycopeptides were analyzed by mass spectrometry. Statistical analysis was performed by PLSDA (partial least square for discriminant analysis), which sharpens the separation between groups of observations to obtain maximum separation between the classes control and patients.
IgG1 of LEMS was characterized by a low degree of galactosylation compared to age-matched controls: next to the biantennary, corefucosylated agalacto N-glycan structure (IgG-G0), also its bisected counterpart (carrying an additional N-acetylglucosamine) was elevated. Likewise, AChR MG patients, but not MuSK MG patients showed a reduced galactosylation of the IgG Fc moiety.
Several IgG glycoforms of LEMS patients showed a correlation with the occurrence of a SCLC. The bisected IgG1-G0 and IgG2-G1 glycoform decorated with a sialic acid residue were elevated, whereas the non core-fucosylated IgG4-G1 glycoform was reduced.
A reduced degree of galactosylation was found in patients with LEMS and AChR MG, but not in MuSK MG patients. IgG glycosylation was also different between LEMS patients with and without SCLC. Current studies aim at corroborating these findings in larger patient cohorts, and at associating IgG glycosylation with clinical disease severity.
14 -B cell regulating activity in patients with myasthenia gravis Myasthenia gravis (MG) is characterized by the presence of auto antibodies (Ab) to acetylcholine receptor (AChR) in 85-90% of patients. Only among anti-AChR negative patients, a subgroup has also been defined with antibodies against muscle-specific kinase (MuSK) and with overlap of these antibody responses. The regulation of the differential Ab production, B cell activity and related inducing T cell activity in disease subgroups is evaluated in this study.
Peripheral blood mononuclear cells are stained for B cell (CD38, CD138, B cell activating factor of the TNF family (BAFF) receptor (BAFF-R) and Th2 cell (CD4 and CCR4) markers and analyzed by flowcytometry. BAFF levels are measured in the sera of MG patients and healthy controls (HC) by ELISA. MG group consisted of 22 generalized patients without thymoma and with 2 anti-MuSK Ab (+) patients, 10 anti-AChR Ab (+) patients and 10 patients with none of these Ab and compared to age-matched 13 HC.
In the peripheral blood of patients, B cells (CD19+) with or without CD38 or CD138 did not differ from the HC. B cells were strongly positive for BAFFR in both groups as well. The BAFF levels were lower in the MG group (0.74 vs. 1.39 pg/ml, p = 0.001) compared to HC. However Th2 subtype, defined by CD4 and CCR4 co-expression, was significantly increased in MG group compared to HC (11.3 vs. 7.9%, p = 0.005).
The reported increase of BAFF levels in some antibody mediated autoimmune diseases is not supported for MG in the present study. However, the increase of B cell regulating T cell subset in MG implicates differential effects in MG subgroups on production of Ab.
The present work was supported by the Research Fund of Istanbul University.
Myasthenia gravis (MG) is an autoimmune disease defined by muscle weakness due to autoantibody mediated depletion of acetylcholine receptors (AChR) at the neuromuscular junction (NMJ). Recently we have shown that C57BL6 mice immunized with LPS and AChR develop MG-like disease which is CD4+ T cell-independent. In LPS-AChR induced EAMG, CD4−/− mice are able to produce significant quantities of high affinity isotype switched anti-AChR IgG, particularly complement binding.IgG2b. Recent studies of patients with MG indicate innate immune signals such as TLR4 and BAFF are up-regulated. To evaluate the role of TLR4 signaling in the development of EAMG, we utilized the LPS-AChR EAMG model because LPS is a TLR4 ligand.
TLR4−/− and WT C57BL10 mice were immunized with LPS and AChR and evaluated for generalized muscle weakness, sera anti-AChR antibodies, and pathologic IgG and C3 deposits at NMJ. Both WT and TLR4−/− mice developed MG-like muscle weakness, sera anti-AChR IgG2b and IgG2c antibodies, and IgG, C3, and MAC deposits at the NMJ. However, TLR4−/− mice developed less severe disease with reduced incidence compared to WT mice which was associated with significantly reduced titers and avidity of IgG2b for AChR. TLR4−/− mice also had reduced muscle C3 deposits M = 58.33 + −19.22 vs M = 91.25 + −6.44 in WT mice and reduced muscle MAC deposits M = 59.86 + − 3.2 vs M = 69.5 + − 9.6 in WT mice. Following boost immunization with AChR and LPS, lymph node cells were stimulated in vitro with AChR or AChR plus LPS and analyzed for proliferation and cytokine production. TLR4−/− cells proliferated in response to AChR stimulation but failed to proliferate to LPS alone. Stimulation of cells with AChR and LPS synergizes to produce significantly higher amounts of IFN-g in WT mice but not in TLR4−/− mice. Both WT and TLR4−/− cells stimulated with AChR alone induced IL-10 but not IL-6 production. However, addition of LPS with or without AChR induced significant production of IL-6 by WT lymph node cells but not TLR4−/− cells.
These data indicate that TLR4 promotes EAMG by increasing anti-AChR IgG2b avidity and enhancing lymph node production of IL-6 and IFN-g. To elucidate the prognostic and pathogenic significance of IL-6 in NMO, another inflammatory disorder with transverse myelitis, CSF IL-6 levels of 6 NMO, 22 relapsing remitting multiple sclerosis (RRMS), 15 optic neuritis patients and 15 neurologically normal controls were measured by ELISA. Indirect immunofluorescence studies showed a vascular staining pattern typical of NMO-IgG in the sera of 3 NMO patients. NMO patients had significantly (p b 0.01) elevated CSF IL-6 levels (33-553 pg/ml; 219.41 pg/ml ± 89.44) as compared to RRMS patients (13-100 pg/ml; 27.85 pg/ml ± 3.85), optic neuritis patients (12-85 pg/ml; 23.29 pg/ml ± 4.89) and controls (2-8 pg/ml; 4.45 pg/ ml ± 1.61). Particularly, NMO IgG positive patients displayed higher CSF IL-6 levels (265-553 pg/ml; 401.09 pg/ml ± 83.6) than NMO IgG negative NMO patients (33-40 pg/ml; 37.73 pg/ml ± 2.25).
Our results suggest that IL-6 is involved in NMO pathogenesis and NMO IgG production. By 2D-PAGE and immunoblotting we found that CSF IgG from HE patients specifically recognized 3 spots, which were identified as dimethylargininase-I (DDAHI) and aldehyde reductase-I (AKRIAI). DDAHI was present in two isoforms recognized respectively by five and four HE patients; immunohistochemistry with anti-DDAHI antiserum depicted endothelial cells in normal human CNS. AKRIAI was recognized by three HE CSF and this enzyme was widely distributed on neurons and endothelia by immunohistochemistry. IgG from HE CSF immunostained both neuronal and endothelial cells in mouse CNS.
The presence of these autoantibodies selectively in the CSF of HE patients may have important diagnostic and pathogenetic implications, since the autoimmune response to DDAHI and AKRIAI enzymes may lead to vascular and/or neuronal damage, two major mechanisms involved in the pathogenesis of HE.
A broad spectrum of neuropsychiatric symptoms including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, are described in association with group A beta hemolytic streptococcal (GABHS) infection. Patients suffering from such symptoms are diagnosed with Sydenham's chorea or PANDAS. It has been suggested that antibodies produced against GABHS, may cross the blood brain barrier and react with neural cells in a process involving molecular mimicry, which might lead to the emergence of neuropsychiatric symptoms in these two disorders. To date there is no animal model for either Sydenham's chorea or for PANDAS.
We have recently found that Lewis rats immunized with a mutanolysin GABHS extract showed increased compulsivity as well as motor disturbances. Sera taken from immunized rats demonstrated strong immunoreactivity to GABHS antigens and to neural tissue compared to sera of control rats. In addition sera from rats demonstrating movement and behavior disturbances induced high levels of a calcium/calmodulin-dependent protein (CaM) kinase activity in human neuroblastoma cells as shown previously in acute Sydenham's chorea and PANDAS. Autoantibodies in immunized rats targeted striatum and thalamus, two brain regions implicated in the pathophysiology of Sydenham's chorea and PANDAS.
We are currently working to identify the brain epitopes involved in antibody binding. These preliminary results support the hypothesis that an immune response to GABHS may in part underlie the behavioral and movement abnormalities reported in PANDAS and Sydenham's chorea, and provide an important step toward identification of the specific mechanisms.
4 -Autoantibodies against GLuR epsilon 2 in adult patients with non-paraneoplastic acute limbic encephalitis Takahashi Yukitoshi⁎ 1 , Kubota Yuko 1 , Yamasaki Etsuko 1 , Nishimura Shigeko 1 , Tsunogae Hisano 1 , Fujiwara Tateki 1 1 National Epilepsy Center, Shizuoka, Japan
The incidence of acute encephalitis is 19.0/1 million/year in Japanese adults and only 22.4% of the patients have the evidence of viral invasion in CNS. We developed the methods of detection of autoantibodies to NMDA-type glutamate receptor (GluR) epsilon 2 and GluR delta 2 using their whole molecules synthesized by PEX system, and found the autoantibodies to GluR epsilon 2 in 13 patients with Rasmussen syndrome and two patients with acute encephalitis (Y Takahashi, Neurology 2003). Thereafter, we found the IgMautoantibodies to GluR epsilon 2 in 6 of 26 CSF samples in acute stage of localized encephalitis, but not in 10 samples in acute stage of widespread encephalitis (Y Takahashi, Future Neurology 2006). Recently, paraneoplastic anti-NMDAR encephalitis associated with ovarian teratoma was reported (Dalmau, Ann Neurol, 2007). We examined the autoantibodies to GluR epsilon 2 in adult patients with non-paraneoplastic acute limbic encephalitis (NPALE).
We examined autoantibodies to GluR epsilon 2 in sera and CSF from 91 adult patients with NPALE, using full-length GluR epsilon 2 molecules by immunoblot, and n-terminal epitope of the autoantibodies by immunoblot using bacterial fusion proteins containing n-terminal peptides of the GluR epsilon 2 (amino acid residues 1 to 48) (NT1).
Autoantibodies against GluR epsilon 2 were found mainly in NPALE patients around 15-34 years old. Autoantibodies to GluR epsilon 2 in sera were detected in approximately 55% of NPALE patients from acute to chronic stages, and the autoantibodies in CSF were detected in 51.8% (acute stage), 41.4% (recovery stage), and 28.6% (chronic stage) of patients. 13 of 19 CSF with IgM-autoantibodies to GluR epsilon 2 were detected within five days from the onset of NPALE. All four CSF with autoantibodies to GluR epsilon 2 included epitope to n-terminal of GluR epsilon 2 (NT1).
These data suggest that autoantibodies against GluR epsilon 2 produced in sera after infection infiltrate into CNS through damaged BBB in acute stages, and affect n-terminal of GluR epsilon 2. In chronic stage, recovery of function of BBB reduces levels of the autoantibodies in CSF. Because BBB in hippocampi and amygdala are vulnerable, autoantibodies against GluR epsilon 2 including epitope to n-terminal may bring the limbic symptoms around onset. Most patients with myasthenia gravis (MG) have IgG1 antibodies against the nicotinic acetylcholine receptors (AChRs) or muscle specific kinase (MuSK). The remaining patients, termed seronegative myasthenia (SNMG) have been shown recently to have antibodies that bind to AChRs when they are densely clustered on the surface of transfected cells (as they are at the neuromuscular junction). MuSK antibodies are thought to be predominantly of the IgG4 subclass. Antibodies against aquaporin-4, a membrane water channel expressed in astrocytes and choroid plexus epithelial cells, are found in a high proportion of patients with neuromyelitis optica (NMO), clinically characterised by optic neuritis and myelitis, and are also IgG1. The pathogenic mechanisms are not always clear and we have established techniques for measuring the IgG subclass and complement activation in vitro.
HEK cells were transfected either with AChRs clustered using rapsyn or with MuSK or with aquaporin-4. After incubating with patients' sera, IgG subclasses or activated complement components (C3b and membrane attack complex, MAC) were visualised using fluorescent labeled secondary antibodies. Flow cytometry was used to quantify some of these results.
Antibodies against clustered AChRs in SNMG were predominantly of the IgG1 sub-type, similar to seropositive MG. Deposition of activated complement components, C3b and MAC, was demonstrated in-vitro in a proportion of patients with SNMG. Antibodies against MuSK were predominantly IgG4, but also had significant amounts of IgG1 antibodies and could activate complement in-vitro. NMO sera had mainly IgG1 antibodies that activated the complement system very strongly. The degree of complement activation correlated significantly with the presence of IgG1 antibodies in all groups of patients.
We show that immunocytofluorescence techniques can be useful not only for identifying the presence of antibodies but also to demonstrate some of their functional properties. This study provides further in-vitro evidence of complement activation in these autoimmune neurological disorders, giving insight into their pathogeneses and making them potentially suitable for targeted therapy using complement inhibitors. Recent studies reported contrasting results with respect to the presence of anti-myelin protein antibodies in Multiple Sclerosis (MS) and their relation with disease activity. This may be due to the heterogeneous specificity of autoantibodies in MS and the inability of most methods to detect pathogenically relevant antibodies. Here, myelin particles were used to detect anti-myelin antibodies in cerebrospinal fluid (CSF) of MS patients. Subsequently, their relation with MRI parameters was evaluated.
Anti-myelin IgG antibody reactivity was determined in CSF of MS patients (n = 82) and clinically isolated syndrome (CIS) patients (n = 37) using a novel flow cytometry-based assay. In addition, CSF of patients with other neurological diseases (OND, n = 17), inflammatory neurological diseases (IND, n = 21) and healthy controls (HC, n = 22) was tested.
Compared to HC, increased anti-myelin IgG antibody reactivity was most frequently found in CSF of CIS patients (46%, p = 0.002), relapsingremitting (RR) MS patients (69%, p b 0.001) and secondary progressive (SP) MS patients (66%, p b 0.001), together constituting 85% of all positive CSF samples. In contrast, elevated anti-myelin IgG antibody responses were present in a minority (12%) of the primary progressive (PP) MS patients and IND patients (30%), marginally present in healthy controls (5%) and absent in OND patients (0%). Most strikingly, anti-myelin IgG antibody reactivity was related to the number of T2 lesions on brain MRI in CIS and relapse-onset MS patients (r =0.37, p =0.007).
CSF anti-myelin IgG antibodies are promising, specific biomarkers in CIS and relapse-onset MS and correlated to MR measures of disease. The spectrum of neurological disorders associated with serum voltage-gated potassium channel (VGKC) antibodies includes neuromyotonia (NMT), affecting the peripheral nervous system; limbic encephalitis (LE), affecting the central nervous system; and Morvan's syndrome (MoS), which combines features of NMT and LE and often additional autonomic dysfunction (e.g. hyperhidrosis and bladder difficulties) and weight loss. These diseases are considered to have an autoimmune basis because they often respond well to steroids and plasma exchange. The wide phenotypic spectrum of VGKC antibodyassociated disorders, however, suggests that there may be different antibody specificities in each condition. We hypothesized that antibodies specific for VGKC subtype or for associated proteins might distinguish these conditions.
We used a variety of biochemical and immunological approaches to investigate the specificity of the antibodies in sera from patients with MoS.
MoS sera immunoprecipitated a subpopulation of VGKCs solubilised from rabbit brain, radioactively-labeled with 125I-alpha-dendrotoxin which binds to Kv1.1, 1.2 and 1.6. Surprisingly, however, MoS sera did not bind directly to HEK-293 cell lines transfected with cDNA encoding any of the Kv1.1, 1.2, 1.6 subunits. Moreover, addition of SDS detergent to the VGKC extract, to try to disengage associated proteins, reduced binding. We therefore tested known associated proteins and found that MoS IgG bound to HEK293 cells expressing a protein known to interact with potassium channels. This was confirmed by direct immunoprecipitation of EGFP-tagged associated protein.
MoS is a rare treatable neurological disease. The clinical spectrum is very broad and the condition can easily be misdiagnosed. The observation that the MoS antibodies bind to a Kv1-associated protein, rather than directly to the Kv1s themselves, should help to explain the varied clinical spectrum of MoS and provide a more definitive diagnostic test. In previous studies we detected complex antibody profiles against retinal antigens in patients with glaucoma. Heat shock proteins play an important role in cell survival under normal and stress conditions. Antibodies against heat shock protein 27 (HSP27) have been identified in these studies in sera of glaucoma patients. The aim of this study was to analyze if immunization with HSP27 can cause retinal ganglion cell loss in rats similar to the loss found in glaucoma patients.
Lewis rats were immunized with 100 mg HSP27 (plus Freund's adjuvant and pertussis toxin) and divided into three groups: group 1 was euthanized after 4 weeks (n = 6), group 2 after 5 weeks (n = 6), and group 3 after 6 weeks (n = 9). Control animals that received no immunization were euthanized after 6 weeks (n = 10, group 4). Intraocular pressure was measured during the course of the study and blood was collected at several time points. Serum was used to detect IgG antibody patterns against retina via Western blotting and Protein G bead techniques. The antibody patterns were analyzed by multivariate statistical techniques. Eyes were harvested the day the animals were euthanized and were fixed and flatmounts were stained with Brn-3a, which detects the retinal ganglion cell (RGC) nuclei. We used a computer assisted quantitation of RGC density.
No significant changes in intraocular pressure were observed during the course of the study. The animals immunized with HSP27 showed a lower number of RGCs in comparison to controls (P b 0.05). All animals showed complex antibody profiles with both detection methods. Through multivariate statistical methods we could show a significant difference between group 1 and 4 (P b 0.05). Group 3 showed a significant difference to the control group 2 and 4 weeks after immunization (P b 0.05).
After immunization with HSP27 animals developed complex antibody profiles against retinal antigens. Previous studies could demonstrate specific antibody profiles against retinal antigens in glaucoma patients. Thus, this animal model might be a beneficial approach to study the development and actions of anti-retinal antibodies and their possible involvement in neurodegenerative processes of the optic nerve e. g. in glaucoma.
Supported by Boehringer Ingelheim Foundation. Ganglioside complexes (GSCs) are target antigens in Guillain-Barré syndrome (GBS). GM1 and GalNAc-GD1a are thought to locate on axolemma of motor nerves and to be antigens associated with pure motor GBS, and they may exist in the vicinity and colocalize on the axolemma. Patients with pure motor GBS may have antibodies to GSC antigens associated with GM1 or GalNAc-GD1a. The purpose of this study is to investigate GBS sera on antibodies to a mixture of GM1 and GalNAc-GD1a and to analyze clinical and electrophysiological findings of patients with antibodies to a GSC, GM1/GalNAc-GD1a.
Sera from 224 patients with GBS were surveyed for antibodies to GSCs consisting of two of nine gangliosides (GM1, GM2, GM3, GD1a, GD3, GT1a, GT1b, GQ1b and GalNAc-GD1a) with ELISA. We analyzed clinical and electrophysiological features of patients with IgG antibodies to GM1/GalNAc-GD1a. Ten GBS patients had IgG antibodies to GM1/GalNAc-GD1a. Six of the anti-GM1/GalNAc-GD1a-positive patients had other anti-ganglioside antibodies such as anti-GD1a, GD1b, GD1a, or GT1b antibodies. Clinical findings of the anti-GM1/ GalNAc-GD1a-positive patients were featured by preserved sensory system and infrequent cranial nerve deficits, in accordance with characteristics of pure motor GBS. According to criteria by Hadden et al. (Ann Neurol 1998;44:780-788), electrodiagnostic findings were classified into demyelinating pattern in four patients and acute motor axonal neuropathy in two, and few abnormalities were found in sensory nerves. Early motor conduction block at intermediate nerve segments of motor nerves without lowering of motor conduction velocity was found in five patients.
GM1 and GalNAc-GD1a may form a complex in axolemma at nodes of Ranvier or paranodes of motor nerves, which may be a target antigen in pure motor GBS, especially in the form of acute motor conduction block neuropathy. Neuromyelitis optica (NMO), or Devic's disease, is a rare welldescribed demyelinating neurological syndrome with distinct clinical features and management approaches. The differentiation of NMO from more common causes of transverse myelitis, in particular secondary to systemic infection, is important for prognostic and therapeutic reasons. The presence of serum NMO-IgG antibodies supports a diagnosis of NMO. The detection of NMO-IgG by indirect immunofluorescence (IIF), on rodent brain and renal tissues1 is the only method currently available for routine use in diagnostic laboratories. Our group has recently observed the presence of atypical NMO-IgG-like IIF staining in a patient with transverse myelitis secondary to systemic melioidosis. As this atypical staining could be mistaken for NMO-IgG, we evaluated the presence of atypical NMO-IgG-like IIF staining patterns in systemic infections.
Sera from patients with serological evidence of acute infection caused by common bacterial and viral pathogens (INF, n = 57) and other controls (CTRL, n = 27) were tested using fresh frozen sections of rodent brain and renal papilla and anti-human IgG conjugate. None of these patients fulfilled clinical or radiological criteria [2] for NMO. IIF staining was interpreted according to the original description of NMO-IgG [1] .
A variety of different NMO-IgG-like IIF patterns were detected in 5/ 57 INF sera (10%), and in 1/27 (4%) of CTRL sera. Although different combinations of staining of the central nervous system (CNS) microvessels, pia, subpia, Virchow-Robin space and tubules of renal papilla were detected, the overall patterns were atypical compared with that defined by Lennon et al. [1] .
This study demonstrates the importance of experience in the interpretation of the NMO-IgG assay. Strict adherence to original description of NMO-IgG is essential for the correct interpretation of NMO-IgG IIF patterns, 1 particularly in patients with systemic infections that may be complicated by transverse myelitis. Given the clinical implications of a diagnosis of NMO, there is an urgent need to standardise the reporting of NMO-IgG IIF patterns and develop a robust confirmatory assay to reduce the risk of reporting atypical NMO-IgG-like patterns as NMO-IgG.
Antibodies against aquaporin-4 (AQP4), a water channel particularly expressed on perivascular astrocytic podocytes, are proposed as a marker for the diagnosis of neuromyelitis optica (NMO). However, a consensus on seroprevalence and optimal detection method has not yet been reached.
We set-up five different assays. Two of them were capable to detect perivascular IgG reactivity on brain tissue by immunofluorescence (NMO-IgG). The other three assays have been set to specifically detect anti-AQP4 antibodies: immunofluorescence and flow cytometry on AQP4-expressing cells, and a radioimmunoprecipitation assay. We assessed sensitivity and specificity of the different assays by interrogating sera of 33 NMO patients, 13 patients at high risk to develop NMO (hrNMO), 6 patients affected by acute partial transverse myelitis (APTM), 20 patients with multiple sclerosis (MS), and 67 age and sex matched healthy controls. As expected, we found that the presence of serum NMO-IgG and anti-AQP4 reactivity is almost exclusively restricted to patients with NMO and hrNMO. Seroprevalence and sensitivity ranged from 30 to 47%, depending on the assay. Specificity ranged from 95 to 100%. Comparing results obtained in the five assays we noticed lack of concordance in some samples.
However, sensitivity increased to over 60% if all assays were used in combination. Detection of NMO-IgG or anti-AQP4 antibodies may represent, in the very near future, a valuable tool to assist neurologists in the differential diagnosis between patients with NMO, hrNMO, APTM or MS. The current lack of a gold standard to detect anti-AQP4 antibodies implies the necessity to standardize the detection of these antibodies. The MS patient used for SAS analysis had oligoclonal antibody bands (OCB) in both CSF and serum with unique OCB in CSF, indicating intrathecal antibody production. Both body fluids were used in parallel to affinity select a MS cDNA phage display library to enrich for antigens recognized by MS immunoglobulins. After 4 selection rounds phage were screened for enrichment by colony PCR and DNA fingerprinting. Phage ELISA testing was used to confirm specific enrichment in CSF and serum.
We identified 14 enriched clones; 6 of these were exclusively enriched by serum selections and 2 were solely identified in selection rounds performed with CSF. Moreover, we identified 6 common cDNA clones, enriched by both serum and CSF selection procedures. Phage ELISA was performed to confirm immunoreactivity against the selected phage clones in both CSF and serum. Six clones showed immunoreactivity in both compartments while 3 other clones showed exclusive reactivity in the CSF. Immunoreactivity in CSF was remarkably higher compared to serum reactivity, consistent with an intrathecal antibody production.
One of the cDNA clones that was extensively enriched and displayed immunoreactivity in both CSF and serum was identified as a member of the Mitogen Activated Protein Kinase (MAPK)-family and has not yet been described in relation to MS. Expression of this cDNA clone was demonstrated in spinal cord of healthy and experimental autoimmune encephalomyelitis mice by Western blot and immunohistochemistry.
The use of SAS to study autoantibody profiles in MS CSF and serum in parallel provides clues regarding intrathecal and systemic autoantibody production in the disease. Antibodies were produced by the immortalization of B cells from CSF and peripheral blood of 4 MS patients and 11 control patients with non-inflammatory or other inflammatory neurological disease (NIND/ OIND). Peripheral blood mononuclear cells (PBMC) or CSF cells were cultured in the presence of Epstein-Barr virus (EBV) and polyclonal growth factor CpG2006, combined with T cell inhibitory and B cell stimulating factors and irradiated allogeneic PBMC. After 2 weeks the culture supernatant was screened for the presence of immunoglobulin G (IgG) and positive cultures were further cloned. The resulting monoclonal antibodies are representative for the enriched B cell populations in vivo and will be compared between CSF and blood.
The B cell immortalization procedure has been optimized. At present, we obtained 18 IgG-positive B cell lines from 2 MS patients, 7 originating from the CSF of 1 patient and 11 from PBMC of another MS patient. From 4 NIND patients, 7 immortalized B cell lines have been isolated, 2 derived from CSF cells of one patient, 3 from PBMC of a second patient and 2 from PBMC of two other patients. To investigate monoclonality, B cell spectratyping analysis was performed on the obtained B cell lines. Most of the IgG-positive B cell lines appeared to be monoclonal.
B cell immortalization has proved to be a useful method for the production of monoclonal antibodies but further optimization is necessary to increase the efficiency of the procedure. In conclusion, we obtained a panel of IgG-positive B cell lines from MS patients and controls. In future experiments, culture supernatant of the monoclonal B cells will be used to screen for antibody binding to a human oligodendroglial cell line (HOG), healthy and experimental autoimmune encephalomyelitis (EAE) brain tissue from rat and rhesus monkey and to viruses such as cytomegalovirus (CMV) and EBV.
14 -Devic's neuromyelitis optica: Prognostic implications of NMO IgG status in Turkish patients Devic's neuromyelitis optica (NMO) is an inflammatory/demyelinating disorder predominantly affecting optic nerves and spinal cord. There has been some debate on the classification, and naming of the disorder; recent findings showed an underlying humoral abnormality in NMO, characterized by the presence of a highly specific serum antibody against aquaporin-4, the most abundant water channel in the CNS (called NMO-IgG or Aqp4-Ab). In this study, we aimed at evaluating the NMO-IgG/Aqp4-Ab status among Turkish patients with NMO, and to find out any clinical significance.
Serum samples from 21 patients with NMO, diagnosed according to the 2006 criteria, who were being followed-up in our Multiple Sclerosis and Myelin Disorders unit were evaluated for NMO-IgG/ Aqp4-Ab using an indirect immunofluorescence. Confirmation was carried out by immunoprecipitation employing recombinant human aquaporin-4. Mean age at onset was 29.1 + 11.2 years, and mean disease duration was 9.9 + 7.0 years. Sex ratio (F:M) was 16:5 (3.2). Disease followed monophasic course in five patients and multiphasic course in 16. First clinical episode was optic neuritis in 14, transverse myelitis in two, and both in five cases. 17 of the sera were taken at an acute attack, four were taken at remission. NMO-IgG/Aqp4-Ab was positive in 12/21 (57%) patients. Among the NMO-IgG seropositive cases, nine had an EDSS of six or more, whereas only two patients in the seronegative group had such severe disability (p b 0.05), and the NMO-IgG positive cases had a mean EDSS score of 5.5 + 1.6 compared with 3.8 + 1.9 in NMO-IgG negative cases (p b 0.05). All seropositive cases were female whereas 5 of the seronegative cases were male (p b 0.05). Disease duration, age at onset and being monophasic or multiphasic did not differ significantly between groups. Three of the seropositive cases and one seronegative case were sampled at remission (difference, not significant). One seronegative case and eight of seropositive cases were under some immunosuppressant treatment at the time of sampling (p b 0.05).
NMO is relatively uncommon among other demyelinating disorders. Our findings in this relatively large single-center cohort suggest that the presence of NMO-IgG/Aqp4-Ab might have a prognostic significance. This should be further assessed in larger series. There are an increasing number of diseases in which specific antibodies to membrane proteins have been identified and new antibodies are being discovered. The associated diseases usually respond well to immunotherapies. There is a need to establish simple quantitative non-radioactive immunoassays for these antibodies and to test for novel antibodies in other patients with unexplained neurological diseases.
We used a new fluorescent immunoprecipitation (FIPA) method for antibodies to membrane proteins using expression of fluorescent protein (FP)-or luciferase-tagged proteins in human embryonic kidney cells, extraction in detergent solution, incubation in patient or control sera, and immunoprecipitation with either Protein A beads or with anti-human IgG. The precipitate is then measured for fluorescence on a plate reader or, after addition of the substrate luciferin, for light with a luminometer.
Acetylcholine receptor (AChR) antibodies can be measured by immunoprecipitation of enhanced green FP-acetylcholine receptor and MuSK antibodies by immunoprecipitation of mCherryFP-MuSK. The results correlate well with those obtained with the standard radioimmunoprecipitation assays. More than 70% of patients with neuromyelitis optica had serum AQP-4 antibodies using either enhanced green FP-AQP4 (Waters et al 2008 in press) or luciferase-AQP4. Finally, we tested the possibility that a patient with a severe form of encephalopathy with rigidity, myoclonus and excessive startle had a novel antibody to the glycine receptor. Using EGFP-glycine receptor, we found high levels of specific antibody at onset and levels fell substantially following treatment, corresponding to clinical remission (Hutchinson et al 2008 in press).
The results show the potential of either fluorescent protein or luciferase constructs to provide novel clinical assays for a range of antibodies to existing and novel targets. This approach is proving useful for identifying new antibodies (see S Irani abstract). Several myelin proteins of the central nervous system (CNS) have been investigated as potential targets for humoral immune response involved in the formation of multiple sclerosis (MS) lesions. In most studies, investigators employed recombinant antigens and/or relevant peptides from pre-selected targets but in spite of these efforts, the precise identity of MS antigens has not been clearly defined yet. In the present study we overcome the traditional approach using a large panel of native protein antigens derived from white matter homogenates of human healthy CNS, resolved by bi-dimensional electrophoresis (2D-PAGE) technique.
We compared the IgG repertoires in sera and cerebrospinal fluid (CSF) from 19 control subjects and 18 MS patients (4 primary progressive, 4 secondary progressive, 10 relapsing remitting) by immunoblot after 2D-PAGE resolution of white matter homogenate.
Nine spots have been specifically recognized by CSF of MS patients with statistical relevance (p b 0.05). They corresponded to isoforms of cytoskeletal proteins (CNPase I, Radixin, Collapsin response mediator protein 2, Tubulin beta-4, NAD-dependent deacetylase sirtuin-2, actin interacting protein 1). Of particular interest is one isoform of Transketolase (TK) specifically recognized by 50% of MS CSF. Interestingly, TK was mostly localized by double immunohistochemistry on mature oligodendrocytes (OL) and less prominently on NG-2 positive OL precursors and neurons in the healthy human CNS. In MS sections, TK immunoreactivity markedly decreased in the core of demyelinated lesions but was still present in a subpopulation of NG-2 positive OL precursors at the lesion border, in reactive astrocytes and macrophages. In addition to TK autoreactivity, CSF IgG from secondary progressive MS patients showed a significant reactivity to the above cytoskeletal proteins. Eight spots have been specifically recognized by seric IgG (p b 0.05). They corresponded to specific isoforms of proteins involved in the regulation of cytoskeleton (Alpha-centractin, Collapsin response mediator protein 5/1, Syntaxin binding protein 1, CNPase I) and in the energy production (TK, Pyruvate kinase). Among these proteins, the most frequently recognized by serum MS IgG was one isoform of CNPase I.
Our immunomic approach sheds new light on the autoimmune repertoire present in MS patients revealing novel oligodendroglial and/or neuronal molecules, with potential important pathogenic, diagnostic and prognostic implications. Neuromyelitis optica (NMO) is an idiopathic, chronic demyelinating and inflammatory disease characterised by predominant involvement of Optic nerves and Spinal cord. Recently, an antibody response against the water channel protein aquaporin-4 (AQP-4) was established as diagnostic biomarker for NMO. Current assay methodologies consist of indirect immunofluorescence and radioimmunoprecipitation assays, respectively.
Here we introduce a cell based flow cytometry assay to measure antibodies to AQP-4; the human glioblastoma cell line LN18 was stably transfected using a lentiviral vector to overexpress human AQP-4. Expression was further confirmed by immnocytochemistry and Western blotting of cell lysates.
We tested antibody reactivity in 160 serum samples (25 healthy controls (HC), 50MS, 50 OND, and 70 NMO) by flow cytometry at 1/100 dilutions. Results were expressed as median fluorescence intensities (MFI) corrected for background binding to the control cell line transfected with an empty vector. Samples are deemed positive if the MFI above cut-off (MFI (HC) +10SD).
70 NMO samples were positive (median MFI 835, range 66-4800), but no MS, HC samples, respectively (p b 0,005; ANOVA for Multiple MFI comparison); median endpoint titres were N1/10,000. The antibodies were confined to the IgG1 isotope and highly dependent on the native conformation of the protein.
We established a novel assay that allows fast and reliable quantification of autoantibodies to AQP-4. This assay improves AQP-4 autoantibody testing and may provide new insights into the pathogenic role of the antibodies in NMO.
Mascaro-Blanco A.⁎ 1 , Alvarez K. 1 Sydenham chorea is a neurological disorder resulting from group A streptococcal infection and acute rheumatic fever. This disorder is characterized by rapid and involuntary movements of the body extremities and face and other neuropsychiatric disturbances. Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) classification is applied to a subgroup of children whom tic disorders or obsessive-compulsive disorders develop following a streptococcal infection. It is believed that streptococcal infection induces a crossreactive humoral response directed against neuronal determinants in basal ganglia. Our goal was to study and compare autoantibody reactivity in acute and convalescent sera of Sydenham chorea, PANDAS and Tics patients against lysoganglioside.
Human monoclonal antibodies (mAbs) against lysoganglioside derived from Sydenham chorea were shown to be high avidity and result in neuronal cell signaling and elevated levels of CaM kinase. Using the ELISA we studied patients sera collected longitudinally and patterned dose response curves of concentration of antibody versus optical density (OD). The results show that anti-lysoganglioside autoantibodies are elevated in sera taken in the acute phase or during flares of disease and correlate with activation of CaM kinase in neuronal cells by serum antibody. Elevation of autoantibodies against lysoganglioside correlated with disease or exacerbations of behavior and movement symptoms.
Our data suggest that assay for levels of anti-lysoganglioside antibodies may be used along with the clinical findings for diagnosis of Sydenham chorea, Tics and PANDAS. Our data also suggest that the anti-lysoganglioside antibodies associated with disease activity and antibody-mediated neuronal cell signaling are high avidity and may contribute to disease pathogenesis. Acute disseminated encephalomyelitis (ADEM) is a rare monophasic CNS demyelinating disease that affects both grey and white matter of brain and spinal cord. Even though ADEM and multiple sclerosis (MS) share pathological characteristics, the clinical course of ADEM is very different from that of MS. Therefore differentiation of ADEM and MS is important for effective treatment.
The role of autoantibodies in CNS demyelinating disease is unknown however the detection of Myelin-Oligodendrocyte Glycoprotein (MOG) reactive antibodies was reported in MS and ADEM. The role of these antibodies for diagnosis and prognosis has remained controversial.
Here we introduce a cell based flow cytometry assay based on surface expression of MOG on human glioblastoma cell line transfected with lentiviral vector containing the MOG sequence. Expression of native MOG on transfected cell line was confirmed by flow cytometry, immunohistochemistry and western blotting.
We tested native MOG reactivity in healthy donors, patients with relapsing remitting (RR-MS), secondary progressive (SP-MS), primary progressive MS (PP-MS) and ADEM. Results were expressed as median fluorescence intensity (MFI) corrected for background binding to a cell line transfected with the empty vector. Samples were deemed positive if the MFI above cut-off MFI (HC) +10 SD were observed. While antibodies to native MOG are significantly elevated in MS patients compared to controls, very high titers are primarily observed in ADEM patients.
The findings confirm previous reports from other and our group with respect to antibodies to native MOG in MS and ADEM. Further studies are warranted to determine whether anti-MOG antibodies are useful to discriminate between ADEM and early MS. We studied AChR, VGKC and AQP4 antibodies serially in samples from a woman with a 30 year history of ocular MG which was treated only with cholinesterase inhibitors. In 2003, she developed rapidly progressive LE with very high levels of VGKC antibodies. She was treated with intravenous immunoglobulins, steroids and plasma exchange. The VGKC antibodies fell rapidly and she made a good recovery. In 2005 she had a short period of transverse myelitis which responded to treatment, but six months later relapsed with quadraparesis and longitudinallyextensive spinal cord lesions. We measured her antibodies to AChR, VGKC and AQP4 in all available samples and correlated the results with her clinical findings.
VGKC antibodies were very high at onset of VGKC-LE (N5000 pM) and fell substantially following treatment, normalising after 1 year. AChR antibodies were present (N60 nM) throughout the period of study but fell appreciably when the patient was treated for VGKC-LE. AQP4 antibodies were present before the onset of myelitis, rose markedly at the time of her major relapse and have fallen after therapy. The clinical features at each time point reflected well the relative antibody titres. This case illustrates well three different antibody-associated diseases in a single patient, and the correlation between antibodies and disease status during serial studies. However, AQP4 antibodies can be present in the absence of overt myelitis. Sydenham's chorea (SC) is a central nervous system (CNS) disorder and sequela of group A streptococcus-induced acute rheumatic fever (ARF) where deposition of antibodies in brain may result in movement and neuropsychiatric abnormalities. Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) represent a subgroup of obsessive compulsive disorder (OCD) and tic disorders which may share a common set of symptoms as well as a similar proposed etiology with SC. The goal of the study was to characterize SC and PANDAS antibody responses to streptococcal and neuronal antigens.
Our study investigated streptococcal and neuronal antigen specificity as well as cytotoxic capabilities of SC and PANDAS antibodies using enzyme linked immunosorbant assay (ELISA) and chromium-51 release assays. Streptococcal M type 5 protein and Nacetyl-beta-D-glucosamine (GlcNAc), the immunodominant epitope of the group A carbohydrate, have been suggested as cross-reactive antigens in ARF. Acute SC sera and cerebrospinal fluid (CSF) showed significantly greater IgG reactivity to M5 protein and GlcNAc than did matched convalescent sera and control CSF. Although matched PANDAS sera did not show a similar pattern of IgG reactivity to intact streptococcal antigens, PANDAS acute sera showed significantly elevated IgG levels to M5 protein N-terminal peptides in comparison to non-PANDAS controls. For SC acute sera and CSF, IgG subclass distribution analysis showed elevated levels of IgG1 and IgG3 to the M5 protein in contrast to PANDAS acute sera which only demonstrated increased IgG1. Both acute SC and PANDAS sera showed elevated IgG2 concentrations to GlcNAc-BSA. Of interest was the increased IgG1 response to GlcNAc by SC acute sera. SC CSF IgG recognized lysoganglioside, a CNS ganglioside associated with signal transduction in the brain, more strongly than did PANDAS CSF. SC and PANDAS sera and CSF were tested for complement-mediated cytotoxicity by chromium-51 release assay. Sera and CSF samples did not induce cytotoxicity against SK-N-SH human neuroblastoma cells. Analysis of IgG subclass distribution of cell surface-bound antibody demonstrated that IgG2 from SC CSF was the predominant subclass to label SK-N-SH neuronal cells.
The new data supports the hypothesis that anti-group A carbohydrate antibody of the non-complement activating IgG2 subclass may play an important role in the pathogenesis of SC and PANDAS. We report a patient suffered from a chronic sensory ataxic neuropathy associated with oculomotor paresis. Immunological studies revealed IgM lambda paraprotein, cold agglutinin and IgM autoantibodies reacting with GD1b, GQ1b, GD3 and GT1b but not GM1, GD1a or GM2 suggesting the presence of antibodies reacting with disialosyl epitope. Sural nerve biopsy revealed demyelinating neuropathy without IgM deposit. All features suggested a CANOMAD. A partial response to intravenous immunoglobulin and plasmapheresis is reported in some cases. Rituximab have gained widespread acceptance in the management of B-cell malignancies. More recently, they have been used to treat a number of autoantibody-mediated diseases, including some neuropathies, with encouraging results.
We evaluated the effect of Rituximab using the protocol recommended for rheumatoid arthritis, i.e. 1000 mg at J1, J15 and M6. We observed a disappearance of disialosyl antibodies at M3 followed by an increase at M6. CD19 numbering in blood was 4.9% at M0, 0.2% at M3 and 0.3% at M6. We observed at M3 a reduction of the ataxy and the handicap scoring ONLS (7 at J0 and 4 at M3) and an escalation at M6 correlating with evolution of the autoantibodies levels.
The results of this case report are very encouraging. Further studies of Rituximab therapy for neuropathy associated with disialosyl IgM autoantibodies are warranted. Sydenham chorea is a central nervous system (CNS) disorder characterized by involuntary movements affecting limbs and face, leading to loss of coordination. Chorea patients may exhibit psychiatric and psychological abnormalities including obsessive-compulsive disorder. Mechanisms of pathogenesis in Sydenham chorea are associated with antecedent Group A streptococcal infection which induces a crossreactive humoral response directed against neuronal determinants. Antibodies (Abs) to Group A streptococcal carbohydrate N-acetyl-beta-D-glucosamine (GlcNAc) are elevated in sera of chorea patients. Chorea-derived human anti-streptococcal monoclonal antibody (mAb) 24.3.1 crossreacted with GlcNAc, tubulin, and lysoganglioside and induced increased Ca++/calmodulin dependent protein kinase (CaM kinase) II activity in a human neuronal cell line. Our objective was to create transgenic (Tg) mice expressing mAb 24.3.1 heavy and light chain variable region (VH and VL) genes. V gene-expressing Tg mice were studied for antibody deposition in the brain to gain insight into the mechanisms of CNS disorders involving anti-brain antibodies.
Tg mice were created by pronuclear microinjection of mAb 24.3.1 VH and VL constructs into C57Bl/6 embryos. Double (VH + VL) and single (VH-only) Tg mice were produced. Gene-integration was verified by tail DNA PCR using V-gene-specific primers. ELISA determined expression of chimeric (human V gene/mouse constant region) IgG1a antibody and its antigen specificities in Tg mouse sera. Experiments were performed to see if the chimeric Ab would deposit in brains of Tg mice if the blood-brain barrier was breeched by administration of lipopolysaccharide (LPS). Brains and organs of sacrificed Tg and non-Tg mice were collected. FACS analysis characterized splenic, lymph node and bone marrow B cells and confirmed chimeric Ab expression. RT-PCR of splenic RNA verified V gene expression. Ab deposition in brains was determined by immunohistochemistry with allotype-specific antibodies.
Tg mice developed crossreactive Ab specificities for GlcNAc, lysoganglioside and tubulin in serum and chimeric chorea Ab expressed in Tg mice penetrated and bound to antigen in the brain in vivo. Deposition of IgG1a was observed in the brain of double Tg but not single VH Tg or non-Tg mice. Anti-brain autoantibodies against lysoganglioside and tubulin may contribute to CNS disturbances in Sydenham chorea and other brain disorders. To clarify a pathological implication of AQP1 in Alzheimer disease (AD), we investigated the possible relationship between amyloid-beta (Abeta) deposition and astrocytic AQP1 expression in the motor cortex and the hippocampus of 11 AD patients and 16 age-matched other neurological disease cases. In all cases, AQP1 was expressed exclusively in a subpopulation of multipolar fibrillary astrocytes. The great majority of AQP1-expressing astrocytes were located on the top of Abeta plaques in AD brains but not in non-AD cases, whereas those independent of Abeta deposition were found predominantly in non-AD brains. By Western blot, cultured human astrocytes constitutively expressed AQP1, and the levels of AQP1 protein expression were not affected by exposure to Abeta1-42 peptide, but elevated by hypertonic sodium chloride. By immunoprecipitation, the C-terminal fragmentbeta (CTFbeta) of amyloid precursor protein (APP) interacted with the N-terminal half of AQP1 spanning the transmembrane helices H1, H2 and H3.
A unique subset of AQP1-expressing astrocytes have a direct contact with Abeta in AD brains, suggesting the possibility that Abeta deposition causes abnormal brain water homeostasis by interfering with astrocytic water channel function. Alzheimer's disease (AD) is a major cause of dementia in humans. The main hallmarks of this disorder are the over production and aggregation of beta-amyloid (Abeta) peptides and the formation of neurofibrillary tangles of hyperphosphorylated tau proteins. The appearance of cognitive decline is linked to the overproduction of the short peptide Abeta in both soluble and aggregate forms. In the brain parenchyma, microglia are known to surround Abeta deposits and to internalize this peptide, and in some circumstances, target it for degradation. Thus, stimulating microgliosis should be beneficial in preventing the development of the disease.
Here, we show that injecting macrophage colony-stimulating factor (M-CSF) to APPSwe/PS1 transgenic mice, a well documented model for AD, on a weekly basis prior to the appearance of learning and memory deficits, prevented cognitive loss. The M-CSF-injected group, tested in the T-water maze task, showed no difference when compared to the wild-type (WT) animals. However, the number of trials needed to achieve the task nearly doubled for the saline-injected APPSwe/PS1 transgenic mice. M-CSF also increased the number of microglia in the parenchyma (2.4 fold and 2.7 fold increase in cortex and hippocampus, respectively) and decreased the number of Abeta deposits. Senile plaques were smaller and less dense in the brain of M-CSF-treated mice compared to littermate controls treated with vehicle solution. Interestingly, a higher ratio of microglia internalized Abeta in the brain of M-CSF-treated animals and the phagocytosed peptides were located in the late endosomes and lysosomes as shown by colocalization with the LAMP-2 marker. Less Abeta40 and Abeta42 monomers were also detected in the extracellular protein enriched fractions of M-CSF-treated transgenic mice when compared to vehicle controls.
Together these results provide compelling evidence that systemic M-CSF administration is a powerful treatment to stimulate microglia proliferation into the brain, degrade Abeta, and prevent the cognitive decline associated with Abeta burden in a mouse model of AD.
Microglia are the immune cells of the brain, they are activated in the brain of Alzheimer's disease (AD) patients and mouse models of AD and they express the innate immune receptor toll-like receptor 2 (TLR2). The present study investigated role of this receptor in the progression of AD-like pathologies.
Here we show that amyloid beta (Aβ) stimulates TLR2 expression in a small proportion of microglia. We then generated triple transgenic mice that are deficient in TLR2 from mice that harbor a mutant human presenelin 1 (PS1) and a chimeric mouse/human amyloid precursor protein (APP) genes. TLR2 deficiency accelerated spatial and contextual memory impairments, which correlated with increased levels of Aβ1-42 and transforming growth factor beta 1 in the brain. NMDA-R1 and NMDA-R2A expression levels were also lower in the dentate gyrus of APP-TLR2−/− mice. Gene therapy in cells of the bone marrow using lentivirus constructs expressing TLR2 rescued the cognitive impairment of APP-TLR2−/− mice. Indeed, lentiGFP/TLR2 treatment had beneficial effects by restoring the memory consolidation process disrupted by TLR2-deficiency in APP mice.
These data suggest that TLR2 acts as an endogenous receptor for the clearance of toxic Aβ by bone marrow-derived immune cells. The cognitive decline is markedly accelerated in a context of TLR2 deficiency. Up-regulating this innate immune receptor may then be considered as a potential new powerful therapeutic approach for AD.
Ron-Harel Noga⁎ 1 , Segev Yifat 1 , Cardon Michal 1 , Schwartz Michal 1 1
The hippocampus is one of the brain regions that are prominently affected by functional decline, even in what is considered "normal aging". Age-related decrease is found in hippocampus-dependent spatial and episodic memory abilities. Our previous studies have shown that immune cells are involved in maintaining various aspects of hippocampal plasticity at adulthood: neurogenesis, BDNF production and performance in the learning/memory task in the MWM are impaired in immune-deficient mice.
In the current study we were able to dissect out spatial learning ability from long-term memory performance and show that peripheral immune cells specifically support hippocampus-dependent spatial memory. Moreover, a sudden imposition of immune compromise in young healthy mice caused spatial memory impairment, whereas immune reconstitution reversed memory impairment in immune deficient mice. Hippocampal mRNA analysis suggested that immune-dependent spatial memory maintenance was, at least partially, mediated by regulation of presynaptic proteins involved in fast neurotransmitter release: reduced expression levels of genes encoding presynaptic proteins were found in immune-deficient mice, and returned to normal levels following immune reconstitution. Finally, the relevancy of our findings to aging was demonstrated: increasing peripheral immune activity in aged mice improved their spatial memory performance.
Our results suggest that age-related memory loss may be reversible, at least to some extent, by a remote controlvia the rejuvenation of the senescent immune system. Cognitive impairments, including spatial working memory deficits, occur even in the early stage of Parkinson's disease (PD) when the motor symptoms are barely observed (pre-motor phase). Most of cognitive deficits concern however developed PD when motor functions are disturbed and the central dopamine content deeply diminished. It is then possible that dopamine level depletion may influence also cognitive function in PD.
In the present study 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and influence of content dopamine depletion on memory impairment have been studied.
One year old C57/BL male mice, received MPTP 40 mg/kg, and control group received 0.9% NaCl. To evaluate learning and memory abilities, the Morris water maze behavioral test was provided.
We didn't notice statistical significant differences in the latency to reach the hidden platform, swim distance to reach the hidden platform, swim speed, percentage time spent in the correct quadrant, annulus crossing during the probe trial between MPTP and the control animals.
The results of swimming speed, latency and swim distance during visible platform test were similar for all groups and did not differ significantly between them. MPTP intoxication does not alter the locomotors activity during the memory tests.
After completion of the behavioral experiments the brain concentration of monoamine: dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT) were estimated by using high-performance liquid chromatography (HPLC). Correlation between the results of the behavioral testing in the probe trial in the water maze and the level of monoamines in the striatum were calculated. The accuracy of spatial memory was not reliably correlated with any monoamine level in the striatum. Dopamine content in the striatum decrease to 27% of the control (p b 0.0000).
Our results suggest that place navigation learning does not critically depend on the dopaminergic system. Probably other distant brain structures degeneration besides nigrostriatal pathway might to be involved in spatial learning and memory process.
More investigation and long-term observation are necessary to check if neurodegenerative changes in distant structures of the brain induced by nigrostriatal degeneration might cause memory deficits in animals with injured dopaminergic system. AAV2-SYN or AAV2-GFP was injected into the SN of male C57BL/6 mice and 2, 4 and 12 weeks post-treatment, microglial activation was examined using immunohistochemical staining for CD68 and quantitative PCR for proinflammatory cytokines. In addition, the participation of components of the adaptive immune response, including Blymphocytes, T-lymphocytes and mouse immunoglobulin (IgG) was studied immunohistochemically using the markers CD45R, CD3 and a goat anti-mouse IgG, respectively. The difference in B and Tlymphocyte infiltration into the SN between the two groups was quantified by counting the cells present using unbiased stereology. The difference in IgG and CD68 immunoreactivity between the groups was evaluated by an observer blinded to the treatment groups. Enhanced immunoreactivity for mouse IgG was detected early in the AAV2-SYN animals, at 2 weeks, and persisted at 4 weeks and 12 weeks. This IgG staining was associated with microglia, neurons, and neuropil. At 4 weeks, increased CD68 stained microglia were present in the AAV2-SYN animals, and there was a marked increase in both B and T cells. At 12 weeks, CD68 staining declined, but B and T cell infiltration persisted. Pro-inflammatory cytokine levels were elevated at both 2 and 4 weeks in AAV2-SYN group compared to the AAV2-GFP group.
These observations suggest that over-abundance of human aSYN can result in both an adaptive immune response and microglial activation. The temporal course, with early appearance of IgG staining, suggests that the humoral immune and microglial responses may be closely linked. This inflammatory process may contribute to dopamine neuronal injury. Modulation of this response may be a useful target for slowing the progression of human PD.
Vascular endothelial growth factor (VEGF) is well known as one of the major regulators of angiogenesis. Recently, there have been reports implicating VEGF as an important trophic factor in the nervous system. For example low levels of VEGF in mice result in an adultonset amyotrophic lateral sclerosis like syndrome. We wanted to examine the role of VEGF in the context of cerebellar degeneration-in particular, the autosomal dominant degenerative disease spinocerebellar ataxia type 1 (SCA1). SCA1 belongs to the family of polyglutamine neurodegenerative disorders that are caused by the increased number of CAG repeats in the coding region of respective proteins. Ataxin-1, the protein mutated in SCA1, is ubiquitously expressed and recent data suggest that it plays role in transcriptional regulation.
In a well-studied mouse model of this disease (SCA1 knock-in mouse), we have discovered that VEGF protein levels are perturbed relatively early in pathogenesis. To begin to understand the mechanism by which this reduction occurs, we performed Laser Capture Microdissection of Purkinje cells, the cell type predominantly affected in SCA1, followed by RNA purification and quantitative RT-PCR. We have found a significant reduction in VEGF mRNA levels in Purkinje cells derived from SCA1 mice.
Employing VEGF promoter luciferase reporter, we were able to show that mutant ataxin-1 inhibits transcription at the VEGF promoter. Furthermore, we could also demonstrate by chromatin immunoprecipitation that ataxin-1 occupies the VEGF promoter, suggesting that ataxin-1 induces repression via a direct effect on the VEGF promoter. These studies raise the exciting possibility that decreased VEGF levels could starve Purkinje neurons of key trophic factor or angiogenic support; conversely increasing VEGF might be able to ameliorate the SCA1 phenotype. To test these ideas, we have taken a genetic approach where we have mated mice that transgenically overexpress VEGF (in neurons) with the SCA1 knock-in model. We have found that overexpressing VEGF indeed improves the SCA1 ataxic phenotype.
Our findings implicate ataxin-1 as a novel regulator of VEGF transcription and suggest a therapeutic role for VEGF in SCA1, expanding its role as an important player in neurodegeneration. Synaptic injury is associated with the memory loss of Alzheimer's disease (AD). Shank proteins are abundant in the postsynaptic density (PSD) of central excitatory synapses. Increasing evidence suggests that Shank promotes the maturation and enlargement of dendritic spines, suggesting an important role for Shank proteins in normal cognitive development. GEPT combination of herbal extracts, also called GETO, consists of four active components: panaxoside, flavonoids, tenuifolin and curcumine. Previous studies show that GEPT extracts work to improve memory decline and to postpone MCI in old patients. This study was to investigate the effect of GEPT extracts on shank1 expression in CA1 subfield within the hippocampus of the APPV717I transgenic mice in the early stage of dementia.
APPV717I transgenic mice were randomly divided into GEPT group by intragastric administration of 0.3 g/kg/d, and a donepezil group by intragastric administration of 0.92 mg/kg/d, a APPV717I transgenic model group and a normal group by intragastric administration of distilled water. A four-month treatment regimen with GEPT extracts was administered to APPV717I transgenic mice. Spatial memory ability was measured in Morris water maze. The total area covered by shank1 and integral optical density in CA1 subfield within the hippocampus was determined using immunohistochemical stains and Image-Pro plus analysis.
After GEPT treatment regimen, the mean escape latency period was significantly shortened (35.3 ± 15.3 vs 59.9 ± 15.5, P b 0.05), and the target quadrant search time was significantly increased (44.5 ± 9.9 vs 29.1 ± 10.4, P b 0.05) compared to the APPV717I transgenic model mice. There was a significant higher level in the expression of shank1 detected in the hippocampal CA1 area of APPV717I transgenic mice treated with GEPT than the levels in the APPV717I transgenic model mice alone. The total area of positive cells covered by shank1 and their integral optical density in the hippocampal CA1 area of the APPV717I transgenic mice treated with GEPT were significantly increased more than those of the APPV717I transgenic model mice (11744.4 ± 2246.5 vs 8119.4 ± 1770.8, P b 0.05; 245.9 ± 55.0 vs 134.0 ± 44.7, P b 0.05).
GEPT extracts can obviously improve the spatial memory ability of APPV717I transgenic mice in the early stage of dementia, and its probable mechanism may be associated with enhancing the expression of Shank1. Multiple sclerosis (MS) is a common inflammatory demyelinating disorder of central nervous system with lesions disseminated in time and space. The diagnosis of MS is supported by evidence of intrathecal antibody synthesis. However, other autoimmune neurological disorders may show a similar laboratory pattern. We present a case with paraneoplastic disorder with immune profile distinct from MS.
We report a 58-year old right handed woman with an episode of post-traumatic acute loss of left central vision. Magnetic resonance imaging (MRI) of the brain showed several small non-enhancing T2 hyperintensity in subcortical and periventricular white matter. Five years later, she developed vertigo and ataxia. Repeat MRI was unchanged. Cerebrospinal fluid analysis showed numerous oligoclonal bands, elevated IgG index and synthesis rate and lymphocytic pleocytosis. Based on the clinical presentation and CSF findings, a diagnosis of MS was made and treatment was commenced with interferon β1-a and later glatiramer acetate. She continued to worsen and developed spastic-ataxic dysarthria. Axial and appendicular ataxia rendered her bedbound. Further workup included a comprehensive paraneoplastic antibody evaluation which was negative except for a low positive P/Q-type calcium channel antibody (80 pmol/L, normal b 20). Additional CSF studies again showed oligoclonal bands and evidence of intrathecal IgG synthesis. By flow cytometry, the CSF lymphocytes were predominantly B cells (52% CD19+) and the T cells were predominantly CD8+ (CD4/CD8 ratio, 0.4). Biopsy of an enlarged inguinal lymph node showed high grade neuroendocrine carcinoma consistent with a primary small cell cancer.
MS is a complex disorder in which helper T-cells play an important role. At least some forms of central paraneoplastic disorders are mediated by the action of cytotoxic CD8 T cells. Both disorders are associated with the inappropriate production of autoantibodies. In MS, the majority of cells in the CSF are T cells. The predominance of B cells and CD8 cells in the CSF seen in this patient may be a unique and diagnostically-useful characteristic of the paraneoplastic neurological syndromes. Paraneoplastic anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a severe, but treatment responsive encephalitis that predominantly affects young women with an ovarian teratoma. Patients usually develop prodromal fever or headache followed by prominent psychiatric symptoms, short-term memory loss, seizures, progressive catatonia-like stage, dyskinesias, autonomic instability and hypoventilation.
We report the immunopathological analysis of the brain and ovarian teratoma specimens of two patients who died of anti-NMDARassociated encephalitis and of the tumor of 9 patients who recovered. Prominent microgliosis and deposits of IgG with rare T and B lymphocyte infiltrates were observed in the hippocampus, forebrain, basal ganglia and spinal cord. Detection of cells expressing markers of cytotoxicity (TIA, granzyme B, perforin, Fas/Fas ligand) was extremely uncommon. Moderate amounts of plasma cells (CD79) detected on brain sections suggested intrathecal antibody production. No complement deposits were identified. All tumor samples showed NMDARexpressing neurons and extensive infiltrates of T lymphocytes, macrophages/monocytes, B lymphocytes and plasma cells. The ovarian teratoma samples from patients without NMDAR associated encephalitis showed similar infiltrates of T cells and macrophages/ monocytes, but few or absent B lymphocytes and plasma cells. In all patients the predominant anti-NMDAR IgG subclasses were IgG1, IgG3 and IgG4.
The antibody immune-response is more relevant than cytotoxic Tcell mechanisms in the pathogenesis of anti-NMDAR-associated encephalitis.
11 -Uptake and cytotoxic specificity of anti-Yo antibody for cerebellar Purkinje cells: Purkinje cells in organotypic culture are killed by anti-Yo IgG but not by IgG from neurologically normal ovarian cancer having other anticerebellar antibodies Greenlee John E.⁎ 3 , Hill Kenneth E. 2 Purkinje cell destruction in paraneoplastic cerebellar degeneration (PCD) in patients gynecological cancer is accompanied by high serum and CSF titers of "anti-Yo", an antibody which reacts with internal antigens of Purkinje cells. The role of anti-Yo antibody in causing Purkinje cell death is not known. We have recently found that Purkinje cells in organotypic culture can incorporate IgG and that uptake of anti-Yo IgG, but not IgG from normal individuals, results in cell death. We and others have also found that occasional neurologically normal ovarian cancer patients exhibit anticerebellar antibodies which do not recognize Yo antigens. An important question in PCD is thus whether the cytotoxic effect for Purkinje cells by anti-Yo antibody is specific or whether it is shared with other anticerebellar antibodies, such as those from ovarian cancer patients who remain neurologically normal.
We studied sera from three neurologically normal patients with ovarian cancer (2 adenocarcinoma and one sarcoma) who had high titers of anticerebellar antibodies reactive with Purkinje cells but who remained neurologically normal over many months until cancerassociated death. None of their sera reacted with Yo antigens in Western blots. Rat cerebellar organotypic cultures were incubated with sera from these 3 patients or with sera from PCD patients exhibiting anti-Yo antibody response. Concentrations of antibody were adjusted to approximate those found in patient CSF. Cultures were harvested at intervals through 144 hours and stained for antibody uptake using Cy-5 conjugated anti-human IgG followed by confocal microscopy. Purkinje cell death was determined using the dead cell stain, SYTOX green. Cultures incubated with anti-Yo antibody showed widespread IgG uptake by Purkinje cells. Death of Purkinje cells containing IgG began by 72 h and was extensive after 96-120 h. Although Purkinje cell uptake of IgG was also seen in cultures incubated with sera from our three neurologically normal patients, Purkinje cell death was not observed.
The cytotoxic effect of anti-Yo antibody on cerebellar Purkinje cells is specific and is not shared by anticerebellar antibodies from ovarian cancer patients who remain neurologically normal.
to further characterize the pathogenesis of PNS. Here we report a new antibody that is co-expressed with Yo antibodies.
Serum samples from three different patients with known paraneoplastic cerebellar degeneration and Yo antibodies were pooled and used for screening a rat cerebellum cDNA library. One of the antibodies found reacted with an uncharacterised protein known as coiled-coil domain-containing protein 104 (ccdc104). This protein has also been found in other species and is conserved. Immunoblotting with protein extracts showed that the ccdc104 protein is widely expressed in the central nervous system, except in the frontal cortex and spinal cord. The protein is also present in other organs, especially in the spleen and testes. Sera from 103 patients with various cancers and onconeural antibodies against Hu, Yo, Ri, CRMP5, amphiphysin or Ma2, as well as sera from 100 healthy blood donors without onconeural antibodies were screened by a sensitive immunoprecipitation technique. Four patients with Yo antibodies (2 with ovarian cancer, one with lymphoma and one with prostate cancer) also had ccdc104 antibodies, whereas these antibodies were not detected in the other sera.
Anti-ccdc104 is a potentially new onconeural antibody that is associated with anti-Yo. The function of ccdc104 is largely unknown.
Myasthenia gravis (MG) is the prototypical autoimmune disorder of the nervous system. Some patients have paraneoplastic MG associated with thymic neoplasm. Thymoma and MG may be associated with other autoimmune neurological disorders including paraneoplastic encephalitis. This report describes a second case of multifocal cortical encephalitis associated with thymoma and establishes an association of this thymoma-related paraneoplastic syndrome with voltage-gated potassium channel (VGKC) antibodies.
A 43-year old right handed woman had a history of seropositive MG and invasive thymoma which was treated successfully with surgery, radiation and chemotherapy. MG was in pharmacological remission with mycophenolate. Four years after thymectomy, she presented with acute onset of confusion and seizure progressing to complete mutism. On examination, she was awake, alert but globally aphasic. She was able to imitate and had normal strength and reflexes.
Magnetic resonance imaging of the brain showed innumerable nonenhancing cortically-based signal abnormalities as well as extensive left mesial temporal lobe abnormality with minimal enhancement. Cerebrospinal fluid analysis showed lymphocytic pleocytosis (70 cells) and elevated protein. Electroencephalogram showed left temporal periodic lateralized epileptiform discharges. On chest computed tomography, there was abnormal pleural thickening of the left lung which proved to be recurrent metastatic thymoma. Serological evaluation was positive for AChR, striational and voltagegated potassium channel antibodies. She had a partial improvement response to immunotherapy and one cycle of chemotherapy, but ultimately died two months later.
Most examples of paraneoplastic encephalitis involve the limbic system and related structures. This is the second case with a unique pattern of multifocal cortical involvement associated with thymoma. Potassium channel antibodies were found in this case while CRMP-5 antibodies were found in the previously reported case. Paraneoplastic encephalitis associated with thymoma should be considered in the differential diagnosis of MG patients who develops new cognitive symptoms.
14 -Induction of TLR expression in the peripheral nerve upon the neurodegeneration Van Avondt K., Goethals S., Jacobs A., Timmerman V., Janssens S.⁎ Peripheral Neuropathy Group, Molecular Genetics Department, VIB, University of Antwerp, Antwerpen, Belgium
In recent years, the field of innate immunity changed dramatically due to the discovery of the families of so-called Toll-like receptors (TLRs) and Nod-like receptors (NLRs). Both induce different signaling cascades, culminating on activation of NF-κB, MAPKs, interferon responsive factor (IRF) or production of IL-1. Interestingly, while originally these receptors were thought to be involved exclusively in the detection of pathogens, more and more data accumulate that they are also important in the recognition of endogenous ligands, like hsp proteins or degraded extracellular matrix breakdown products, all indicative of some kind of stress. Therefore these receptors are now categorized as "danger" receptors and known to be pivotal to elicit a proper stress response.
Also the peripheral nervous system contains a defense mechanism to alert the immune system in cases of danger. In this defense response, Schwann cells play a major role. Upon neurodegeneration, they adopt a macrophage like phenotype and start secreting factors that are important in the recruitment of other inflammatory cell types. How Schwann cells become activated by neurodegeneration is not clear yet. We would like to test the hypothesis that certain released components of dying axons can trigger Toll like receptors expressed on the surface of Schwann cells.
Though recent data report the expression of various TLRs on glial cells in the central nervous system, the situation in the peripheral nervous system is less studied and remains limited to the study of selected TLRs. Therefore, we first analysed basal expression levels of all TLRs in primary Schwann cells. Schwann cells appear to express mainly TLR3, TLR4 and TLR5 while TLR6 and TLR9 are hardly detectable in unstimulated cells. Through the use of NF-κB signaling as readout, we could show that most TLRs are functional, with especially TLR2 showing a strong response.
Next, we focused on a comprehensive qPCR analysis of innate immune receptor components in unstimulated versus activated peripheral nerve. We induced neurodegeneration through axotomy of the sciatic nerve in mice. Control and axotomised nerves were isolated at several time points upon injury and processed for RNA isolation and qPCR analysis. In the unstimulated nerve, TLR3, TLR4, TLR5 and TLR7 were mostly expressed, which is comparable to the situation in primary Schwann cells. We detected a major induction of TLR1, TLR2, TLR6 and TLR9 upon nerve injury, pointing to a possible role of these receptors in the context of neurodegeneration. While TLR1, TLR6 and TLR9 reached peak expression levels 32 h upon injury, TLR2 was already induced at earlier time points.
In conclusion, our data show a major shift in TLR expression pattern upon neurodegeneration, most likely illustrating specialized functions of TLRs in basal versus activated conditions of the peripheral nerve. Through the use of wild type versus knockout mice, we would like to find out which receptor is of main importance in the triggering of the innate immune response in Schwann cells versus sciatic nerve upon neurodegeneration. Genetic factors are believed to be of importance for outcome of traumatic brain injury (TBI). However, so far mainly allelic variation in apolipoprotein E4 has been studied in human TBI. A strategy for the identification of genes regulating complex traits is to perform linkage analysis in large intercrosses of susceptible and resistant strains. Previously we have identified several gene regions that regulate different aspects of inflammation and neurodegeneration induced by spinal ventral root avulsion (VRA) in inbred DA and PVG rats. These quantitative traits (QTL) include Vra1, which regulate axotomyinduced nerve cell loss, and Vra4 that regulate expression of MHC class II on microglia. It is, however, not known if these QTLs regulate general aspects of neuroinflammation and cell death after traumatic CNS injuries.
In order to study the role of genetic factors in experimental TBI, we examined parental DA and PVG strains, as well as Vra1 and Vra4 congenic strains on DA background. A standardized weight drop injury was used, inflicting a parietal cortical contusion by letting a weight fall onto a rod with a flat end diameter of 1.8 mm from a height of 6.0 cm on the exposed dura. The rod was allowed to compress the tissue a maximum of 3 mm. Some rats received only craniotomy and served as controls. The pericontusional area was dissected 6 days after TBI and real time PCR was used to evaluate differences in apoptotic and inflammatory mRNA expression.
There was a significant upregulation of inflammatory molecules, such as complement factors C1q and C3, the class II invariant chain CD74, and apoptotic factors such as the transformation related protein trp53, after TBI as compared to controls. Levels of several of these transcripts were higher in DA compared to PVG. The increase in CD74 was attenuated in Vra4 congenic rats, consistent with a prominent regulatory effect of allelic differences in the Mhc2ta gene located in the congenic fragment. Vra1 congenics displayed a significant difference in trp53 levels, indicating differences in the regulation of apoptosis.
These results provide further evidence for a genetic component in TBI and also support the notion that QTLs regulating VRA are also implicated in the regulation of inflammation and apoptosis after TBI. These strains now constitute an important tool for the study of genetic influences after TBI. 48 children with autistic regression fulfilling the diagnostic criteria according to the DSM-IV were included. A questionnaire for assessment of cognitive/behavioral regression, detailed neurological and psychiatric evaluations and Childhood Autism Rating Scale (CARS) were performed. In our study, DNA isolation was accomplished by using standard methods. IL-10, IL-6, TNF-alpha, TGF-beta1b, and IFNgamma were genotyped by using PCR with sequence-specific primers (PCR-SSP) method. The sera of 48 patients diagnosed as autism and 75 healthy individuals were included. The allele frequencies between groups were determined. HLA typing for A, B, DR alleles was performed by PCR-SSO method. When the patient group and control group were compared as regards the frequency of the HLA antigens, significant difference was found only in HLA A2 as higher in the patient group. Though not significant statistically, the ones with odds ratio over 2 included A11, B38, B39, B48, B52, B53, B55, B58, DRB1⁎14, DR14 was more frequent in the patients; A29, B27, B40, B41 was more frequent in the controls. The controls had A69; B37; B54; B57, DRB1⁎09; DR9 while they did not exist in the patient group. One of the patients had B58 while none of the controls had. In the TGF-beta gene region, TGF-beta1 (C/T codon 10, C/G codon 25) polymorphism was examined and the TT-GG genotype frequency was found high in controls while TC-GG genotype frequency was high in the patient group.
This study showed various immunological abnormalities in autistic children, which might be markers of a special condition, that is, autistic regression seen in autism spectrum disorders. Chemokines and chemokine receptors (CCR) have been detected in normal human central nervous system (CNS). Recent evidence suggests that chemokines and CCR are involved in the pathogenesis of immune-mediated inflammation of chronic neurodegenerative disorders as Alzheimer's (AD) and Parkinson disease (PD). For instance, increased expressions of CCR5 and CCR2 have been reported in the CNS of patients with AD and PD, which has been associated with pathological changes that characterize both neurodegenerative diseases.
In order to establish the importance of this polymorphism in neurodegenerative disorders, 100 Venezuelan patients diagnosed with sporadic AD according to NINCDS-ADRDA criteria (mean age 71-10 years), 50 patients with PD according to the PD Society Brain Bank criteria, and 100 healthy individuals (mean age 71-10 years) without any kind dementia based on the Mini-Mental State Examination were studied. The CCR5 59029 A/G polymorphism was analyzed by PCR-RFLP. Genotype frequencies were determined by direct counting. Statistical significance of genotype frequency was estimated by Fisher's exact test and p values were corrected according to Bonferroni. Odds Ratio (OR) with 95% confidence intervals was calculated to measure the strength of associations. Statistically significant differences were found when the AD and PD patients were compared with the control group. The frequency of the A/A genotype was observed decreased in both group of patients compared with healthy individuals (OR b 1; p b 0.05). In contrast, the frequency of the A/G genotype was observed increased in both neurodegenerative disorders compared with healthy individuals (OR N 1; p b 0.05).
The results suggest a possible involvement of CCR5 gene in the development of late-onset Alzheimer's disease and Parkinson disease. In 2007, various reports identified and confirmed for the first time non-HLA susceptibility genes for multiple sclerosis (MS). SNPs in the IL2RA and IL7RA receptor genes were found to constitute heritable risk factors for multiple sclerosis with odds ratios of around 1.2. Though numerous earlier studies have reported associations or lack thereof between cytokine (receptor) genes and multiple sclerosis, a systematic screen covering genetic diversity at multiple cytokine loci has never been performed. Using a haplotype-tagging approach based on selection of SNPs from the CEU-HapMap, we have analysed association between MS and around 380 SNPs in the major different functional classes of cytokines, their receptors and associated cytokine signal transduction factors (gamma-c-dependent cytokines; interleukin-17 group; gp130-dependent IL-6-like cytokines; IL-12-like cytokines; members of the IL-1 family; SOCS family members). In total, 49 genes or chromosomal gene clusters were included for 60 discrete cytokine or cytokine-related genes. The majority of included genes have never been analysed before in relation to MS.
Wherever possible, haptag-SNPs were selected to coincide or be complemented with non-synonymous SNPs. In addition, our screen included 8 glutamate receptor genes from the GRIA, GRIK and EAAT subfamilies. The latter selection was motivated by a growing body of evidence for a significant role of glutamate signalling in glial cell death. Genotyping was performed using Golden Gate technology (Illumina) in around 1000 MS patients and controls using a 2-stage design (Basurto Hospital, Bilbao). Exhaustive single-and multiplemarker data analysis was performed with HaploView, PLINK, HelixTree and MDR software. Following the first stage, we have identified 2 associated SNPs, one in suppressor of cytokine signaling-1 (SOCS1) and one in oncostatin M receptor (OSMR) with significant values withstanding permutation correction, and found modest evidence for cytokine gene interaction in MS. A suggestive trend for association with non-synonymous polymorphisms in glutamate receptor GRIA1 and GRIA2 was observed. The second stage of the study is being completed. We will present the results from the combined two stages on the occasion of the conference.
While our data contribute to furthering our understanding of cytokine genetics in MS, it is clear that confirmatory studies in additional cohorts will be needed to substantiate any findings arising from our screen. Neurodegeneration is an important feature of many neurological disorders. So far the degree of genetic regulation on axotomy-induced nerve cell loss has been unknown. In previous studies we have described substantial differences in the degree of loss of motoneurons after spinal ventral root avulsion (VRA) across different inbred rat strains. We now use an unbiased experimental approach using genetic dissection to elucidate relevant mechanisms. In a first step a whole genome scan in a F2 DA x PVG intercross showed evidence of linkage of nerve cell loss VRA to quantitative trait loci (QTLs) on chromosomes 8 (Vra1) and 5 (Vra2).
An advanced intercross line (AIL) was created by reciprocal breeding of DA and PVG-RT1av1 (PVGav1) rats. Subsequent crossings of offspring from 50 breeding pairs were carried out for eight (G8) or ten (G10) generations. We here studied 126 and 186 male rats subjected to VRA with 14 days post-operative survival in the G8 and G10 population, respectively. Linkages to both Vra1 and Vra2 are reproduced and reduce the confidence intervals of linkage peaks to 13.1 and 29.3 Mb, respectively. A pure additive effect of the two loci is seen, and both significantly contribute to the additive LOD. Individual rats in the AIL homozygote for DA or PVG alleles at both Vra1 and Vra2 display neuronal survival similar to the corresponding parental strain, suggesting that strain differences are mainly regulated by these two QTLs. Overlapping congenic strains with PVGav1 alleles on DA background demonstrate that a 9 Mb Vra1 fragment significantly attenuated the loss of motoneurons after VRA. These congenic strains are now used to dissect mechanism and preliminary evidence suggest that increased microglia activation early after injury correlate with increased nerve cell loss later on.
These results demonstrate that naturally occurring allelic variability affects susceptibility to axotomy-induced nerve cell death, where Vra1 and Vra2 represent the first QTLs to be characterized and fine mapped using AIL and overlapping congenic strains. Exact positioning of the gene(s) in the QTLs will be of importance to understand how genetic susceptibility to neurodegeneration is regulated in diseases such as progressive forms of MS. Many human autoimmune diseases, including multiple sclerosis (MS), are regulated by the MHC complex and the susceptibility to experimental autoimmune encephalomyelitis (EAE) maps to the MHC class II region. This supports the notion that qualitative differences in class II mediated antigen presentation to be of pivotal importance in these conditions. Previously we have described large differences in MHC II expression on microglia after a standardized mechanical nerve injury (ventral root avulsion; VRA) in a panel of inbred rat strains. Using a DAxPVG F2 intercross and an advanced intercross line (AIL) differences in MHC II expression was positioned to a genetic variation in Mhc2ta (encoding the class II transactivator). Recently we demonstrated that congenic strains carrying allelic variations of Mhc2ta display variable susceptibility to EAE, suggesting that quantitative aspects of MHC II expression may also be of importance for regulation of autoimmune neuroinflammation.
The BN rat displays higher constitutive MHC II expression compared to DA and LEW.1N although BN has the same Mhc2ta allele. In this study we performed a genome-wide linkage analysis in a F2(LEW.1NxBN) intercross (n = 151) in order to map additional genetic influences on the expression of MHC II after VRA. The mRNA levels of CD74 (invariant chain), co-stimulatory molecule B7-2 and beta2microglobulin (b2m) were determined using quantitative RT-PCR. In addition, Ox6 immunostaining was recorded. Identified QTLs were subsequently studied in a DAxPVG.1AV1 G12 AIL. Several regions were found to regulate MHC II, with the two strongest QTLs being located on chromosomes 1 and 7. The chr 1 QTL was reproduced in the G12 AIL. In addition, there were evidence of epistatic and gender effects. Two regions were found to regulate several phenotypes; Ox6, C1q, Il-1beta and C3 receptor on chr 10, and B7-2, b2m, C3 and C3 receptor on chr 11.
These results demonstrate a high degree of genetic heterogeneity in the expression of MHC II on microglia after nerve injury. Differences in MHC II across strains are regulated mainly by one dominant QTL containing Mhc2ta, but with additional effects from several other gene regions. Identification of the genes underlying these additional QTLs will be of importance for the understanding of the genetic background to autoimmune diseases such as MS. We present two separate studies focus on the role of IRF5 gene and chromosome 19q13 in the susceptibility to multiple sclerosis (MS). IRF5 is a transcription factor involved both in the type I interferon and the toll-like receptor signalling pathways. Previously, IRF5 has been found to be associated with systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel diseases. Here we investigated whether polymorphisms in the IRF5 gene would be associated with yet another disease with features of autoimmunity, MS.
Previous studies have suggested a role of allelic variation on chromosome 19q13 in multiple sclerosis (MS) susceptibility. Three subregions of 19q13 were tested for association with MS.
In the first study we genotyped nine single nucleotide polymorphisms (SNPs)and one insertion-deletion polymorphism in the IRF5 gene in a collection of 2337 patients with MS and 2813 controls from three populations: two case-control cohorts from Spain and Sweden, and a set of MS trio families from Finland. Two SNPs (rs4728142, rs3807306) and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF5 gene, showed association signals with pvalues b 0.001 when the data from all cohorts were combined. The predisposing alleles were present on the same common haplotype in all populations. Using electrophoretic mobility shift assays we observed allele-specific differences in protein binding for the SNP rs4728142 and the 5 bp indel, and by a proximity ligation assay we demonstrated increased binding of the transcription factor SP1 to the risk allele of the 5 bp indel.
In the second study we tested 14 markers, both SNPs and microsatellites across three subregions of 19q13 in 440 Finnish MS trio families. The marker D19S876 on 19q13.1 showed nominally significant association with MS (p = 0.007). Another association was found with an APOE haplotype (p = 0.007) on 19q13.2. Both associations were not replicated in another independent dataset of 317 families. Previous suggestions on ILT6 deficiency or association with D19S585 on 19q13.3-q13.4 were not replicated in this study.
These findings add IRF5 to the short list of genes shown to be associated with MS in more than one population. Our study adds to the evidence that there might be genes or pathways that are common in multiple autoimmune diseases, and that the type I interferon system is likely to be involved in the development of these diseases.
The formation of antibodies to Interferon beta (IFNβ), a proteinbased disease-modifying agent for multiple sclerosis (MS), is a crucial problem in clinical practice. These antibodies may neutralize the biological effects of the protein drug, potentially decreasing its therapeutic effects. The goal was to find HLA alleles which are associated with the development of antibodies against interferon-beta therapy in multiple sclerosis.
HLA genotyping for HLA-A, -B, -C, DRB1 and DQB1 was performed by hybridization of sequence-specific oligonucleotide probes immobilized on microspheres with amplified genomic DNA samples followed by flow fluorescence intensity analysis according to the instructions of the manufacturer (One Lambda, Canoga Park, CA, USA). By high resolution HLA classes I and II typing we identified two HLA class II alleles associated with the development of antibodies to IFNβ. In two independent continuous and binary trait association studies, HLA-DRB1⁎0401 and HLA-DRB1⁎0408 (odds ratio: 5.15)but not other HLA alleleswere strongly associated with the development of binding and neutralizing antibodies to IFNβ. The associated HLA-DRB1⁎04 alleles differ from not associated HLA-DRB1⁎04 alleles by a glycine to valine substitution in position 115 of the epitope-binding alpha-helix of the HLA class II molecule.
The peptide binding motif of HLA-DRB1⁎0401/⁎0408 might promote binding and presentation of an immunogenic peptide which may eventually break T cell tolerance and facilitate antibody development to IFNβ. This is the first study that identified genetic factors determining the immunogenicity of a protein-based diseasemodifying agent. Toll-like receptors (TLRs) play an essential role in host defense responses. Following recognition of pathogen-associated molecules and self molecules TLR generally trigger pro-inflammatory factors and enhance antigen-presenting cell functions. In our previous studies, we demonstrated the presence of various TLRs in and on human astrocytes, microglia and oligodendrocytes. Marked differences, however, separate TLRs on either type of glial cell. In microglia, a rather diverse set of TLRs are expressed and at least TLR3 and TLR4 can be found only inside endosomal vesicles. Astrocytes and oligodendrocytes on the other hand, express a more limited set of TLRs, and only on their cell surface. Upon activation of astrocytes a selective and strong induction of TLR3 was observed. Gene profiling and functional data indicated that TLR3 activation on astrocytes acts as a repair mediator.
The aim of this study was to investigate the expression, regulation and the function of TLRs in adult human microglia and rat oligodendrocyte progenitor cells (OPCs) using real time PCR, antibody array and in the case of oligodendrocytes morphological evaluation, viability assays as well immunofluorescence staining for differentiation markers.
Adult human microglia in culture express all TLRs with TLR1, 2, 3, 4 and 10 at relatively high mRNA levels. TLR expression is also controlled by TLR ligation itself and by treatment with different cytokines. Remarkably, TLR3 was the strongest induced TLR, especially by poly I: C. In addition, in vivo expression of TLRs was examined using mRNA extracted from both control and multiple sclerosis brains, revealing enhanced expression of TLR3 and in some cases of TLR1 and TLR2. OPCs on the other hand express a limited set of TLRs with high levels of TLR2 and to a lesser extent TLR3 and TLR4. Profiling of 42 cytokines/ chemokines responses indicated that TLR agonists elicit broad and similar cytokine/chemokine response in human adult microglia. In highly purified oligodendrocyte precursors, poly I:C was a potent inducer of oligodendrocytes death and demyelination. LPS and zymosan, in contrast, promoted survival and myelin sheet formation.
Together, these data indicate that TLRs in the CNS play important roles in the regulation of innate immune responses and in oligodendrocyte development and that especially TLR3 acts as a dominant regulator in the CNS. Alzheimer's disease (AD) is a neurodegenerative disease, which is characterised by deposition of amyloid-beta (A-beta) containing plaques and also by neuroinflammatory changes, typified by an increase in pro-inflammatory cytokine expression in the brain. Activated microglia are considered to be the major source of proinflammatory cytokines in AD, and recent studies have highlighted the importance of adaptive immune responses, in particular T cells, in the pathology of this disease. Clinical trials involving active immunisation with A-beta in adjuvants resulted in development of meningoencephalitis in a subset of patients, which was associated with increased infiltration of T cells into the brain. Moreover, A-beta-specific T cells have been identified in AD patients. However the interaction of these A-beta-specific T cells with microglia is poorly understood.
In this study, we have examined the role of Th1, Th2, IL-17producing T cells (Th17) and regulatory T cells (Treg) on A-betainduced microglial activation. We developed methods for generating A-beta-specific T cell subtypes ex vivo. We then assessed the effects of the different T cell subtypes and their secreted cytokines on microglial activation. Microglial activation was determined by measuring the production of the pro-inflammatory cytokines IL-1beta, IL-6, and TNFalpha by ELISA and by quantifying cell-surface expression of MHC class II, CD80, CD86, and CD40 by flow cytometry. The data show that Th1 and Th17 cells enhanced A-beta-induced production of IL-1beta, TNF-alpha and IL-6 from glial cells. They also increased surface expression of MHC class II, CD80, and CD86 on microglia. Th2 and Treg cells induced significantly smaller increases in TNF-alpha and IL-6 production and microglial expression of CD80 and CD86 when compared with Th1 and Th17 cells. Recombinant IFN-gamma and IL-17 stimulated activation of microglia in a manner similar to that of Th1 and Th17 cells respectively, albeit to a lesser extent, while IL-10 suppressed A-beta-induced production of IL-6 and TNF-alpha and CD86 expression.
Our findings demonstrate that both Th1 and Th17 cells can promote microglial activation through production of IFN-gamma and IL-17 respectively and that these inflammatory responses can be suppressed by the regulatory cytokine, IL-10. Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. In the ethidium bromide (EB) demyelinating model, Schwann cells are known to invade and contribute to myelin repair in the spinal cord. As Schwann cell remyelination in the central nervous system is a well known event in this gliotoxic model, the aim of our investigation was to observe the behaviour of such cells after local EB injection in the spinal cord of streptozotocin diabetic rats.
Forty-nine adult Wistar rats were used, from which 28 received a single intravenous injection of streptozotocin (50 mg/kg), being submitted 10 days after to a local injection of 1 μl of 0.1% EB (group I, n = 21) or 0.9% saline solution (II, n = 7) into the spinal cord. A laminectomy was performed on the first lumbar vertebra to allow 1 μl of 0.1% EB solution or 0.9% saline solution to be injected into the dorsal columns by means of a Hamilton syringe. One microlitre of EB was also injected in non-diabetic rats (III, n = 21). Rats from all groups were anaesthetized, perfused through the heart at 7, 9, 11, 15, 17, 21 and 31 days after EB or saline injection and spinal cord sections were collected and processed for light and transmission electron microscopy studies. Comparison between the final balance of myelin repair in groups I and III at 31 days was assessed using a semi-quantitative method for documenting the extent and nature of remyelination in semithin sections from the EB-induced lesions.
Diabetic rats presented delayed macrophagic activity, with greater amounts of myelin-derived membranes in the center of the EBinduced lesions, and lesser remyelination at peripheral areas in comparison to non-diabetic rats. Although oligodendrocytes contributed to myelin reconstruction, invading Schwann cells were the major sources of remyelination and they firstly appeared at 9 days in nondiabetic rats and by 15 days in diabetic rats. Astrocyte behaviour did not seem to be affected by diabetes. Evidence of glial loss and primary demyelination was not noted in rats from group II.
Results indicate that short-term streptozotocin-induced diabetes hindered both Schwann cell and oligodendrocyte remyelination (mean remyelination scores of 2.02 for Schwann cells and 0.71 for oligodendrocytes) in comparison to non-diabetic rats (3.89 and 1.57, respectively). Microglia are resident immune cells of the central nervous system (CNS). Microglia are usually isolated from primary cultures to study their function. This procedure is time consumable and inefficient due to low cell numbers. The microglia-derived oncogenically transformed cell line BV2 was successfully applied in certain experiments, but BV2 cells are limited in several microglial functions. Therefore, we established several microglial cell lines from mouse embryonic stem (mES) cells, which are suitable to study microglia function.
Two mES cell lines (ES-C57BL/6 and MPI-II; derived from 129Sv mice) were differentiated into microglia cell lines. In total, 7 independent cell lines were generated which exhibited a stable microglial phenotype. ES cell derived microglia (ESdM) cell lines were characterized by flow cytometry, immunocytochemistry and functional assays. ESdM were immunoreactive for IBa1, CD11b, CD45, F4/80, CD49d, CD29, CXCR4, MHC class II and B7.2. Furthermore, stimulation with IFN gamma and LPS showed upregulation of proinflammatory cytokines (i.e. iNos, IL1beta and TNFalpha) and downregulation of anti-inflammatory cytokines (i.e. TGFbeta) at comparable levels to primary microglia. ESdM expressed CX3CR1 and showed a concentration dependent migration towards fractalkine. The capacity to migrate and phagocytose Abeta or microsphere beads of ESdM was comparable to primary microglia and superior to BV2 cells. For in vivo characterization, ESdM were lentivirally transduced with green fluorescent protein (GFP) and transplanted into the CNS of neonatal mice. Analysis of engrafted cells after 4 weeks showed a typical ramified microglial-like structure of ESdM and colocalization with the marker protein IBa1. Functionality of ESdM in vivo was shown after transplantation into the entorhinal cortex of aged transgenic APP mice. Sites of transplantation showed decreased plaque size and less amyloidbeta peptide load compared to the non-transplanted contralateral site.
In summary, ESdM have many characteristics of primary microglia making them a suitable tool to study microglial function in vitro and in vivo. Insult to the central nervous system (CNS) leads to initiation of a glial response that is innate to the CNS. This glial response includes the induction of inflammatory cytokines and chemokines, which promote recruitment of leukocytes to the CNS. The transcription factors, signal transducer and activator of transcription (STAT), nuclear factor-KB (NF-KB) and interferon regulatory factors (IRF), are associated with cytokine responses, and signal for cellular growth, proliferation, differentiation, apoptosis and antiviral functions. We have studied the involvement of these transcription factors in glial responses in the hippocampus following transection of entorhinal afferents.
Axonal lesioning induced upregulation of STAT1/2 and IRF7 gene expression in the hippocampus. STAT1/2 upregulation was localized precisely to zones of synaptic degeneration in the dentate gyrus. Whereas STAT1/nuclear STAT1 colocalized with both microglia and astrocytes, STAT2/phosphoSTAT2 staining was restricted to astrocytes within denervated hippocampus. Notably, the induction of IRF7 in lesion-reactive hippocampus was dependent on STAT1 and Type I interferon receptor, and astroglial STAT2 upregulation was NF-KB-dependent. In addition, lesion-induced CCL2 mRNA expression and leukocyte infiltration into the hippocampus were significantly reduced in mice with inactivated NF-KB signalling in astrocytes.
Our findings indicate the involvement of transcription factors in lesion-induced glial responses in the CNS. Neuroinflammatory changes are now acknowledged to be a characteristic feature of neurodegenerative diseases like Alzheimer's disease. These changes are typified by activation of microglia and increased production of proinflammatory cytokines, like Interleukin-1beta (IL-1beta), which are released from activated microglia; in experimental models, these changes are coupled with deficits in cognitive function.
It is now recognized that interaction of microglia with other cells contributes to maintenance of microglia in a quiescent state. We have shown that the interaction between the glycoprotein CD200 which is expressed on neurons, and its receptor, CD200R which is expressed on microglia, is important in modulating microglial activation. The evidence indicates that the anti-inflammatory cytokine, IL-4 increases neuronal expression of CD200 but CD200 expression is decreased in neurons prepared from IL-4 knockout mice (Lyons et al., 2007) .
Here we report that, while addition of neurons prepared from wild-type mice attenuated the LPS-induced release of pro-inflammatory cytokines from glia, neurons prepared from IL-4 knockout (KO) mice failed to do so; we propose that this is due to the decreased expression of CD200 and consequently the decreased interaction between neurons and glia. However addition of CD200Fc attenuated the LPS-induced increase in IL-1beta to a similar extent in mixed glia prepared from wild-type and IL-4 KO mice; this is consistent with the finding that CD200R expression was similar in wild-type and IL-4 KO mice.
Treatment of young and aged, WT and IL-4 KO mice with LPS, resulted in an exaggerated increase in expression of CD40, IL-1beta and IL-6 in hippocampal tissue prepared from aged, compared with young, mice and this was further exacerbated in IL-4 KO mice. These changes were coupled with a reduction in CD200 expression.
These data suggest that IL-4 plays a central role in modulating neuronal expression of CD200 and we propose that the decrease in CD200 expression in tissue prepared from IL-4 KO mice contributes to the accentuated LPS-induced neuroinflammation observed in these animals. To mimic axonal transection in early lesions in MS, T-cells reactive to myelin basic protein (MBP) were transferred to female SJL/N mice, that subsequently were subjected to perforant pathway (PP) lesion in the entorhinal cortex resulting in a dense anterograde axonal and terminal degeneration in the dentate gyrus. Brains from mice with: 1) T-cell transfer and PP-lesion with 7 days survival post lesion, 2) T-cell transfer alone, 3) PP-lesion alone, or 4) unmanipulated control mice, were processed immunohistochemically for T-cells, microglia-macrophages and for MBP to evaluate clearance of myelin debris.
Axonal lesion resulted in a massive infiltration of CD3+, CD4+ and to a smaller extent CD8+ cells in the deafferented dentate gyrus of the PP-lesioned T-cell recipient mice compared to PP-lesioned mice, T-cell recipient mice, and control mice. PP-lesioned T-cell recipient mice also showed enhanced activation of CD11b+ microglia, and signs of enhanced macrophage recruitment. Observation of reduced amounts of MBP+ particles in PP-lesioned T-cell recipient mice compared to PPlesioned mice indicated that the presence of T-cells in deafferented dentate gyrus enhanced microglial-macrophage clearance of myelin debris. This was confirmed by increased numbers of CD11b+ microglia-macrophages containing intracellular MBP+ material as a sign of phagocytosis in PP-lesioned T-cell recipient mice compared to PP-lesioned mice.
Axonal and terminal degeneration distal to sites of axonal transection may precipitate the development of new inflammatory lesions along the anterogradely degenerating axons. Enhancement of microglial-macrophage phagocytotic capacity by T cell infiltration could prove beneficial to regenerative processes following an MS attack. A possible role for inflammation in the onset and progression of neurodegenerative diseases has been suggested. Using the mouse ME7 prion model, we previously showed that systemic inflammation causes "primed" microglia to switch to a pro-inflammatory phenotype. This study aimed to investigate the molecular mechanism associated with this phenotype switch.
Mice infected with the ME7 prion agent were systemically challenged with lipopolysaccharide or saline in the late stage of the disease (18 weeks) and compared to control mice. Hippocampal tissue was collected and subjected to microarray analysis, quantitative-RT-PCR and immunohistochemistry for selected receptors.
In ME7-infected mice a large number of molecules that have a role in the immune response and signalling pathway showed increased expression. These included all four Fc receptors (FcR), which signal through either activating (ITAM) or inhibitory (ITIM) motifs. Systemic inflammation induced a further increase in the activating FcRIII and FcRIV, while FcRI did not change and the inhibitory FcRII was downregulated. The changes in FcR expression were accompanied by entry of IgG into the parenchyma and we have preliminary data showing the presence of (auto)-antibodies in sera of ME-7 infected mice and their capacity to bind neurons.
Systemic inflammation during chronic neurodegeneration changed the ratio of activating and inhibitory FcR expression on microglia, lowering the signalling threshold for cell activation. We hypothesize that cross-linking of activating FcR results in excessive microglia activation and production of cytotoxic molecules that are detrimental to degenerating neurons within the demented brain. The situation may be compounded by entry of circulating IgG (auto) antibodies into the parenchyma and their binding to neurons. This study was supported by Wellcome Trust. Microglia are resident cells of the central nervous system (CNS) that belong to the myeloid lineage. In experimental models of neuroinflammation, microglia have demonstrated limited capacity to function as professional antigen presenting cells (APCs) when compared to dendritic cells (DCs). Human peripheral blood monocytes can differentiate into immature DCs when exposed to granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 and then into mature DCs if subsequently stimulated with lipopolysaccharide (LPS), thereby becoming highly efficient APCs. We addressed the potential of human adult microglia to acquire phenotypic and functional properties of DCs.
Primary cultures of human adult microglia treated with GM-CSF + IL-4 with or without subsequent LPS activation were compared to human monocytes treated under similar conditions for DC phenotypic and functional characteristics. Analogous to monocytes, human microglia modified their surface phenotype upon GM-CSF + L-4 treatment by downregulating CD14, a monocyte marker, and by acquiring CD209, an adhesion receptor of DCs. Unlike monocytes however, treated microglia decreased major histocompatibility complex (MHC) class II expression and did not acquire CD1a, a lipid presenting molecule. Upon LPS stimulation, microglia lacked CD83, a mature DC marker, and only slightly increased costimulatory molecules. LPS-activated monocyte-derived DCs efficiently promoted CD4 T cell proliferation in an allogeneic mixed leukocyte reaction whereas similarly treated adult microglia had a decreased ability to stimulate CD4 T cell proliferation compared with their untreated counterparts. Monocyte-derived DCs produced high levels of tumor necrosis factor (TNF), IL-12 and IL-10 whereas microglia produced IL-10, relatively low amounts of TNF and no detectable IL-12. Monocytes differentiated into DC downregulated MafB mRNA, a transcription factor associated with macrophage differentiation, whereas similarly treated microglia maintained high levels of MafB. However, such microglia remained CD14-and had a low phagocytic activity, suggesting that they did not acquire a complete macrophage or a DC phenotype.
Our data show that conditions inducing efficient professional APC from monocytes generate microglia with more modest APC functions and lesser inflammatory properties, suggesting a potential microglial role in dampening local inflammation within the CNS.
an autoimmune response can be directed against Schwann cell components. Whether Schwann cells promote this response by presenting autoantigens to T-cells remains controversial.
Here, we show by immunohistochemistry that human Schwann cells express the intracellular machinery required for processing and presenting antigens to autoreactive T cells both in vitro and in vivo. Addition of interferon-gamma results in increased expression of antigen processing and presenting molecules in cultured human Schwann cells. Furthermore, Schwann cells in sural nerve biopsies from GBS patients show increased expression of antigen associated transport proteins and antigen presenting MHC classes I and II complexes compared to controls. In coculture experiments rodent Schwann cells expressing ovalbumin as model antigen activate proliferation of ovalbumin specific CD4+ T cells when pre-treated with proinflammatory cytokines.
These data suggest that glia cells of the peripheral nervous system have the ability to process and present antigen and activate autoreactive T cells. Schwann cells may therefore promote local autoimmune responses in inflammatory neuropathies. Specifically targeting this pathological function of Schwann cells may in the future extend our therapeutic options in GBS. Astrocyte and microglial responsiveness to TLR ligands have been addressed in vivo and in vitro. Astrocytes were purified by flow cytometric cell-sorting from transgenic mice expressing EGFP under the control of a GFAP promotor. Microglia were sorted as CD45-dim cells. Quantitative real-time PCR was used to measure gene expression by astrocytes and microglia isolated from naive mice and from mice that received ligands for TLR3 (Poly-I:C) and TLR4 (LPS) by intrathecal injection. As expected naive microglia expressed a wide range of TLR receptors and co-receptors (e.g. TLR's 1, 2, 3, 4, 6, and CD14), and naive astrocytes predominantly expressed message for TLR3 and TLR6. At 18 h following injection of LPS or Poly-I:C, both astrocytes and microglia showed significant upregulation of TLR2 message. Consistent with published reports astrocytes expressed relatively low levels of TLR4 and CD14 mRNA. However, at the dosage used, LPS was as effective as Poly-I:C in induction of TLR2 expression by astrocytes.
To examine the role of microglia in the astrocyte response, we measured LPS responsiveness of astrocytes in primary cultures containing variable proportions of both cell types. Astrocytes in cultures containing N30% microglia responded to LPS by upregulating TLR2 3-fold, whereas in cultures with b5% microglia, TLR2 was not induced on astrocytes, although in both cases expression was strongly upregulated on microglia. Astrocytes were shown to respond to Poly I:C stimulation by a 2-fold induction of TLR2 in cultures containing b5% microglia and 6-fold induction in cultures with N30% microglia. This suggests that astrocytes upregulate TLR2 in response to direct stimulation and to factor(s) released from TLR-signaled microglia. Absence of TLR signals in the sterile injury model could explain the lack of TLR2 on astrocytes. We are currently investigating the factor(s) and signalling pathways involved. Microglia, the resident immune cells of the central nervous system, are normally quiescent but become activated after infection or injury. Their properties then change and they promote both repair and damage processes. The extent of microglial activation is regulated, in part by activation-induced cell death (AICD). Although many apoptotic aspects of the microglial AICD mechanism have been elucidated, little is known about the connection between the activation step and the death process.
Employing a DNA microarray approach we identified the ectoenzyme CD38 as a potential mediator of microglial activation and AICD. Using mouse primary microglial cultures, we show that the CD38, via its calcium-mobilizing metabolite cyclic-ADP-ribose (cADPR), helps promote microglial activation and AICD induced by LPS plus IFNgamma (LPS/IFNg), suggesting that CD38 links the two processes. Accordingly, CD38 expression and activity, as well as intracellular calcium concentration ([Ca2+]i) in the primary microglia were increased by LPS/IFNg treatment. Moreover, CD38 deficiency or treatment with cADPR antagonists conferred partial resistance to LPS/IFNg-induced AICD and also reduced [Ca2+]i. Microglial activation, indicated by induced expression of nitric-oxide-synthase-2 mRNA and production of NO, secretion and mRNA expression of TNFα and IL-12 p40, and expression of IL-6 mRNA, was attenuated by CD38 deficiency or cADPR-antagonist treatment. The observed effects of CD38 on microglial activation is probably mediated via a cADPRdependent increase in [Ca2+]i, and the effect on AICD by regulation of NO production.
Our results thus suggest that CD38 significantly affects regulation of the amount and function of activated microglia, with important consequences for injury and repair processes in the brain. In the adult mammalian brain, multipotent and self-renewing neural progenitor cells (NPCs) may generate new neurons, astrocytes and oligodendrocytes. NPCs may thus serve as a regenerative tool by which brain damage could be compensated. However, repair processes in response to inflammatory (and other) forms of brain injury are characterized by the failure of neuronal regeneration and the predominant occurrence of astrocytes (known as astrogliosis). Moreover, inflammation, which generates an oxidative milieu, has recently been reported to directly inhibit neurogenesis within the brain. We therefore intended to clarify possible molecular pathways underlying inflammation-mediated inhibition of neurogenesis and enhancement of astrogenesis in chronic autoimmune neuroinflammation.
We systematically investigated the effects of redox modulation in neural stem cells in vitro and in vivo, as well as in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We found that subtle (non-toxic) oxidation suppressed proliferation of NPCs and directed their differentiation towards the astroglial lineage at the expense of the neuronal (and vice versa). Our findings indicate that oxidation plays an instructive role in directing uncommitted NPCs to differentiate towards the astroglial lineage by altering their "default" development program. According to our data, Sirt1, a class III NAD+-dependent histone deacetylase involved in various cellular processes that comprise energy metabolism, transcriptional silencing, and DNA repair, mediates the observed redox effects, as mild oxidation in NPCs leads to increased Sirt1 deacetylase activity at the promoter of the proneuronal transcription factor Mash1. Furthermore, ongoing analysis of Sirt1 regulation in the course of EAE will be presented.
Our results provide evidence for an as yet unknown metabolic master switch which has an impact on the fate of neural progenitors under inflammatory conditions.
14 -Role of CD38 in IL-1beta-induced astrocyte glutamate clearance impairment Astrocytes are numerically the most abundant cell-type in the central nervous system (CNS). One of the most essential functions of astrocytes is to regulate the level of extracellular glutamate, the major excitatory neurotransmitter in the mammalian CNS. Astrocytes express glutamate transporters, most prominently excitatory amino acid transporter 2 (EAAT2), which performs the vast majority of synaptic glutamate clearance. This prevents excitotoxicity, the overactivation of glutamate receptors on the cell surface of neurons. However, during neuroinflammatory conditions, cytokines have been shown to inhibit astrocyte glutamate uptake. CD38 is a 45 kD ectoenzyme involved in the synthesis of potent calcium mobilizing agents, cyclic adenosine diphospho-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP+). Our previous studies demonstrated that astrocyte CD38 expression was upregulated upon immune or viral stimulation and, it played an important role in astrocyte inflammatory responses upon cytokine stimulation. We performed this study focusing on the effects of IL-1beta, a proinflammatory cytokine on the expression of EAAT2 in astrocytes and its results on astrocyte glutamate uptake ability. The potential role of CD38 in this effect was also tested.
Primary human astrocytes were cultured and treated with IL-1beta. The glutamate uptake ability of astrocytes was determined by performing an Amplex Red Glutamic Acid/Glutamate Oxidase Assay. The astrocyte IL-6, IL-8, and MCP-1 production was examined by ELISA. EAAT2 expression in astrocytes was tested by real-time PCR and immunocytochemical analyses. Our results showed that IL-1beta induced astrocyte activation as indicated by significant increases in production of cytokine/chemokines such as IL-6, IL-8 and MCP-1. Furthermore, we detected that IL-1beta time-dependently reduced glutamate uptake ability of astrocytes, which was completely reversed by co-incubation with 8-Br-cADPR, a specific cADPRantagonist. We also found a decrease in astrocyte EAAT2 expression induced by IL-1beta, which was reversed by co-incubation with 8-Br-cADPR.
These results indicate that the glutamate clearance capability of astrocytes is impaired by cytokine activation and the decreased expression of EAAT2 in astrocytes contributes to this impairment. In addition, we provide new evidence that CD38 upregulation in immune-activated astrocyte contributes to its functional dysregulation during neuroinflammatory conditions. In HIV-1-infected patients in the post-HAART era, a new syndrome, Immune Reconstitution Inflammatory Syndrome (IRIS) has emerged. In some patients it may rapidly progress toward the development of severe encephalitis, and ultimately death, even in the absence of opportunistic infections. CNS-IRIS is largely mediated via activated-T cells, which differentiates it from HIV-1 encephalitis that is accompanied by macrophage infiltration and a paucity of T cells. This paradoxical infiltration of the brain with T cells in patients who are immune suppressed, with low peripheral CD4 cell counts due to HIV-1 infection, represents a diagnostic challenge and a treatment dilemma.
To determine the mechanism of T cell activation in IRIS, lymphocytes from normal donors were seeded into a 96-well plate at 2 × 105 cells/well, and treated with 200 nM HIV-1-encoded Tat (transactivator of transcription) in the presence or absence of 10 μM TPCK (NFkB blocker), 100 nM RO-32-0432 (pan-PKC inhibitor), 10 μM chloroquine, 10 μM pyramethamine, or CD3/CD28 antibodies. To map the epitope of Tat that activates T cells, lymphocytes were exposed to 15-mer Tat peptides derived from HIV-1 clade B (BRU strain) overlapping by 10 amino acids. As an indicator of T cell activation, granzyme B levels were measured in culture supernatants by ELISA. The mean + SEM was determined from 5 donors and analyzed by ANOVA with Dunnet correction. The data indicate that Tat activated T cells in a dose-responsive manner. This activation required the NF-kB pathway (p = 0.032), but was independent of PKC activation. The endocytotic pathway was important in Tat-mediated activation of T cells, as treatment with chloroquine decreased T cell activation by Tat (p = 0.021). No effect was seen with pyramethamine. The cysteine rich region of Tat was critical in activation of CD4+ and CD8+ T cells (p = 0.0039; p = 0.0134, respectively).
HIV-1 Tat activates T cells to secrete effector molecules, as exemplified by granzyme B release. The mechanism of Tat-induced activation is dependent on endocytosis of Tat and NF-kB signaling, which may represent novel therapeutic targets to treat IRIS. The cysteine rich region of Tat is critical for mediating these effects. The neurologic symptoms associated with dengue fever are many and have been recognized for more than a century. Symptoms range from drowsiness, irritability, short term memory, and depression, through mononeural palsies, to encephalitis seizure and death. Optic spinal syndrome following Monophasic neuromyelitis optica (NMO) has been observed in Dengue virus infection, suggesting an influence of the genetic background in the susceptibility to post-NMO. Despite the high prevalence of tropical/subtropical countries there is no vaccine/medicine available for the disease.
Heat Shock Proteins (Hsps) are highly conserved molecules and expressed in all cell types under various stressful conditions. The objective of the present study was to determine the effect of Dengue virus infection on relative expression of Hsps and their role in the progression of the infection.
As macrophages are the primary host for Dengue, human promonocytic myeloblastoma U937, monocytic THP1 cells and blood derived macrophages were infected with Dengue virus type 2 for the evaluation of Hsp expression. A significant expression of Hsp60 was observed in virally infected U937 cells where as Hsp70 was predominantly expressed in blood derived macrophages and THP1 cells. In order to determine the correlation between Hsp60/70 expressions and viral multiplication in infected cells, expression of Hsp60 and 70 was down regulated by RNA interference. Accumulation of activated macrophages and microglia is a hallmark of viral encephalitis, however mechanisms of recruitment and function during infection of the brain are poorly defined. While leukocyte infiltration is critical for virus eradication, these cells can cause irreparable damage to neurons, contributing to increased morbidity and mortality of the host. West Nile Virus (WNV) encephalitis has a significant immunopathological component, however this is poorly understood. This study investigates the role of inflammatory monocytes in WNV infection.
Intranasal inoculation of WNV resulted in a 3-fold increase in CD11b+/ CD45lo-int/CD11c-microglia and a 5-fold increase in CD11b+/CD45hi/ CD11c-/Ly6G-macrophages in the brain at d7 post infection (p.i.) by flow cytometry. Bromodeoxyuridine pulsing showed that few microglia had undergone proliferation in situ. Depletion of GR1+/CD11b+/CCR2+ peripheral inflammatory monocytes during infection abrogated increases in both microglia and macrophages, suggesting that they share a circulating precursor. At d7 p.i. C57BL/6 chimeras recon-stituted with c-fms/EGFP bone marrow showed large numbers of GFP+ peripherally-derived CD45lo-int microglia and CD45hi macrophages, expressing a GR1+ (Ly6C+) phenotype in the brain, suggesting that these cells have a common inflammatory monocyte precursor. Furthermore, adoptively transferred inflammatory monocytes migrated to the brain and expressed microglial and macrophage phenotypes during WNV infection, confirming this. Inflammatory monocyte-derived macrophages and microglia utilised different adhesion molecules to enter the infected brain. Blockade of Very Late Antigen-4 (VLA-4) reduced macrophage immigration by 66% and resulted in 10% long term survival in this lethal model. Lymphocyte Function-associated Antigen-1 (LFA-1) blockade reduced macrophage immigration by 33%, but did not improve survival. Importantly, VLA-4 or LFA-1 blockade had no impact on numbers of immigrant microglia. The majority (75%) of macrophages infiltrating into the brain produced nitric oxide (NO). Treatment with aminoguanidine on d6 p.i. resulted in a 6-fold reduction in numbers of NO-producing macrophages, with an increase in mouse survival of up to 4 days.
Together these data indicate a role for NO-mediated immunopathology by infiltrating macrophages in WNV encephalitis. Herpes simplex encephalitis (HSE) is the most commonly identified cause of acute, sporadic non-epidemic encephalitis and is characterised by severe swelling of the brain tissue. The purpose of inflammation is to restore homeostasis by eliminating invading pathogens and promoting recovery from tissue damage. Cytokines and other immune mediators play an essential role in CNS inflammation through the activation of chemokines and adhesion molecules, recruitment of immune cells into the parenchyma and stimulation of endogenous glial cells. If this inflammatory response however, is not regulated adequately it can lead to chronic tissue damage caused by the infiltration of peripheral lymphocytes into the CNS and collagen deposition.
The aim of this study was to provide a controlled in vitro system for measuring the cytokine response upon HSV infection by means of a cytometry-based assay allowing for multiplexed analysis, and to finally investigate the effect of anti-inflammatory agents on cytokine levels.
A cell model comprising pure murine microglia, astrocytes, neurons and a co-culture of these cell lines was established to explore the cytokine response upon infection with the moderately neurovirulent HSV-1 strain VR3 and the highly neurovirulent strain HSV-1 SC16. The cell culture supernatants taken at indicated time points were subsequently probed for a total of 23 cytokines utilising a commercially available Bio-Plex assay and compared with each other.
Cells infected with SC16 generated a more vigorous cytokine response compared to cells infected with VR3 with microglia producing by far the highest cytokine levels. Contrary to previous studies the experiment clearly indicated neurons and astrocytes to sense infection however, synthesis was restricted to certain types of cytokines at low concentrations.
In addition, compared to pure microglia the co-culture revealed a shift in cytokine expression, with a marked decrease in KC (functional IL-8) levels suggesting cross-talk between individual cell types.
As the cytokine cascade represents a vast inter-dependent network the study of multiple cytokines provides an improved insight into their role in the pathophysiology of HSV CNS infection and the Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) during the early stages of infection where it triggers a profound humoral immune response distinguished by plasmablast infiltration and intrathecal IgG synthesis. The aim of our study was to characterize the memory B cell receptor usage in HIV-1 cerebrospinal fluid (CSF).
Single-cell RT-PCR was used to sample CSF CD27+CD19+ memory B cell repertoires, including CD27^high plasmablasts, from a treated and an untreated HIV patient. Sequencing of amplified variable region heavy-(VH) and light-chain (VL) products identified the rearranged variable region and J segment and determined the degree of homology to germline sequence. Expanded memory B cell clones were detected in the CSF of both HIV-1 subjects. Both HIV-1 repertoires demonstrated a similar distribution of VH and VL family germline usage: VH3 family heavy chains and VK1 family light chains dominated their respective repertoires. Treated and untreated HIV-1 repertoires, however, showed significant differences in the amount of B cell clonal expansion (21.2% vs. 32.3%, p = 0.02) and the degree of VH germline homology (95.7% vs. 92.3%, p = 0.0002). The increased memory B cell clonal expansion and somatic hypermutation observed in the untreated subject were associated with the presence of intrathecal HIV-1 virus and increased numbers of CSF plasmablasts.
HIV-1 infection elicits a prominent CNS memory B cell response. Clonal expansion and somatic hypermutation are enhanced in the presence of intrathecal virus consistent with a targeted antibody response against HIV-1. Rearranged VH and VL sequences in clonally expanded HIV-1 B cells may be used to generate recombinant antibodies that recreate the epitopic specificity of the humoral immune response in treated and untreated disease. with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in extensive OPC migration and remyelination. As OPC are transplanted into an environment in which a persistent virus is present, it is possible that these cells may be susceptible to infection. Therefore, the experimental goals of this study were to determine if OPC were able to support JHMV replication and if cytokine treatment affected viral growth.
The present study demonstrates that JHMV is able to infect and replicate within primary cultures of OPC derived from neural progenitor cells. In addition, treatment of infected OPC with recombinant mouse IFN-gamma inhibits viral replication in a dosedependent manner. Although IFN-β production (mRNA and protein) is muted in JHMV-infected OPC, IFN-β is produced by IFN-gammatreatment of JHMV-infected cells and recombinant IFN-β inhibits viral replication. Finally, IFN-gamma is unable to control JHMV replication in OPC derived from type I receptor knock-out mice.
These data indicate that JHMV is capable of infecting OPC generated from neural progenitor cells and IFN-γ-mediated control of viral replication is dependent on IFN-β secretion.
identified as a cellular receptor for some strains of MV. Inhibition of cell proliferation through engagement of MV with CD46 has been proposed as an important mechanism of MV-induced immunosuppression. Additional cells exhibiting immuno-suppressive activity may be involved in the mechanisms responsible for MV-induced SSPE as well.
In this study, MV-induced immuno-suppression has been evaluated by the characterization of peripheral blood cells (PBC) from SSPE patients compared to controls. PBC of totally 70 SSPE patients (31 girls/39 boys, mean age: 8.3 years) and 55 children (20 girls/ 35 boys, mean age: 10.4 years) with non-inflammatory or inflammatory diseases were stained and phenotypical cell subsets have been These results implicate that CD46 may also be used as a cell entry receptor by some strains of MV which cause SSPE. The lower presence of the inhibitory NK receptors on CD8+ cells in SSPE may be caused by altered regulation of CD8+ T cells as well.
The present work was supported by the Research Fund of Istanbul University (Project #: 192).
Maingat Ferdinand 1 , Viappiani Serena 1 , Zhu Yu 1 , Afkhami-Goli Amir 1 , Power Christopher⁎ 1 1
Infection by the human immunodeficiency virus (HIV) results in neurodegeneration caused by aberrant activation of glial cells although the impact of concurrent systemic infections on neurological disease remains uncertain. We studied the role of recurrent immune activation on neurological function and viral replication in the brain using an HIV/ AIDS model, feline immunodeficiency virus (FIV) infection.
We administered lipopolysaccharide (LPS), a component of the membrane of gram-negative bacteria, to cats infected with FIV at 7, 9 and 11 weeks post-infection. Following serial neurobehavioral assessments, protein and mRNA were extracted from cerebral cortex and basal ganglion for immunoblotting of neuronal markers and RT-PCR analyses of inflammatory genes. FIV-infected and uninfected animals differed significantly on multiple neurobehavioral tasks of gait and memory over time, which was associated with reduced weight gain and immunosuppression in the FIV-infected group. FIVinfected cars treated with LPS developed less severe neurobehavioral deficits (mean deficit score 1.4 +/− 0.2 in FIV-LPS group versus 3.6 +/ − 0.2 in FIV group. CD4+ T cell levels in blood were suppressed in both FIV infected groups compared to uninfected animals. However, CD8+ T cell levels in blood were also lower in blood which was accompanied by reduced CD3e transcript abundance in brain among the LPS-treated FIV-infected animals relative to untreated FIV-infected animals. The presynaptic protein, synaptophysin, and the vesicular acetylcholine transporter were reduced in FIV-infected animals together with upregulation of mRNA for chemokine, IP10, in neurons of both basal ganglia and cortex of FIV-infected animals but these effects were also reversed by LPS treatments.
Repeated LPS exposure improved neurological function and rescued synaptic architecture in FIV-infected animals through a mechanism involving reduced viral replication and concomitant T cell infiltration of the brain. Our findings underscore the beneficial aspects of selective activation of innate immunity in neurological disease.
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, United States Microglia and astrocytes are important effector cells of the CNS innate immune response to parenchymal infections. The Toll-like receptor (TLR) family of pattern recognition receptors (PRR) facilitates the recognition of conserved antigenic motifs of various pathogens, leading to the downstream production of inflammatory mediators. TLR2 is pivotal for glial recognition of S. aureus, a prevalent CNS pathogen, and eliciting proinflammatory mediator release. Recent studies from our laboratory have revealed a correlation between the loss of TLR2 signaling and aberrant Th17 infiltrates in the S. aureus brain abscess model, which may provide insights as to how TLR2 interfaces with adaptive immunity during CNS parenchymal infections. Since IL-17 exerts many proinflammatory activities, we propose that the exaggerated release of IL-17 in TLR2-deficient mice is a compensatory mechanism to mount an effective anti-bacterial immune response in the face of the loss of this important PRR. In other studies, signaling via the TLR/IL-1R adaptor MyD88 was essential for the induction of CNS innate immunity in the experimental brain abscess model. Specifically, the absence of MyD88 signaling significantly impaired the production of a wide range of proinflammatory molecules, attenuated neutrophil and macrophage influx into the CNS, and resulted in the exaggerated necrosis of infected tissue. Collectively, these studies reveal important roles for the TLR/IL-1R family of molecules in regulating both innate and adaptive immune responses during gram-positive infections in the CNS parenchyma.
Sydenham chorea is the major neurologic sequelae of group A streptococcal-induced acute rheumatic fever (ARF) and is most likely due to autoimmunity and molecular mimicry between host and pathogen. Our first clue to the pathogenesis of the disease was the identification of human monoclonal antibodies(mAb) derived from Sydenham chorea that demonstrated crossreactivity and specificity for group A streptococcal antigen and reacted with striatal tissue and brain antigens lysoganglioside and tubulin. mAb and acute Sydenham chorea sera and CSF targeted the surface of human neuronal cells and caused induction of calcium-calmodulin dependent protein (CaM) kinase II activity and dopamine release. Sera from other streptococcal diseases or sequelae and convalescent Sydenham chorea sera did not demonstrate such increased antibody reactivity with brain or antibody mediated signaling of CaM kinase II. Primary neuronal cells from striatal tissue demonstrated increased CaM kinase II activity when reacted with acute Sydenham chorea sera or CSF. New preliminary data suggest that dopamine D1 and D2 receptors are targeted by the crossreactive anti-brain mAbs and sera. Serum antibodies from related disorders such as Pediatric Autoimmune Neurologic Disorder Associated with Streptococci with obsessive compulsive behaviors, vocal tics or tic-like movements demonstrated that the level of the CaM kinase II activity was increased at an intermediate level and the reactivity with lysoganglioside was not as evident although present in some patients. However, comparison of matched acute and convalescent PANDAS sera taken before and during the exacerbation demonstrated an elevation in CaM kinase II activity associated with disease. Study of an animal model of Sydenham chorea revealed that immunization with group A streptococcal antigen led to behavioral changes which correlated with antibody deposition in the striatum as well as reactivity of serum IgG with dopamine receptors and the induction of CaM kinase II activity. These data are consistent with the current hypothesis that disinhibition of basal ganglia-thalamo-cortical pathways may be involved in Sydenham chorea and with its treatment with haloperidol, a D2 receptor antagonist.
Department of Neurology, Johns Hopkins University, Baltimore, MD, United States Some patients with HIV infection when aggressively treated with antiretroviral drugs develop a paradoxical subacute encephalitis. The development of this syndrome coincides with a drop in viral load and recovery of T cell counts in the blood. Left untreated it may result in death over a few weeks. Neuropathological evaluation shows massive infiltration of T cells with a predominance of cytotoxic and CD8 cells. This is associated with glial cell activation and neuronal injury but relative sparing of myelin. To study the pathophysiology of this syndrome, we first determined the mechanism of activation of T cells by an HIV protein, Tat that is not impacted by antiretroviral drugs. Tat-mediated activation of T cells occurs independent of T cell receptors but is NF-kB dependent. Using human neuronal cultures, we found that supernatants from these activated T cells are neurotoxic. Granzyme B is a major neurotoxic substance released by the activated T cells. Granzyme B causes neuronal injury independent of perforin and the mechanism of neuronal injury by granzyme B sequentially involves the activation of a Gi-protein coupled receptor, notch-signaling pathways and over expression of potassium channel, Kv1.3 followed by neurite trimming and neuronal cell death by apoptosis. Pharmacological or molecular blockade of Kv1.3 results in protection against T cell mediated neuronal injury. Previous studies have suggested a role for Kv1.3 in activation of T cells. Thus, Kv1.3 represents a novel therapeutic target for this syndrome. Bone marrow stern cell in the rescue of brain diseases
Microglia are the immune cells of the central nervous system. They patrol the brain environment with their ramifications and they respond quickly in the presence of pathogens and brain damages. Bone marrow derived cells (BMDCs) have the ability to populate the CNS and differentiate into functional parenchymal microglia as well as perivascular microglia. Even though BMDCs can enter the brain parenchyma throughout the CNS in normal mice, it seems that they are preferentially attracted to regions afflicted by neurodegeneration or neurological insults. Of great interest is the fact that blood-derived microglial cells are associated with amyloid plaques and these cells are able to prevent the formation or eliminate the presence of amyloid deposits in mice that develop the major hallmark of Alzheimer's Disease (AD). These cells are also recruited in the brain of other mouse models of brain diseases and acute injuries. The exact mechanisms involved in these events have yet to be fully characterized. It is likely that stressed or damaged neurons send signal to resident microglia or produce specific chemokines to cause recruitment of BM-derived microglia. A number of proteins can act as endogenous ligands for tolllike receptors (TLRs) expressed on the surface of microglia and then trigger NF-kB signaling and gene transcription. This would necessarily lead to a subtle inflammatory response and consequently proliferation and chemoattraction of myeloid cells. Of interest is the fact that there is no need of a massive neurodegeneration and BBB breakdown. We can therefore propose that neurons, especially those in danger, have the ability to interact with microglia and stimulate their proliferation and recruitment, although the physiological relevance of this phenomenon is still largely unknown. We can speculate that this natural process takes place to provide survival signals and help to stressed, injured or infected neurons. A timely controlled innate immune response may also limit CNS toxicity by eliminating foreign materials and debris, thus contributing to create an environment that is more permissive for regeneration and recovery. Since toxic and secreted proteins are frequently the cause of many brain diseases, such a cellular therapy may prove to be quite effective to prevent and remove the main trigger of neurodegeneration. Hematopoietic stem cells represent therefore a fantastic new vehicle for delivering key molecules to improve recovery, repair and elimination of toxic proteins.
The Canadian Institutes in Health Research (CIHR) supports this research. Transforming growth factor-beta TGF-beta) has pleiotropic effects, both beneficial and harmful in the central nervous system (CNS). Transgenic mice overproducing this cytokine from astrocytes have higher numbers of T cell in their brains without showing overt signs of inflammation. However, when immunized against an endogenous or ectopically expressed CNS antigen, T cell infiltration and inflammation are aggravated. Consistent with these observations, TGF-beta1 synthesis in glial cells and TGF-beta signaling in the CNS were activated several days before the onset of paralysis in mice with autoimmune encephalomyelitis. Sequentially, after glial cells were modulated, TGF-β1 signals then were induced in infiltrating T cells in inflammatory lesions. Pharmacological inhibition of TGF-β signaling ameliorated the paralytic disease, reducing the accumulation of pathogenic T cells and expression of IL-6 in the CNS. Priming of peripheral T cells was not altered, nor was the TH17 pathway, indicating that this effect was directed within the brain, yet affected the immune system. The early production of TGF-β1 in the CNS creates a permissive and dangerous environment for the initiation of autoimmune inflammation. Inhibition of TGF-β signaling may have benefits in the treatment of the acute phase of autoimmune CNS inflammation. Late pregnancy is beneficial in MS. The third trimester has been associated with an approximate 80% reduction in relapse rate. Estriol, the estrogen unique to pregnancy, reaches highest levels in the third trimester. Estriol treatment was previously shown to ameliorate experimental autoimmune encephalomyelitis (EAE) when used at a dose equivalent to what occurs naturally during late murine pregnancy. Numerous mechanisms are involved in estriol mediated protection from disease. These include both anti-inflammatory as well as neuroprotective mechanisms. Anti-inflammatory mechanisms have previously included the induction of favorable changes in cytokines, chemokines and T regulatory cells. Here we will present our recent observations of effects of estriol treatment on matrix metalloproteinase-9 (MMP-9). Neuroprotective effects include a sparing of axon densities and myelin white matter, as well as neuronal staining in gray matter. Estrogen receptor (ER)-alpha and ER-beta differentially mediate these anti-inflammatory and neuroprotective effects. Together these observations in EAE provide a rationale for a clinical trial of treatment with estriol, at a pregnancy dose, in relapsing remitting multiple sclerosis (RRMS). This trial will include both antiinflammatory as well as neuroprotective outcome measures. A distinct female predominance exists for a variety of human autoimmune disorders, including multiple sclerosis (MS). This differential gender susceptibility has also been observed in many experimental animal models of autoimmune disease, including experimental autoimmune encephalomyelitis (EAE). The increased levels of sex hormones produced during pregnancy are reported to reduce the clinical symptoms of MS. Interestingly, the clinical symptoms of MS worsen post-partum, which is marked by reduced sex hormone levels. The work from our laboratory illustrates the large number of complex processes affected by estrogen that might contribute to the striking ability of 17-beta estradiol (E2) and its derivatives to inhibit clinical and histological signs of EAE in mice. These effects require sustained exposure to relatively low doses of exogenous hormone and offer better protection when initiated prior to induction of EAE, although oral ethinyl estradiol (EE) could partially reverse clinical EAE when given after the onset of disease. The main areas to be discussed include E2-mediated inhibition of encephalitogenic T cells, the role in E2 regulation of the newly described GPR30 membrane receptor (mER) and the E2 agonist G1, inhibition of inflammatory cell migration into central nervous system tissue, neuroprotective effects that promote axon and myelin survival, and finally new data showing synergistic treatment effects of E2 in combination with T cell receptor peptide therapy in MS patients. E2 effects on EAE were mediated through Esr1 (alpha receptor for E2) and GPR30, but not Esr2 (beta receptor for E2) and resulted in the dramatic down-regulation of IL-17 recently demonstrated to be the major pathogenic cytokine in EAE. Chimera studies demonstrated that E2 does not directly inhibit pathogenic T cells, but rather mediates its suppressive effects through other cell types, including macrophages and dendritic cells. Of mechanistic importance, immunosuppression that can prevent EAE also involves E2-dependent up-regulation of Foxp3 and PD-1 that contribute to the activity of CD4+CD25+ regulatory T cells (Treg). New findings indicate that G1, an E2 agonist that binds selectively to mER (GPR30), can mediate full protection against EAE by upregulating the PD-1 pathway in Treg cells as well as the antiinflammatory cytokine, IL-10. The protective effects of E2 and E2 agonists in EAE suggest their potential use as therapy for MS.
Fred Hutchinson Cancer Research Center, and the University of Washington, Seattle, WA USA Microchimerism (Mc) refers to harboring a small number of cells (or DNA) that originated from another genetically distinct individual. Some cells traffic between the mother and fetus during pregnancy and have been found to persist many years later in respective hosts. Hematopoietic cell transplantation (iatrogenic chimerism) is often complicated by chronic graft-vs.-host disease which is a condition that resembles some autoimmune diseases and is strongly determined by the donor-recipient HLA-relationship. Considering these observations together led to the hypothesis that naturally acquired Mc from pregnancy and the childmother HLA relationship impacts risk of subsequent autoimmune disease. Mc can also be acquired from a twin or from a blood transfusion, and possibly from an older sibling transferred via the maternal circulation to a later pregnancy. Antibodies and the brain: Lessons from lupus Betty Diamond B. Diamond, P. Huerta, C. Kowal, J. Lee and B Volpe A subset of anti-DNA antibodies present in lupus patients crossreacts with NMDA receptors (NMDARs) and modulates NMDAR function, mediating excitotoxic death of neurons both in vitro and in vivo. These antibodies are preset in serum of 30-50% of lupus patients and can also be found in their cerebrospinal fluid and brain tissue. We have demonstrated that these antibodies, when present in the circulation cause no brain injury until there is a compromise to the integrity of the blood-brain barrier. They then may cause a memory deficit or behavior disturbance depending on whether they penetrate hippocampus or amygdala, respectively. Histopathologic analysis displays a non-inflammatory loss of neurons.
These antibodies, via transplacental transmission, can cause abnormal fetal brain development, leading to a thin cortical plate, delayed acquisition of normal reflexes and impairments in adulthood in isolated tasks that depend on an intact cortex.
Overall, these studies demonstrate the capacity of antibodies to cause acquired changes in cognition and behavior and to cause congenital nongenetic alterations in fetal brain development. We speculate that brainreactive antibodies may often be present and, therefore, be a frequent threat to optimal brain function or development.
In myasthenia gravis (MG), a T-cell dependent antibody response is directed against acetylcholine receptor (AChR) at the neuromuscular junction. To study immune tolerance to this autoantigen, we generated a transgenic mouse in which the AChR alpha chain was expressed in the normal physiological pattern (muscle N brain ≫ thymus) and used this model to assess the mechanisms regulating tolerance to AChR. Since AChR is expressed in the thymus, we expected that central tolerance would dominate, but several lines of evidence suggest that peripheral mechanisms may play a more significant role. First, T cell tolerance is maintained with age in spite of thymic involution; responses to the self determinant, but not to foreign antigens, were still seen in very old mice (28-30 months of age). Second, T cell tolerance remains functional in Aire knockout animals carrying the transgene. Third, when crossed to mice carrying a transgenic TCR specific for this ligand, tolerance was easily broken. Fourth, adoptive transfer of TCR transgenic T cells labeled with CFSE revealed a slight decrease in the number of cell divisions seen in an AChRα transgenic host as opposed to a non-transgenic host. Fifth, using thymus transplant approaches, we demonstrated that T-cell tolerance to AChRα is dependent on transgene expression in the muscle, but not in the thymus. Lastly, the tolerant state could be overcome by treating the mice with anti-CD25; this is consistent with a mechanism of tolerance that is regulated peripherally by regulatory T (T reg ) cells. To explore this further, a second transgenic founder line was generated in which the AChRα transgene is expressed at a slightly higher level (2-3 fold) in both muscle and thymus. Preliminary thymus transplant and adoptive transfer experiments with this new founder line suggest that central tolerance may also be involved. Ongoing studies with the second transgenic line are designed to assess whether Aire and/or T reg cells play a role and if so, whether the level of expression and/or the distribution of the ligand among thymic epithelial subsets is critical. This work was supported in part by an APC Subcontract, pilot grants from the San Antonio Area Foundation and the Myasthenia Gravis Foundation of America, a UTHSCSA ERC grant, and NIH R36 AG029667.
Classical complement pathway and IL-6 in autoimmune myasthenia gravis pathogenesis P. Christadoss Mice deficient for complement factors C3, C4 or C5 are resistant to CD4 cell-dependent antibody mediated autoimmune myasthenia gravis (EAMG) induced by CFA-acetylcholine receptor (AChR) immunization. AChR immune C3 deficient mice lymph node cells (LNC) produced less IL-6 and EAMG resistant IL-6 deficient mice had less serum C3. Moreover, increased serum C1q-circulating immune complex (CIC) levels correlated with EAMG disease severity in RIIIS/J mice. The CIC promotes EAMG severity by stimulating the production of IL-6, C1q and C3 via Fc gamma receptor interaction. However, MBL and C5a receptor deficient mice were equally susceptible to EAMG as wild-type mice, implicating that MBL pathway and C5a are not involved in EAMG development. Both complement accumulation and IL-6 up-regulation were also observed in the recently discovered CD4-independent EAMG in C57BL6 mice induced by LPS-AChR immunization. Anti-C1q antibody administration before and following AChR immunization suppressed EAMG by reducing LNC IL-6 production and neuromuscular junction deposits of IgG, C3 and C5b-C9 complexes. Treatment with anti-C1q antibody twice a week for four weeks in mice with ongoing clinical EAMG reduced the clinical severity of disease and LNC IL-6 production. Anti-IL6 antibody administration suppressed EAMG by reducing anti-AChR Ig2b antibodies which bind complement. Therefore, agents which block the CCP factors C1q, C2 or C4 and/or IL-6 could be used in the targeted therapy of MG.
Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan MuSK antibodies were found in seronegative MG patients, but the experimentally autoimmune MG model was required to show the pathogenicity of MuSK antibodies in MG. We have provided the evidence that active immunization with MuSK protein reproduces myasthenia gravis in rabbits. Next we focus on how MuSK antibodies cause MG. The pathogenic roles of MuSK antibodies in MG have been questioned as the number of AChRs is not reduced and complement is not deposited at the NMJ of biceps brachii muscles MuSK-positive patients with MG. The mechanisms of MG caused by AChR antibodies are well delineated, but the revealed mechanisms are not able to simply apply to MG with MuSK antibodies. MuSK antibodies have been identified as predominantly IgG4 subclass, which does not activate complement. We found that agrin-induced clustering of AChR was strongly blocked in the presence of MuSK antibodies, whereas absorption of the antibodies with purified MuSK products prevented this blocking effect. These results showed that the MuSK antibodies effectively inhibited the formation of agrininduced AChR clustering. Intriguingly, the monovalent Fab fragments of MuSK antibodies from rabbits with EAMG also inhibited AChR clustering by agrin on C2C12 cells, indicating that complement-mediated mechanisms are not necessarily required for such inhibition. We then examined the reduced expression of AChR at NMJ in soleus muscles of paretic and normal rabbits by fluorescence microscopy. In addition, the structure of NMJ in our paretic rabbits, as well as the size and branching of the motor terminals, were significantly reduced. Electron microscopic observations of NMJ in rabbits with EAMG induced by injection of MuSK protein demonstrated a significant loss of complexity of convoluted synaptic folds but no destruction, and EAMG model cited here resembles the phenotype of humans with MG and MuSK antibodies. Our results showed that MuSK antibodies inhibit both anterograde and retrograde signals required for maintaining the synaptic structures in mature NMJ. The EAMG model with MuSK antibodies will facilitate the understanding of the pathogenic mechanisms of MG with MuSK antibodies and functional roles of MuSK molecules in mature neuromuscular junctions.
T cells constitutively expressing the immuno-tolerogenic human histocompatibility leukocyte antigen G (HLA-G) have recently been described as a new type of human natural thymus-derived regulatory T cells (HLA-Gpos Treg). Treg suppress immune responses against foreign and self-antigens and play a key role in the mechanisms of autoimmunity. We here challenge the role of HLA-Gpos Treg with respect to their function and role in multiple sclerosis.
HLA-G positive (HLA-Gpos) T cells can be found in the cerebrospinal fluid (CSF) and in tissue specimens from MS patients. In contrast to other noninflammatory neurological diseases (OND), CD4+ HLA-Gpos T cells are enriched in CSF from MS patients. A substantial population of CD3+ T cells co-expresses HLA-G and localize to inflammatory lesions from MS patients, suggesting that HLA-Gpos T cells accumulate at the sites of CNS inflammation. CSF-derived HLA-Gpos T cells revealed a clear predominance of central memory T cell (CD45RA-CD27+) phenotype, showed markers of activation (ICOS), and had significantly higher expression of the inflammatory chemokine receptor CCR5. Using an in vitro model of human blood brain barrier we show that HLA-Gpos T cells have a propensity to migrate and are vigorously driven by inflammatory chemokines such as MIP1a and RANTES (ligands of CCR5), but not to MIP3b (ligand of CCR7). In contrast to CD4+CD25+FoxP3+CD127dim Treg, purified peripheral blood CD4 HLA-Gpos Treg cells don't differ in their suppressive capacity from healthy controls. Most importantly, CD4+ HLA-Gpos T cells directly isolated from the CSF of MS patients potently suppress proliferation of autologous responder cells.
This work characterizes the pathophysiological role of HLA-Gpos Treg, a novel population of human Treg, with respect to MS. Our data show that HLA-Gpos natural Treg have a chemokine-dependent propensity to transmigrate the blood-brain barrier, and accumulate in the CSF and in CNS lesions from MS patients. Importantly, patientderived HLA-Gpos Treg are functionally active and suppress autologous T cell activation at the site of inflammation. It is therefore assumed that these natural regulatory cells are specifically recruited to the CNS in order to counterbalance autoimmune inflammatory activity. Our work contributes to the understanding of HLA-Gpos natural Treg and provides an important pathophysiological example of beneficial T cell inflammation in MS, implicating both from pathophysiological/therapeutical points of view. The blood-brain barrier (BBB) plays a crucial role in protecting the central nervous system (CNS) by restricting entry of cells and molecules into the brain. In the CNS disorder multiple sclerosis (MS), breakdown of the BBB allows activated leukocytes to infiltrate the brain parenchyma, leading to the formation of the characteristic demyelinated lesions. Interleukin (IL)-17-secreting lymphocytes (TH17) appear to be essential in the pathogenesis of numerous inflammatory diseases, including MS. In spite of the intensified focus on TH17 lymphocytes, their contribution to the disruption of the BBB and leukocyte infiltration into the CNS, both important early events in the development of MS, remains unclear.
We developed and optimized a method to successfully generate human TH17 lines in vitro from peripheral blood CD4+CD45RO+ memory lymphocytes of healthy donors. We demonstrate that in response to IL-23, human TH17 memory CD4+CD45RO+ lymphocytes, but not naive CD4+CD45RA+ lymphocytes, produce IL-17 and IL-22, with a subset of cells co-expressing IL-17 and IFN-gamma. We show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo and that receptors for these cytokines are upregulated on human endothelium during inflammation. Moreover, human TH17 cells transmigrate efficiently across brain endothelial cells via adhesion molecules ALCAM and ICAM-1 and promote CD4+ lymphocyte recruitment across the BBB, through the concerted action of IL-17 and IL-22. Interestingly, we also note an increase in the percentage of IL-17+ and IL-22+ lymphocytes, as well as in the subpopulation of cells co-producing IL-17 and IFN-gamma in TH17 cell lines expanded from the peripheral blood of women with active MS than in those generated from healthy men and women controls.
Our study further refines the phenotype of human TH17 lymphocytes and emphasizes the importance of TH17 lymphocyte infiltration into the CNS and their consequent involvement in lesion formation in MS. In this study, we examined the role of MOG-specific Th17 cells in microglial activation. We also assessed the ability of neurons to modulate Th17-induced microglial activation and investigated the possible role for CD200 ligand-receptor interactions. Microglial activation was assessed by measuring cell surface expression of MHC class II, CD40, CD80 and CD86 by flow cytometry and the production of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6).
The results demonstrate that Th17 cells were potent activators of microglial cells, upregulating cell surface expression of MHC class II, CD40, CD80 and CD86 and inducing the production of TNF-alpha (⁎⁎p b 0.01), IL-1beta and IL-6 (⁎⁎⁎p b 0.001). Neurons attenuated the Th17 cell-induced production of TNF-alpha, IL-1beta and IL-6 (⁎⁎p b 0.01) from microglia but had no effect on of MHC class II, CD40, CD80 and CD86 expression. The inhibitory effect of neurons on Th17 cell-induced proinflammatory cytokine production from microglia was reversed by co-incubation with an anti CD200 receptor antibody (⁎⁎p b 0.01).
These findings show that Th17 cells are potent activators of microglia and that this activation can be attenuated by CD200 ligandreceptor interactions between neurons and microglia. Experimental autoimmune encephalomyelitis (EAE) is a well known animal model of multiple sclerosis. Upon activation, myelin reactive T cells invade the central nervous system (CNS) to initiate disease. It remains unknown whether myelin reactive T cells may also infiltrate into the CNS during disease recovery. We investigate whether the CNS is still susceptible for the infiltration, activation and proliferation of resting myelin reactive T cells during the recovery phase of EAE.
Resting myelin reactive T cells were obtained by isolating the draining lymph nodes of immunized animals followed by in vitro restimulation with MBP. Subsequently, cells were incubated with IL-2 during 24 h and then rested on IL-2 deprived medium during 72 h. Flow cytometric analyses were performed to characterize the resting T cells. At time of transfer, cells were labeled with SPIO or CFSE and intravenously injected at different stages during EAE. At 24 h and 72 h post transfer, either MRI was performed or the CNS and immune organs were isolated and analyzed for the expression of CFSE and 7-AAD.
Flow cytometric analysis showed that resting myelin reactive T-cells generated in vitro and used for transfer were CD25low, CD45RC negative and CD62L low. When transferred at recovery, cells could be detected in the CNS as soon as 24 h after transfer using in vivo MRI and remained present in the CNS for at least 3 days. Flow cytometric analysis showed that more than 90% of the transferred cells proliferated in the CNS 72 h after transfer. Proliferation of these cells was less abundant in the periphery. A low number (19%) of the cells in the CNS were apoptotic or necrotic. The amount of cell death was much higher in the periphery compared to the CNS. Finally, transfer of resting myelin reactive T cells during the recovery phase also led to a clinical disease relapse. This is in contrast to the normal monophasic disease course in these animals.
These results show that during clinical recovery, the CNS in EAE is still susceptible to the infiltration and the inflammatory activities of myelin reactive T cells. This could indicate that immunoregulation of myelin reactive T cells during recovery is primarily mediated in the peripheral immune organs. Intravenously injected T cells can possibly escape this control by a direct extravasation from the blood and exert their pathogenic function in the CNS.
Flow cytometric analysis of infiltrating immune cells during inflammation or after injury of the Central Nervous System (CNS) offers valuable insights into mechanisms of disease and regeneration. Due to the limited number of these cells isolated from individual animals during disease models, it has been necessary to limit the characterization to a small number of markers and/or to pool several samples. We have now established a 7-color flow cytometric approach, which allows us to identify infiltrating immune cell types and their differentiation/maturation-status with only four stainings.
The combination of markers for CD4+ and CD8+ T cells, NK and NK T cells, B cells, macrophages, microglia and dendritic cells in one staining gives us the opportunity to dissect subsets of immune cells in a very precise way. Additionally, fluorescent labelled beads can be used to quantify absolute numbers of these cell populations in the CNS. During Experimental Autoimmune Encephalomyelitis (EAE), we could show that even before onset of disease, certain cell types already infiltrate in considerable amounts, amplify after onset of symptoms and peak at the maximum disease score. After relapse, the immune response is constricted, and the composition of immune cell types is altered substantially compared to before onset of disease. By using markers for specific cell subsets (naive/effector/ memory cells, regulatory cells) in combination with intracellular cytokine staining, we can follow the transition stages of CD4+ T cell differentiation in the inflammatory lesion. Finally, the presence of antigen-presenting cells (APC), like macrophage or dendritic cell subsets, and changes in their maturation can be monitored over the course of the disease. This protocol has also successfully been applied to the study of brain-infiltrating cells in a mouse model for stroke.
Combining this flow cytometry data with histological analysis in the context of disease course allow for a very well defined representation of the mechanisms involved in disease pathology. We report here the paradoxical development of spontaneous experimental autoimmune encephalomyelitis (EAE) in a myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor (TCR) transgenic mice (2D2) in the absence of the target autoantigen. We show that in these animals T cells cross-react against an alternative, self-mimicking autoantigen, neurofilament-M (NF-M).
In the transgenic 2D2 strain, more than 90% of all CD4+ T cells express transgenic TCR recognizing MOG p35-55 in context of I-Ab. Between 10-20% of these mice spontaneously develop EAE that involves optic nerve and spinal cord. We now report the same proportion of 2D2 mice come down with spontaneous EAE in the absence of the cognate target autoantigen i.e. MOG. EAE in MOGsufficient (MOG+/+) and MOG-deficient (MOG−/−) 2D2 mice was indistinguishable in its clinical expression, in number and location of lesions and in their cellular composition.
We isolated proteins from MOG−/− CNS material and found that a fraction containing predominantly NF-M protein induced strong proliferative activation of 2D2 T cells. The salient epitope sequence shared with MOG p35-55 essential TCR contact residues. The response of 2D2 T cells to recombinant NF-M protein and synthetic peptides was heteroclitic with regard to MOG p35-55, resulting in higher proliferation activity and cytokine production. It appears that cross-reaction between MOG and NF-M is not a private response pattern limited to the 2D2 TCR, but was also noted in oligoclonal MOG reactive CD4+ T cells freshly isolated from primed C57BL/6 mice. This is the first report on immunological self-mimicry involving two distinct autoantigens within the same target organ. Neurodegeneration is an important feature of multiple sclerosis (MS) while the underlying mechanisms of axonal loss are poorly understood. We investigated whether two-pore-domain potassium channel TASK1 contributes to neurodegeneration in experimental autoimmune encephalomyelitis (EAE) using TASK1 knockout mice and pharmacological approaches.
We used ELISA, proliferation assays, FACS analysis and electrophysiological measurements to characterize T lymphocytes of TASK1 knockout mice. EAE was induced with MOG peptide in wildtype mice and knockout mice. Neurodegeneration was measured by immunohistochemical staining of spinal cord sections and by magnetic resonance imaging (MRI) of mouse brain. TASK1 knockout mice showed a dramatic amelioration in EAE disease course. As shown by immunohistochemical staining for SMI-31/-32 and subsequent counting of axons in predefined areas of the spinal cord TASK1 knockout mice also were less susceptible to neurodegeneration compared with wildtype mice (29%-43% less axonal loss). The immune system of TASK1 knockout mice showed a normal phenotype. The endogenous cannabinoide anandamide which is known to block TASK1 failed to reduce INFgamma production and proliferation rate in isolated T lymphocytes from knockout mice. Anandamide also showed no effect on the potassium outward current in TASK1−/− CD4+ T lymphocytes as demonstrated by electrophysiological measurements. Daily application of anandamide in wildtype mice undergoing EAE reduced disease severity and brain atrophy as assessed by repeated MRI scans of individual mice (34% less atrophy).
TASK1 channels are members of the two-pore domain potassium channel (K2P) family that are widely expressed within the brain and contribute to maintaining and regulating the resting membrane potential of various neuronal cell types. Efflux of K+ ions plays a major role in apoptotic cell death in neurons suggesting a role of TASK1 channels in neurodegeneration as it occurs in MS. Modulation of TASK1 channels may provide neuroprotective potential, therefore further studies on leak potassium channels in neuroinflammatory disorders are urgently awaited. In multiple sclerosis (MS) disease susceptibility has been tied to the major histocompatibility complex (MHC) HLA-DR2 haplotype containing both the DRB5⁎0101 (DR2a) and DRB1⁎1501 (DR2b) alleles. Exhibiting almost complete linkage disequilibrium, recent studies have suggested differential roles for the gene pair in driving or modifying autoimmunity. To characterize the specific contribution of DR2a to disease pathogenesis, humanized transgenic (Tg) mice carrying the DR2a allele were created and subsequently crossed to Tg mice carrying an MS patient-derived myelin basic protein (MBP) 83-99 specific T cell receptor (TCR).
Two lines expressing different levels of DR2a showed differential skewing in the generation of CD4 versus CD8 populations. In Rag+/+ as well as Rag−/− animals, MBP-autoreactive T cells can escape deletion and drive spontaneous autoimmunity at low rates (1 to 5% in Rag+/+ animals) depending on the level of DR2a expression. Inflammatory infiltrates, demyelination and associated neuronal damage were present throughout the spinal cord consistent with a classical MBP EAE disease, in addition to minor involvement of the brainstem and peripheral nerve roots in both spontaneous as well as actively induced disease. Positional scanning combinatorial libraries (PSCL) demonstrated the degeneracy of this TCR with recognition of epitopes within several bacterial, viral, and human proteins that can serve as molecular mimics of MBP83-99. In vivo studies are ongoing both in TCR/HLA DR2a and HLA DR2a transgenic mice to determine the pathological relevance of these cross-reactivities and their relationship to antigenic spreading in vivo.
These studies emphasize the pathologic contribution of the HLA-DRB5⁎0101 allele to the development of autoreactive T cells when expressed as the sole MHC class II molecule, defining a pathological role for DR2a in CNS autoimmunity. Multiple sclerosis (MS) is a complex disease and one of the most common chronic inflammatory disorders of the central nervous system (CNS). A model commonly used for dissection of genes in neuroinflammation is myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE) that mimics the human disease to a large extent both in the chronicity and the pattern of histopathological lesions.
A genome-wide linkage analysis previously performed in an F2 cross between the EAE-susceptible LEW.AV1 and the MHC identical but EAE-resistant PVG.AV1 rat strains identified a quantitative trait locus (QTL) on rat chromosome 1 regulating LPS-induced production of proinflammatory cytokines, namely TNF and IL-6. Using a congenic line between these two rat strains, we confirmed a role of this locus not only in regulation of proinflammatory cytokines but also in regulation of MOG-EAE. Further fine-mapping of this region was performed in an advanced intercross line (AIL). Influence on EAE was dissected in G10 generation of AIL and influence on LPSinduced IL-6 and TNF production and pristane-induced arthritis (PIA) was dissected in G12. The initial EAE QTL was resolved into two loci. The first locus regulates clinical phenotypes in EAE and overlaps two QTLs regulating anti-MOG IgG2b and IL-6 production. This locus of 5 Mb harbors approximately 10 genes, including RGMa, a gene playing a role in spinal cord injury in mice. The second locus regulates EAE and overlaps a QTL that regulates PIA, anti-MOG IgG1 and TNF production. Among the interesting genes in this QTL are IL-4R, IL-21R, IL-27, NFAT and LAT are of particular interest due to their involvement in immune regulation, autoimmune diseases or in activation of T-cells. Functional studies showed differential expression of IL-4R, IL-4 and IL-21R in spleens of congenic and parental PVG rats.
We were thus able to define two important QTLs in the rat that affect the outcome of both experimental encephalomyelitis and arthritis, which associate with a regulation of cytokines production. Association studies are being performed in a large material of MS patients and controls in addition to more functional studies in the congenic rats with the aim of defining the gene and the pathogenic mechanisms of EAE and MS. Multiple sclerosis (MS) is a sexually dimorphic, chronic, demyelinating autoimmune disease of the central nervous system resulting in disability and paralysis. In the murine model of MS, experimental allergic encephalomyelitis (EAE), young male SJL/J mice have been reported to be resistant to EAE while older males and all aged females are susceptible.
By comparing a wide age range of mice immunized with PLP 139-151 and CFA on D0 and D7, we found that female disease severity remains constant with age. In contrast, EAE disease severity increases with increasing age of immunization in males with young males having significantly less severe EAE and older males having significantly more disease than equivalently aged females. Cumulative disease score, peak severity, days affected and severity index all increased with age of immunization in males but not in females. Incidence was not affected by age or sex but there was a seasonal effect. Since castration of young SJL/J mice restores relapsing remitting EAE susceptibility, we compared testes gene expression and hormone levels in young susceptible (B10.PL) and resistant (SJL/J) male mice. Serum progesterone levels remained constant in SJL/J males while levels in B10.PL males fluctuated over time. Specifically, serum progesterone was significantly lower in SJL/J mice from 4-8 wks and converged with B10.PL levels at 10 wks. This time frame correlates with previously published changes in susceptibility to EAE and delayed-type hypersensitivity responses. Microarray analysis of 4 wks old testes found 3-beta-hydroxysteroid dehydrogenase, the enzyme that converts pregnelone to progesterone, was significantly lower in SJL/J mice compared to B10.PL suggesting that lower serum levels of progesterone are due to reduced progesterone synthesis in the testes. To verify that progesterone levels are related to EAE susceptibility, we planned to implant progesterone pellets and induce EAE. When placebo pellets were implanted and EAE induced, the previous sexual dimorphism was eliminated. Implantation of the sham pellet increased EAE severity to equal female EAE severity while non-implanted males had significantly less severe EAE suggesting stress of implantation abrogates the sexual dimorphism.
These studies show that age, season and stress can alter the EAE sexual dimorphism in SJL/J mice illustrating the importance of gene-by-environment interactions in EAE disease severity and susceptibility. 36 male Wistar rats were randomly divided into 2 groups: the normal group (18 rats), and the EAE group (18rats). First, we detected the functional and morphological changes by brain stem auditory evoked potential (BAEP), somatosensory evoked potentials (SEP), routine neuropathological methods and immunohistochemical examinations. Second, we analyzed on miRNA expression using miRNA expression microarrays. Third, oligonucleotide microarray was carried out, and the data were correlated with published data on protein and gene expression.
The results showed that clinical onsets in the EAE group were sudden, and the time of onset was average 10.6 ± 0.58 days. The incidence rate was 100%. Clinical scores were 3.2 ± 1.12. The pathological manifestations included significant inflammatory cell infiltration of cerebrospinal tissues, formation of vessel gloves but no apparent amyelination zone on myelin staining. Immunohistochemical examination found that there were a large number of activated T cells in brain and spinal cord. Compared with the normal group, 60 pieces of miRNAs up-regulation more than twice, 86 pieces down-regulation more than twice. In addition, the results correlated with relevant DNA microarray data, some of the miRNAs that control the expression of 3 genes (aquaporins-4, interleukin-10 and matrix metalloproteinase-9) known to be important in the progression of MS were identified.
The miRNAs participate in posttranscriptional gene regulation in pathophysiology on EAE. Some miRNAs might be molecular features that become biomarkers in MS and related pathologies.
We have previously shown that T cell autoreactivity directed against epitopes in the second extracellular loop of myelin proteolipid protein (PLP) correlates strongly with the development of brainstem and cerebellar lesions in multiple sclerosis (MS) patients and in an animal model of MS. PLP is present throughout compact myelin and on the external face of the myelin membrane, and the second extracellular loop would therefore potentially be accessible to antibody. In the current study, we investigate the putative functional properties of auto-antibodies directed against the same region of PLP, and the role that they play in lesion localization.
By ELISA, patients with MS (n = 100) were found to have increased serum levels of antibodies specific for myelin proteolipid protein (PLP) compared to healthy controls (n = 40) and patients with other disorders of the CNS (CND patients) (n = 30). Whole human PLP (h-PLP) and multiple PLP peptides were tested: the most consistent reactivity was directed against peptides from the second extracellular loop of PLP. Anti-PLP antibodies from MS patients were usually a mixture of IgM and IgG1 isotypes, with occasional IgG2, whereas the majority of control sera were of the IgM isotype. PLP-specific antibodies were also tested for their ability to opsonize myelin for phagocytosis by macrophages. Sera that contained high titres of PLP peptide-specific antibodies were pre-adsorbed with the PLP peptides or with control peptides before being used in the opsonization assays. PLP peptides could specifically absorb out the opsonizing activity, to some degree, in all samples tested. There was a positive correlation between the ability of sera to opsonize myelin and the EDSS score of the MS patients. Anti-PLP antibodies were then tested for their ability to affect where lesions develop and the degree of demyelination in the animal model. When PLP-specific T cells alone were injected into C3H/ HeJ mice, lesions developed in the brainstem, but not in the cerebellum. Anti-PLP190-209-specific antibodies injected together with the T cells resulted in lesions in both the brainstem and cerebellum.
The results strongly suggest that PLP-specific antibodies could be of functional relevance in the pathogenesis of MS. To determine frequencies and functions of natural killer (NK) cells in untreated patients with multiple sclerosis. Background: NK cells are multicompetent lymphocytes of the innate immune system, whose central role in host defense, immune regulation and autoimmunity has been increasingly recognized over the last years. Notably, expansion of CD16-CD56bright NK cells correlates with treatment response in daclizumab (anti-IL-2Ra)-treated MS patients.
Frequency, phenotype and expansion of blood-derived NK cell and T cell subsets were investigated by flow cytometry ex vivo, following activation with the classical NK cell mitogen interleukin (IL)-2 and limiting concentrations of the monokine IL-12 in 20 untreated patients with clinically isolated syndrome (CIS) and relapsing remitting MS compared to 25 healthy blood donors. In addition, we determined the cytolytic activity of resting and cytokine-activated NK cells and cytolytic T cells towards a CD4+ T cell line (CEM) and allogeneic mitogen-stimulated CD3+ T cell blasts by a flow cytometry-based cytotoxocity and degranulation assay. Ex vivo frequencies of NK cells and T cells were similar in patients and controls. In contrast, MS patients showed an impaired expansion of both CD16+CD56+ and CD16-CD56 bright NK cells but not CD4+ or CD8+ T cells following IL-2 and IL-12 stimulation. Expression levels of activation markers (CD25, HLA-DR, NKp44) were consistently higher on both NK cell subsets, but similar in T cells from MS patients compared to healthy controls. Resting NK cells from MS patients showed an increased cytolytic activity towards both CEM T cells and primary allogeneic T cell blasts, which was not further increased upon cytokine activation.
Phenotype and function of CD16+ and CD16-NK cells are altered in MS. The increased activation and cytolytic potential towards T cells but impaired expansion suggest that NK cells are involved in regulating antigen-driven autoimmunity in MS. Natalizumab is a humanized recombinant monoclonal antibody against very late activation antigen-4 (VLA-4) approved for the treatment of patients with multiple sclerosis (MS). A phase II study failed to demonstrate a difference between natalizumab treatment groups and the placebo group with regard to gadolinium enhancing lesions on magnetic resonance imaging (MRI) three months after discontinuation of therapy. The objective of this study was to assess clinical MS disease activity, surrogate disease markers on MRI, immunological parameters in peripheral blood and CSF, as well as safety in MS patients after discontinuation of natalizumab therapy.
This study is a longitudinal and serial cross-sectional assessment, in which 23 patients who were treated with natalizumab in the context of two phase III clinical trials were originally enrolled. A subgroup of patients was followed over 14 months. The annual relapse rate, neurological disease progression assessed by the Expanded Disability Status Scale, disease surrogate markers on magnetic resonance imaging (MRI), cellular and humoral immune markers in peripheral blood and CSF, and adverse events of the drug were monitored.
With regard to clinical disease activity, neuroimaging, and immune responses, the majority of patients in our cohort were stable. Decreased lymphocyte cell numbers and altered cell ratios returned to normal 14 months after cessation of natalizumab. No infectious complications were observed. This is the first long-term follow-up of patients who discontinued natalizumab. We did not observe a clinical, radiographic, or immunological rebound phenomenon after discontinuation of natalizumab therapy.
Stoop Marcel 1 , Verbraak Evert⁎ 1 , Jafari Naghmeh 1 , van Meurs Marjan 1 , Wierenga Annet 1 , Luider T 1 , Laman Jon 1 , Hintzen Rogier 1 1
Multiple sclerosis (MS) is a complex disease of which the underlying mechanisms remain poorly understood. Systematic comparison of differentially occurring peptides in the cerebrospinal fluid (CSF) in MS patients versus reference groups allows identification of disease related secreted molecules and tissue debris. We therefore performed state of the art MALDI-TOF Mass Spectrometry on large series of CSF samples, as CSF is the closest source of brain secretory products and tissue debris. This allowed the identification of differentially occurring peptides between MS patients and the selected control group. One of the peptide sequences differentially detected was clusterin (apolipoprotein J). The normal adult brain is one of the major clusterin expressing sites while during chronic inflammation and injury of the brain clusterin expression is further increased. Several functions have been ascribed to clusterin i.e. phagocyte recruitment signaling, cell aggregation, complement inhibition and lipid transport. This study aimed at further unraveling whether clusterin sequences found in the CSF are expressed at the mRNA and protein level within MS brain tissue.
Quantitative PCR was used for the analysis of Clusterin mRNA expression within MS brain, normal appearing white matter and non-demented control brain. Clusterin mRNA expression was significantly increased in MS brain tissue compared to brain tissue of non-demented controls. Moreover, in situ analysis showed elevated clusterin protein expression in active and in preactive lesions as compared to non-demented controls. Preliminary data suggest that the main cells expressing clusterin are astrocytes. In addition cells within the perivascular infiltrates also occasionally express clusterin.
Collectively the data show that clusterin is actively expressed on both the mRNA and protein level in MS brain. These results support the hypothesis that clusterin is actively involved during inflammation in MS. Myelin T cells reactive may play a pathogenic role in MS. We and others have shown that regulatory CD4+CD25+FOXP3+CD127low T cells (Tregs) are functionally disturbed in relapsing-remitting (RR) MS patients but not in secondary progressive (SP) MS patients. The goal of our current study was to clarify this difference in Treg activity between early and chronic disease stages in MS by studying Treg function and homeostasis in MS.
We analyzed the functional capacity and homeostatic parameters of (precursor) naive Tregs (nTregs) and memory Tregs (mTregs). We also measured MBP and MOG proliferative responses of CD4total T cells (including Tregs), naive (CD45RAhigh) and memory (CD45RA-) CD4+CD25-CD127high T cells (no Tregs included) of RR-and SP-MS patients and HC by means of a CFSE dilution assay and measured cytokine production of the myelin reactive T cells. The suppressive capacity of FACS-sorted nTregs was impaired in both early and chronic MS patients, whereas only the latter group showed a restored mTreg function. Chronic MS patients had increased numbers of mTregs as compared to age-matched early MS patients whereas nTreg frequencies did not differ. T cell receptor excision circle (TREC) numbers were reduced in nTregs of early MS patients indicating a diminished nTreg thymic output. MBP and MOG proliferative responses of CD4total T cells but not of Treg-depleted naive and memory CD4+CD25-CD127high T cells were significantly higher in RR-MS patients as compared to HC and SP-MS patients. A significantly higher proportion of myelin reactive memory T cells from MS patients as compared to HC produced IL-17.
These data provide evidence for a disturbed thymic nTreg development and function in early MS disease stage. This may play a role in the peripheral activation of myelin antigen reactive T cells leading to CNS pathology. Future therapeutic immune interventions for MS may focus on an early restoration of Treg function and suppression of pathogenic myelin reactive Th17 cells.
6 -Inflamed blood-brain barrier promotes recruitment of effector memory CD8+ T lymphocytes However the phenotype of migrating CD8+ T lymphocytes and the mechanism by which such cells cross the BBB remain largely unknown. Our objective is to evaluate the phenotype of and mechanism by which CD8+ T lymphocytes access the CNS.
Using CSF obtained from MS patients and spinal cord material from MOG (35-55)-induced EAE, we demonstrate that CD8+ T lymphocytes are mostly of the effector memory (EM) phenotype (CD8+, CD62L-, CCR7-, CD28-, GranzymeBhi). We further show that purified human CD8+ TEM lymphocytes transmigrate more readily across human BBB endothelial cells than ex vivo un-fractionated CD8 lymphocytes and that BBB endothelium promotes the selective recruitment of CD8+ TEM lymphocytes. Furthermore, we provide evidence for an active and 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 the migration of CD8+ T lymphocytes across BBB-ECs is dependent on VLA-4, but independent of ICAM-1/LFA-1, ALCAM and P-selectin.
Our study thus provides evidence for an active role of the BBB in the recruitment of potentially auto-aggressive CD8 TEM lymphocytes to the CNS. The small 27 kD heat shock protein (HSP27) is induced in a variety of cells following stress and inflammation, where it promotes cell survival and inhibits inflammation. Little is known on the localization of HSP27 and its phosphorylated form in the central nervous system tissues after autoimmune or irradiation injury. In this study, we examined the expression of both HSP27 and phosphorylated form of HSP27 (phospho-HSP27) in the spinal cords with experimental autoimmune encephalomyelitis (EAE) and gamma ray irradiation.
Western blot analysis showed that both HSP27 and phospho-HSP27 was weakly expressed in normal spinal cords, significantly increased in the spinal cord with EAE (P b 0.01). Immunohistochemistry showed that both HSP27 and phospho-HSP27 were constitutively expressed in neurons and some fibrous astrocytes in the spinal cords of normal rats; its immunoreactivity was increased primarily in fibrous astrocytes of the white matter in EAE-affected spinal cords. A similar finding was also recognized in the spinal cords with irradiation. However, few inflammatory cells in EAE were immunopositive for both HSP27 and phospho-HSP27.
Collectively, we postulated that increased phosphorylation of HSP27, which is constitutively present in the neurons and glial cells, rescues host cells including neurons and glial cells against autoimmune attack or radiation induced injury in the central nervous system. ⁎This work was supported by a program of the Basic Atomic Energy Research Institute (BAERI), which is a part of the Nuclear R&D Programs funded by KOSEF in South Korea. An important neuropathological feature of Multiple Sclerosis (MS), but also other lesions within the brain, is the formation of an astroglial scar. Firm interaction of the astrocytes with extracellular matrix proteins is likely to be involved in astrogliosis. Indeed, an increased appearance of various extracellular matrix proteins, such as fibronectin, at the lesion sites in MS has been described (Sobel and Mitchell, 1989; van Horssen et al., 2005) . This astrogliosis represents a major mechanical impediment to remyelination and axonal regeneration in MS. Tissue transglutaminase (TG2) is a multifunctional enzyme, well known for its protein crosslinking activity. At the cellular level, TG2 has been implicated in adhesion and migration of fibroblasts and monocytic cells onto extracellular matrix proteins, including fibronectin (Akimov and Belkin, 2001; Balklava et al., 2002) . These data suggest a pivotal role of TG2 in cell-matrix interactions. As we observed that TG2 is present in astrocytes in active MS lesions, we questioned whether TG2 plays a role in astrocyte-fibronectin interactions in vitro.
To this end, intracellular and surface expression of TG2 in primary cultured rat astrocytes was detected. In addition, we observed that inflammatory mediators, present in the CNS of MS patients, regulate TG2 production in astrocytes. Indeed, combinations of IFNγ, TNFα and IL-1β, enhance TG2 production (mRNA and protein) in primary cultured rat glial cells, particularly astrocytes. In contrast, TG2 surface expression is downregulated, particularly by TNFa. Furthermore, adhesion of astrocytes onto fibronectin is, at least, partly mediated by TG2 as determined by siRNA studies and pharmacological inhibition of TG2 activity.
We conclude that TG2 present in and on astrocytes is differentially regulated by inflammatory mediators, suggesting that cellular pools of TG2 molecules exists with probable separate functions. TG2 is clearly involved in the interaction of astrocytes with the extracellular matrix protein fibronectin. Thus, TG2 contributes to astrogliosis and thereby possibly impairs regeneration of the Astrocytes play a number of important physiological roles in Central Nervous System (CNS) homeostasis. Inflammation stimulates astrocytes to secrete cytokines and chemokines that guide macrophages/microglia and T-cells to sites of injury/inflammation. Herein, we describe how these processes are controlled by the Suppressor Of Cytokine Signaling (SOCS) proteins, a family of proteins that negatively regulate adaptive and innate immune responses. The objective of this study was to determine how SOCS expression is regulated in primary astrocytes, and the functional implications of expression.
In this study, we describe that the immunomodulatory cytokine IFN-β induces SOCS-1 and SOCS-3 expression in primary astrocytes at the transcriptional level. SOCS-1 and SOCS-3 transcriptional activity is induced by IFN-β through GAS elements within their promoters. Studies in STAT-1a deficient astrocytes indicate that STAT-1a is required for IFN-βinduced SOCS-1 expression, while STAT-3 siRNA studies demonstrate that IFN-β-induced SOCS-3 expression relies on STAT-3 activation. Specific siRNA inhibition of IFN-β-inducible SOCS-1 and SOCS-3 in astrocytes enhances their pro-inflammatory responses to IFN-β stimulation, such as heightened expression of the chemokines CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1β), CCL5 (RANTES) and CXCL10 (IP-10), and promoting chemotaxis of macrophages and CD4+ T-cells.
These results indicate that IFN-β induces SOCS-1 and SOCS-3 in primary astrocytes in order to attenuate its own chemokine-related inflammation in the CNS.
As such, SOCS proteins may serve as therapeutic targets to attenuate unwanted inflammatory responses within the CNS. Microglia and infiltrating macrophages are considered major producers of tumor necrosis factor (TNF), which plays a key role in ischemic tissue injury and brain inflammation. We report that TNF produced by microglia, but not macrophages, is neuroprotective in experimental stroke in mice. We found that cortical infarction was significantly exacerbated in TNF-knock out mice compared to wildtype mice. Bone marrow-chimeric TNF-knock out mice grafted with wildtype bone marrow cells developed larger infarcts than bone marrow chimeric wildtype mice grafted with TNF-knock out bone marrow cells, identifying a neuroprotective role for microglial-but not macrophage-derived TNF. Increased infarction in TNF-p55 receptor (R)-knock out mice compared to TNF-p75R-knock out and wildtype mice suggested that microglial-derived TNF exerted neuroprotective effects through TNF-p55R. The absence of TNF was associated with reduced microglial population size and expression of Toll-like receptor 2 in unmanipulated brain, and enhanced proinflammatory gene expression in unmanipulated and ischemic brain. Our findings identify a neuroprotective role for microglial-derived TNF in the acute phase after focal cerebral ischemia and suggest that microglia determine to what extent endangered cortical neurons survive after ischemic stroke. The pathogenesis of neurogenerative disorders, including Human Immunodeficiency Virus (HIV)-1 associated dementia (HAD), is exacerbated by an imbalance between metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). As the TIMPs have exhibited diverse non-classical effects including anti-apoptotic effects, the induction of TIMP-1 in neuroinflammation likely serves multiple functions in addition to modulating MMP activity. Our published studies demonstrated differential TIMP-1 expression in acute versus chronic activation of astrocytes and HAD brain tissues, specific to TIMP-1.
Laboratory systems were used to study the mechanisms of astrocyte-TIMP-1 regulation and neuroprotective effects. Primary human neural cells, neurotoxins, virus/viral proteins and molecular manipulations such as TIMP-1 promoter-luciferase reporter constructs were used. Our results indicated that while acute interleukin (IL)-1beta activation upregulates TIMP-1 through multiple mechanisms, chronic downregulation of TIMP-1 is primarily under transcriptional control. We show a novel CCAAT displacement protein (CDP) repressor element involved in astrocyte TIMP-1 regulation. Further, we used staurosporine, macrophage-tropic HIV-1 virus and glutamate to induce cell death in cultured human neurons. In these assays, TIMP-1 had equivalent neuroprotective effects as brain-derived neurotrophic factor (BDNF), a classical neurotrophin. Neuroprotective effects were also seen with TIMP-1-T2G, a mutant with no MMP binding, indicating that TIMP-1 neuroprotection is independent of MMPs. Neurotrophic signaling through Bcl-2/Bcl-xL has emerged as a possible pathway in our recent studies. Adenoviral injections in the SCID mouse HIVE model led to astrocyte expression of TIMP-1, suggesting a potential therapeutic approach.
Taken together, our results demonstrate that astrocyte-TIMP-1 regulation in HAD is complex and may play a critical role in neuroprotective functions of astrocytes. These data are important for unraveling the mechanisms underlying astrocyte responses during chronic neuroinflammation and have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance.
Over the past decade many studies have focused on neuroinflammation as a harmful feature of AD, with the implication that antiinflammatory therapy may be beneficial. More recently, immunization with amyloid beta-peptide (Abeta) has been proposed as a treatment for AD. Experimental models have shown that Abeta accumulation in the brain, a key feature of the disease, can be reversed by immunotherapy mediated, at least in part, by phagocytosis by microglia. We are coordinating a collaborative clinical and neuropathological follow-up of the patients who were in the first trial of active Abeta immunization with full length Abeta42 peptide (Elan Pharmaceuticals).
To date we have neuropathology on 9 immunized AD patients (iAD) who died between 4 and 64 months after first immunization dose. In these cases we are exploring the expression of microglial proteins in relation to plaque removal, and compared with unimmunized AD controls. CD68 immunostaining was observed in cerebral cortex of both the iAD cases and AD controls indicating phagocytic activity in both groups. However, the iAD cases showed Abeta within the lysosomes of activated microglia, indicating that Abeta had been phagocytosed; whereas the phagocytic activity in the AD controls was not associated with Abeta within microglia, suggesting that microglia are qualitatively different after immunization.
These observations highlight the point that, rather than simply demonstrating that microglia have been activated, it is the specific way in which microglia are activated that is important in determining their role in AD pathology. To what extent the changes in the activation of microglia are reflected in changes in cognitive function, remains to be determined. However, our clinical data so far suggest that despite clearance of plaques, active Abeta immunization did not benefit cognitive function. Stressful life events are known to increase the risk of multiple sclerosis (MS) exacerbations. This phenomenon is thought to be caused by inadequate levels of the anti-inflammatory hormone cortisol after stress. Cortisol is the end product of the hypothalamus-pituitary-adrenal (HPA)-axis, also known as the stress-axis, and is routinely used in its synthetic form, prednisone, to treat relapses of MS. Post-mortem and in vivo studies clearly show that the stress-axis is chronically activated in MS. However, in a postmortem study of 16 MS brain donors, we recently found a highly significant inverse relationship between the responsivity of the stress-axis and severity of MS (Huitinga et al., Ann. Neurol. 2004 ). Therefore, we performed a follow-up study of 31 female MS brain donors to more specifically determine the consequences of stressaxis activity for severity of MS.
Clinical, endocrine and neuropathological data of all included MS brain donors were analyzed using statistical software and clearly confirmed our earlier data. Moreover, levels of tau and glutamate in the cerebrospinal fluid (CSF) correlated with the amount of corticotropin releasing hormone (CRH), which drives the stress-axis. Furthermore, our analysis of the neuropathological reports revealed that increased demyelination and decreased remyelination was present in donors with low cortisol and severe MS.
Our results emphasize the importance of high cortisol levels in overcoming MS inflammation, a phenomenon observed in pioneer animal studies in the early nineties (Tonelli et al., Immunol. Rev. 2001). The observed correlation of both tau and glutamate CSF levels with CRH suggests that neurodegeneration contributes to chronic activation of the stress-axis in MS. Based on these data, we postulate that low stress-axis responsivity, possibly as a consequence of genetic predisposition, leads to more severe MS. Currently, we are performing studies in MS lesions obtained from the Netherlands Brain Bank, to identify glucocorticoid driven mechanisms affecting the pathogenesis of MS lesions, using immunohistochemistry and laser dissection microscopy in combination with Q-PCR analysis (Koning et al., Ann. Neurol. 2007). A number of immunological functions are dependent on circadian rhythms and regular sleep. This has impact on the type and magnitude of immune responses following antigenic challenge, for example in vaccination. Little is known about the underlying mechanisms. One possibility could be the circadian and sleep-dependent modulation of immune responses by CD4+CD25+ natural regulatory T cells (nTreg). These cells have been shown in a variety of studies to negatively regulate adaptive immune responses. We could show that during normal sleep, the suppressive activity of nTreg was highest at night and almost no suppressive activity was present in the morning. Deprivation of sleep abrogated this rhythm. We now studied whether nTreg suppress T helper 1, 2 and 17 cytokines to the same extent and how this suppression is modified by sleep and circadian rhythm.
In a within-subject cross-over design, six healthy young men were examined under defined conditions on two occasions, i.e. during sleep and sleep deprivation. Venous blood was drawn periodically every, 5 h, nTreg function was explored in vitro.
nTreg suppress T helper 1 cytokine production and there is almost no suppression of T helper 2 or 17 cytokine production in the sleep and wake condition. In the wake condition suppression of T helper 1 cytokine production was decreased in the afternoon. Responsible for this nTreg functional rhythm might be the sleep dependent hormone prolactin. In preliminary in vitro experiments we could show that prolactin increased nTreg suppressive function.
In this study we show that nTreg suppress T helper 1 cytokine production, and that this suppression is disturbed by sleep deprivation. Hence, nTreg contribute to a sleep-dependent modulation of immune responses. Although classically regarded as a disorder of the white matter, alterations of the neuronal compartment of the central nervous system (CNS) are precocious and largely independent of demyelination in multiple sclerosis (MS). Glutamate excitotoxicity has been proposed as a major determinant of neurodegeneration in MS and in experimental MS. However, a physiological study addressing synaptic transmission during immuno-mediated aggression of the central myelin is still lacking.
By means of neurophysiological recordings from single neurons in slices, we investigated neuronal and synaptic functioning in major oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. Striatal neurons, which are particularly prone to degenerate in MS, exhibited complex and dynamic alterations of glutamate-and GABA-mediated transmission, starting in the presymptomatic phase of the disease and evolving independently of inflammatory infiltrates, demyelination or axonal injury. Changes in the activity of NMDA and non-NMDA glutamate receptors, of Na+/Ca++ exchanger and of voltage-dependent Na+ channels, all contributed to up-regulate excitatory inputs to striatal neurons, while GABA-mediated transmission was persistently down-regulated.
These data reveal an important role of synaptic dysfunction in the pathological process of EAE, and provide a rationale for the use of neuroprotective strategies since the very early stages of MS. Here we found that during Experimental Autoimmune Encephalomyelitis (EAE), MG cells specifically accumulated at the dorsal SVZ niches. Starting at pre-clinical stages, blood derived MG/macrophages and resident MG cells infiltrated the dorsal SVZs and initiated to secrete both IFNgamma and TNFalpha. Moreover, over-expression of either IFNgamma or TNFalpha by ependymal cells was sufficient to direct the mobilization of MG cells toward the SVZs. This finding prompted us to define the crosstalk between inflammatory cues and resident aNPCs. Thus, by using laser capture microdissection (LCM), we found the over-expression of a subset of chemokines (Ccl2, Cxcl9, Ccl19 and Cxcl10) within the EAE SVZ. Moreover, IFNgamma or TNFalpha in vitro stimulation induced aNPCs to over-express some of the abovementioned chemokines. Thus, suggesting that aNPCs are capable of chemo-attracting blood derived cells during inflammation.
The constitutive chemokine expression by aNPCs makes neurogenic niches a preferential site for inflammatory cells extravasation, thus impairing the physiological function and might explain the preferential affection of peri-ventricular areas by neuroinflammation. The crucial roles of cytokines in shaping T cell responses have been documented in numerous autoimmune diseases. IL-27, a recently described cytokine, has been shown to exhibit both pro-and antiinflammatory properties. IL-27 favors naïve CD4 T cells differentiation into Th1 cells to the detriment of Th17 or Th2 differentiation. In the absence of IL-27 receptor, mice had enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE), due to the absence of IL-27 inhibitory signaling on Th17 development. Conversely, IL-27 has been demonstrated to promote CTL functions of mouse CD8 T cells, but no information on human cells exists. Moreover, no human CNS source has been identified. Our goals are to investigate the impact of IL-27 on human CD8 T cell functions and to determine the potential human CNS sources of IL-27. CD8 T cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based assays. Addition of IL-27 to anti-CD3 activated CD8 T cells led to a significant dosedependent increase of proliferation, and IFN-gamma and Granzyme B production. IL-27 had less impact on human CD4 T cells but significantly boosted their proliferation. Expression of both IL-27 subunits p28 and EBI3 was assessed by real-time PCR. Resting human adult microglia expressed very low/undetectable p28 and basal levels of EBI3 mRNA. Stimulation with TLR3 or TLR4 ligands significantly induced p28 and moderately boosted EBI3 mRNA levels suggesting that these local antigen presenting cells could produce IL-27 within the CNS upon environmental stimuli.
These results underscore the pro-inflammatory impact of IL-27 on human CD8 T cells and show that this cytokine could be locally provided in the CNS by human microglia. We are currently investigating how CD8 T cells from MS patients react to stimulation with IL-27. burden that affects 1% people in world-wide. Several evidences indicate abnormalities in dopamine system in both SCZ and BPD. The neuronal calcium sensor NCS-1 is a protein that has many functions such as dopamine D2 receptor desensitization inhibition and involvement with many aspects of neurotransmission as regulation of ionic channels and neurotransmitters release enhancement and exocytose. Also, the NCS-1 protein and mRNA expression levels was found increased in prefrontal cortex (PFC) of SCZ and BPD patients. It is well documented the NCS-1 expression in neural and neuroendocrine cells; recently, it was shown that NCS-1 is also expressed in mast cells and neutrophils. NCS-1 has important functions in mast cells as it stimulates Fc epsilon RI-triggered exocytose and the release of arachidonic acid metabolites. Then, we sought to investigate whether NCS-1 could be expressed in peripheral blood mononuclear cells (PBMC) and if there is a correspondence of the expression on those cells with the levels in PFC of SCZ and BPD subjects.
Using flow cytometry, our results have shown that NCS-1 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. These results showed that there is no correlation with NCS-1 expression in PFC and PBMC of SCZ and BPD patients.
However, these results showed that NCS-1 might be a putative biomarker for both psychiatric disorders. More work is necessary to investigate the NCS-1 functions in lymphocytes and monocytes.
Financial Support: CNPq, FAPEMIG. Thymectomy was first reported as a treatment in non-thymomatous myasthenia gravis (MG) in 1941. However, indications for the use of thymectomy in this population based on randomized trial data are lacking. Over the last 30 years, corticosteroids and other pharmacological immunotherapies have been used increasingly in MG, either alone or with thymectomy. Over this time period, outcomes for MG have improved dramatically. Whether thymectomy has contributed to this positive development remains a point of contention, and recent data will be reviewed.The MGTX study group received funding by NINDS for a 5-year single-blinded, two-arm trial. Approximately 70 centers are participating worldwide with the intention of enrolling 200 generalized MG subjects. The primary study aims to answer three questions: does extended transsternal thymectomy (ETTX) combined with a strictly defined prednisone protocol, when compared with the prednisone protocol alone (1) result in a greater improvement in myasthenic weakness, (2) result in a lower total dose of prednisone, thus decreasing the likelihood of concurrent and long-term toxic effects, (3) enhance the quality of life by reducing adverse events and symptoms associated with the therapies? Inclusion criteria are MG Foundation of America Classes 2 to 4, acetylcholine receptor antibody positivity, age at least 18.0 years and b65.0 years, and disease duration b5 years. Patients can be prednisone naïve or not. Recruitment for the study commenced in late 2006. Diverse regulatory obstacles have been encountered due to the international nature of the trial. The average time for sites to satisfy local and U.S. federal regulations has been 14.5 months. As of July 2008, 61 centers were ready for enrollment and were screening all MG patients encountered. A total of 4575 MG patients have been screened, the vast majority ineligible. Fifty-one patients have been enrolled. The pace of recruitment has increased in 2008. An additional 17 sites are in various stages of regulatory approval. Entry criteria have been modified slightly to favor inclusionincreasing the upper age limit from 60 to b65 years and disease duration from 3 to b5 years. A result favoring thymectomy would establish its benefits unequivocally; failure to show a difference would suggest that thymectomy is an unnecessary procedure in this non-thymomatous population and should lead to cost savings. Thus the results will impact on clinical practice. Transcription of arginase-1 correlated with the presence and number of alternatively aaMph. iNOS transcription and protein expression was pronounced in WT mice but was absent in IL-13T/+ mice.
These data provide first evidence of the development of aaMph in a CNS infectious disease model and highlight the essential role of IL-4 or IL-13 in immunopathology during C. neoformans meningoencephalitis.
Agrawal Smriti⁎ 1 , Yong Wee 1 1 University of Calgary, Calgary, Canada MMP members have been implicated in detrimental roles in neuroinflammatory diseases such as multiple sclerosis (MS). However, the fact that MMP family members interact with, compensate for, and may even be inhibited by each other has made the biology of MMPs in MS a difficult one to examine. Several MMP members are simultaneously elevated in MS and in EAE, an animal model of MS, and this is thought to be the result of the activation of many cell types in these conditions. Another possibility is that a general inducer of several MMPs is engaged during the disease process. If so, a novel approach to affect MMPs in MS could be to alter the activity of such an inducer.
The extracellular matrix metalloproteinase inducer (EMMPRIN, also known as basigin or CD147) is found on the surface of various cell types in an inactive state. EMMPRIN induces fibroblasts and tumor cells to produce and activate various MMPs. Whether EMMPRIN plays a similar role in the immune system is unclear. Our specific objective is to determine a role for extracellular matrix metalloproteinase (EMMPRIN) in MMP regulation in mouse EAE.
We utilize a mouse model of EAE, wherein animals develop a clinically disease with infiltration of leukocytes into the CNS, resulting in demyelination. Various tissues from these mice are dissected out and utilized in flow cytometry (FACS), histological staining, Western blotting, and gelatin zymography. We report that the activation of (MOG35-55) specific T-cells in culture leads to their secretion of high levels of glycosylated EMMPRIN. In vivo, we find increased numbers of EMMPRIN positive cells in the CNS of mice from the onset of EAE signs, compared to controls, and these continue to rise with disease progression. EMMPRIN immunoreactivity is detected in the CNS in both infiltrating and resident cellular populations. Finally, EMMPRIN null mice, although susceptible to EAE, showed a slightly delayed onset and reduced severity in EAE disease versus wildtype controls.
Taken together, our results indicate that the early rise of EMMPRIN is consistent with subsequent evolving pathology in EAE, and may be attributed to its role as an MMP inducer in the disease. If so, targeting EMMPRIN could help influence the marked elevation of MMPs and disease process of MS. Cytotoxic T lymphocytes (CTL) are considered as important effector cells contributing to CNS damage in neuroinflammatory disorders. We here challenge the mechanisms how CD8 T cells injure neurons and how major histocompatibility complex (MHC) class I restricted, cellcontact-dependent interaction of CTL with neuronal cells is determined. Specific emphasis is placed on the interdependency for perforin and granzyme B in immune-neuronal interactions.
We used hippocampal neuronal cells in combination with T-cell receptor (TCR) transgenic CD8 T cells (OT-I) as a paradigm for MHC class I restricted immune-neuronal interaction. Cocultures were analyzed by time-lapse videomicroscopy and two-photon microscopy in conjunction with immunohistochemistry, electrophysiological recordings and molecularbiological assays.
Time-lapse video-microscopy showed that CTL kill neurons fast and depending on cell-cell contact as well as antigen (appr. 30% over 4 h). Real-time electrophysiological recordings of basal neuronal parameters upon direct contact with effector CD8 T cells revealed immediate changes in membrane capacitance and resistance (appr. 60% decrease; b=10 min). With a similar kinetic and MHC restriction, neurons showed a rapid influx of Ca2+ upon CTL interaction. Assuming that observed effects most likely reflect the consequences of a TCRdependent poreformation via the granzyme/perforin pathway, we next studied the effects of OT-I cells deficient for granzyme B or perforin. Interestingly, quantification of neuronal killing revealed that CTL deficient for granzyme B or perforin maintain the same neurotoxicity, albeit migratory velocity was significantly slowed in the absence of granzyme B. Single-cell recordings however showed, that perforin deficient T cells had lost their capability to induce neuronal membrane changes or Ca2+ influx, while these immediate effects were maintained in the absence of granzyme B.
CTL-mediated injury of MHC class I induced neuronal cells is a fast, antigen and cell-cell contact dependent process. CD8+ effector cells immediately affect basal cellular parameters and induce Ca2+ influx in a MHC I restricted way. While acute electrophysiological consequences of immune-neuronal interaction require perforin, perforin or granzyme B are redundant for long-term neurotoxicity.
Fleisher-Berkovich Sigal⁎ 1 , Ben-Shmuel Sarit 1 1 Ben-Gurion University, Beer-Sheva, Israel
Increasing evidence suggests that inflammation plays a crucial role in the deterioration of neurodegenerative diseases. The inflammatory response is sustained by chronic microglial activation that includes the over-production of toxic factors by microglial cells, such as proinflammatory cytokines, prostaglandins and nitric oxide (NO). Although NO has many physiological roles, it can turn harmful when produced at high levels (under inflammatory conditions) and lead to apoptosis.
Brain inflammation can be regulated by neuropeptides, such as bradykinin (BK) and other members of the kinin family, a group of short-lived peptides formed in response to pathophysiological events like inflammation. BK exerts its effects via two kinin receptors: B1 and B2 receptors (G-protein coupled receptors). While B2 receptor is constitutively expressed in various cells, B1 receptor is induced after cell activation by pro-inflammatory factors. Although BK's pro-inflammatory functions in the periphery have been broadly studied, its role in the brain is not yet clear. Recently, a novel neuroprotective role for BK was suggested via inhibition of lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines from microglial cells.
The goal of the present study was to examine the long term effect of BK receptors agonists on basal and LPS-induced NO release from microglial cells.
Nitrite (a stable end-product of NO oxidation) levels were measured using the Griess assay. 24-48 h exposure of BV2 to BK (1, 10 nM) reduced basal NO release by about 75%, and 100 nM respectively. BK decreased NO production by over 90% from control levels. B1 receptor agonist, [Lys-des-Arg9]-BK (10, 100 nM), lead to 70% reduction in NO synthesis while B2 receptor agonist, (1, 10, 100 nM), reduced basal NO release by 40%. Also, BK (1, 10 and 100 nM) attenuated LPS-induced NO production, in a dosedependent manner, with a maximum decrease of 50% at a concentration of 100 nM compared with LPS treatment alone. Similarly, B1 and B2 receptors agonists dose-dependently attenuated LPS-induces NO production by a maximum of 40% and 45%, respectively.
These results imply on a possible neuroprotective role of BK via attenuation of NO released from microglial cells under both basal and inflammatory conditions.
Lindblom Rickard⁎ 1 , Piehl Fredrik 1 , Diez Margarita 1 1 Karolinska Institutet, Stockholm, Sweden
The complement system has been implicated in several neurodegenerative disorders with inflammatory components, e.g. Multiple Sclerosis (MS), Alzheimer, Huntington and Amyotrophic Lateral Sclerosis (ALS), and in experimental systems neurons have proven to be extremely sensitive to lysis by activated complement. So far the degree of genetic heterogeneity in the regulation of the complement system has been unknown. We have here characterized expression of complement components in different rat strains after mechanical nerve injury.
As injury model we used lumbar ventral root avulsion (VRA), which is a well characterized model of nerve injury-induced inflammation and neurodegeneration. A kinetic study was performed in inbred DA and PVG rats using several post-operative survival times up to 14 days. A linkage study was performed in a F2 BN × LEW intercross with 151 animals studied 5 days after VRA. Quantitative RT-PCR and immunohistochemistry was used to determine level of expression of C1q and C3, as well as the complement inhibitors clusterin and CD59.
The kinetic study demonstrated increased expression of both C1q and C3 early after VRA, with significantly higher levels in DA compared to PVG. Clusterin mRNA was also increased after injury, but induction was slower with the highest expression at 14 days after injury in both strains. In contrast, expression of CD59 was reduced after VRA. Whereas levels had normalized in PVG by 14 days, levels in DA rats remained lower at all studied time points. A whole genome scan for expression of C1q and C3 was performed in the F2 (BN × LEW) intercross, showing linkage to several gene regions. One quantitative trait locus (QTL) regulating C1q on chr. 10 reached the threshold for genome-wide significance.
The DA strain, which is more susceptible to nerve cell loss than PVG, displays higher levels of C1q and C3 and lower levels of CD59, supporting the notion that a dysregulated local inflammatory response involving complement activation contributes to nerve cell loss. Gene mapping in an F2 cross demonstrate a polygenic influence, with several suggestive QTLs. A large F2 (DA × PVG) intercross is currently under study in our laboratory. Exact positioning of the responsible genes will contribute to our knowledge about the role of the complement system in conditions of neuroinflammation and neurodegeneration. Microglial-driven inflammation is a feature of the aged brain and neurodegenerative conditions. The inflammatory changes are typified by cytokine release such as interleukin-1beta (IL-1b) and tumour necrosis factor alpha (TNFalpha), and an upregulation of the corresponding signalling pathways. Previous evidence from this laboratory has shown that rosiglitazone, a peroxisome proliferator activated receptor gamma (PPARg) agonist, has the ability to attenuate pro-inflammatory cytokine release and restore the age related deficits in LTP.
The present findings suggest that pre-treatment of rosiglitazone did not affect PPARg activation since CD36 mRNA expression, a downstream indicator of activation, was unchanged; rosiglitazone also exerted no effect on PPARg protein expression. These findings concur with previous evidence suggesting that the anti-inflammatory action of rosiglitazone is independent of PPARg. Previous evidence from this laboratory has shown that the anti-inflammatory action of rosiglitazone is mediated by its ability to increase IL-4 expression. In this study the effect of rosiglitazone on mixed glia, astrocytes and microglia was mimicked by IL-4 pre-treatment. This study also showed that rosiglitazone treatment induced an increase in IL-4 from astrocytes (p b 0.05) but not from microglia.
The evidence suggests that rosiglitazone attenuates the LPSinduced increase in IL-1b via IL-4 release from astrocytes. Many neurodegenerative disorders are characterized by microglia activation. Such a response must be tightly regulated because unrestrained activation can generate a chronic inflammatory milieu that might lead to autoimmunity. Extracellular adenosine can modulate cellular activation through G-protein coupled, adenosine receptor (ADORA)-mediated signaling. The cellular response to extracellular adenosine is orchestrated by the expression pattern of four different ADORA subtypes: A1, A2a, A2b and A3, which are characterized by their capacity to enhance or inhibit cAMP levels.
To study the effects of adenosine during neuroinflammation, we characterized the ADORA expression profile on resting and on Toll-like receptor (TLR)8-activated primary rhesus microglia. Resting microglia expressed mRNA for A2a, A2b and A3. Upon activation, microglia strongly upregulated A2a and A2b and downregulated A3 expression levels. Since A2a and A2b-mediated signaling results in opposite effects than A3-mediated signaling, the predicted relative contribution of A2a and A2b over A3 to adenosine-mediated signaling would be enhanced by N50 fold in TLR-activated microglia. As a consequence, adenosinemediated inhibition of TLR-induced TNFa and IL-12p40p70 was much more potently inhibited in activated than in resting microglia. We could demonstrate that both the sensitivity to adenosine-mediated inhibition, as well as the overall inhibitory potential were enhanced in activated microglia. Microglia acquired this enhanced inhibitory phenotype b8 h upon TLR-mediated activation, and the enhanced inhibitory potential was sustained for N72 h. By using specific antagonists, we determined that the simultaneous increase in A2a, but not that of A2b, mediated signaling, and decrease in A3-mediated signaling were responsible for the described effects. In addition, we demonstrated that the induced ADORA profile on activated microglia was able to crossinhibit cytokine production induced by a variety of TLR.
Our data reveal important novel mechanistic aspects of an endogenous brake on inflammation and would suggest exploration of partial A2a agonists and A3 antagonists as therapeutic possibilities to dampen microglial activation in ongoing inflammation. MicroRNAs form a family of several hundred short non-coding RNA molecules predicted to regulate the expression of about 30% of all protein-coding genes. MiRNA-mediated gene regulation is therefore expected to influence the pathogenesis of many diseases.
In order to study this form of gene regulation in multiple sclerosis lesions, we followed a two-step strategy.
In a first step, we isolated MS lesions of different types, along with control white matter, by macrodissection of fixed and frozen brain sections. Total RNA extracted from these samples was amplified by realtime PCR. Among the examined 367 microRNAs, 269 could be detected which formed lesion specific expression profiles.
In a second step, we combined Laser capture microdissection of individual cells with analysis of purified cultures of immune cell subsets and astrocytes, to assign the microRNAs regulated in MS lesions to specific cell lineages.
Our data indicates that some of the microRNAs which had been upregulated in MS lesions were inducible in vitro during activation of T cells monocytes/macrophages or astrocytes.
This work provides a first insight into miRNA mediated gene regulation in multiple sclerosis lesions. The study had two main parts. First, the numbers of Treg cells identified as CD3+CD4+CD25highFoxp3+ cells were determined using 4-color flow cytometry examinations of CSF and blood samples obtained from patients with acute viral meningitis or encephalitis (n = 7), or those experiencing acute relapse of MS (n = 5), as well as patients with non-inflammatory neurological diseases, used as controls (n = 11). Those Treg cells were significantly increased in the blood of MS patients as compared to those of CNS infection and the controls, while those in the CSF were also significantly increased in patients with CNS infection as compared to the controls, with an increasing trend seen in the CSF of active MS patients. Next, both the numbers of Treg cells identified as CD4+CD25highCD127dim cells and those of recently activated helper T cells with a surface CD4+ CD25lowCD127dim phenotype were determined in CSF and blood samples obtained from another series of MS patients in acute relapse as well as from the controls. Recently activated helper T cells were shown to be significantly increased in the blood of those MS patients. In addition, a follow-up study of a patient with tuberculous meningitis showed that Treg cells in the blood increased with improvement of infection, while those in the CSF were increased during early and peak stages of the disease.
Treg cells in the CNS and systemic circulation in patients with CNS infection seem to have a regulatory role, thus their measurement is useful for monitoring disease status. In contrast, their numbers may not be relevant for monitoring MS disease activity. It is well established that neurodegenerative disorders are associated with evidence of microglial-driven inflammation. These inflammatory changes include enhanced activation of microglial cells and pro-inflammatory cytokine release. Recent data show that activation of the orphan receptor single Ig IL-1R-related molecule (SIGIRR) inhibits proinflammatory cytokine signalling but the ligand for SIGIRR and downstream signalling events triggered by receptor activation are unknown.
In this study, we set out to investigate the effects of lipopolysaccharide (LPS) on microglial activation and cytokine secretion in hippocampal tissue prepared from SIGIRR knockout mice (SIGIRR−/−). We also assessed whether interleukin-1F5 (IL-1F5), a recentlycharacterised member of the IL-1 ligand superfamily which has anti-inflammatory properties, might modulate LPS-induced changes. Microglial activation was assessed by measuring cell surface expression of CD40, and intracellular adhesion molecule (ICAM) by polymerase chain reaction (PCR) and proinflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) by PCR and ELISA. The interaction of IL-1F5 and SIGIRR was assessed by immunofluorescent confocal microscopy. The results demonstrate that the LPS-induced increase in expression of CD40 (p b 0.05), ICAM (p b 0.001), IL-1beta (p b 0.05), IL-6 (p b 0.05) and TNF-alpha (p b 0.05) mRNA was enhanced in SIGIRR−/− mice compared with wildtype mice. IL-1F5 attenuated the LPS-induced increase in IL-1beta (p b 0.01) in wildtype mice and these antagonistic effects were not observed in SIGIRR−/− mice.
These findings show that SIGIRR has anti-inflammatory actions in the CNS since SIGIRR−/− mice showed enhanced responsiveness to LPS treatment. Furthermore, that IL-1F5 is a possible ligand for SIGIRR, since the antagonistic effects of IL-1F5 on LPS-induced signalling were not observed in SIGIRR−/− mice and IL-1F5 was co-localised with SIGIRR. Taken together, the data suggest that the interaction of SIGIRR and IL-1F5 is a significant immunomodulatory system in the CNS. Despite the advances in antibiotic management acute bacterial meningitis remains a life threatening neurological disease if not managed rapidly. The estimated incidence is 2-5 per 100 000 people per year in developed countries and up to ten times this in developing regions. The cellular immune response in the CNS plays a major role in controlling the infection but the study of cytokines and any protective or harmful role they may play in the CNS is poorly understood. Glial cells, the main immune cells in the CNS are known to release cytokines and other immune mediators forming a network of stimulatory and inhibitory effects.
The time and dose dependant release of 23 cytokines by microglia, astrocytes, neurons and mixed cultures were tested in each cell type following pneumococcal infection. Cell cultures were infected with 2 clinical isolates of Streptococcus pneumoniae; the first was isolated from CSF and the second from the oropharynx. Cytokine release into culture supernatants was measured at various time points using a commercially available Bio-Plex assay.
Mixed cultures showed more controlled release of cytokines in comparison to pure microglial cultures. In contrast to findings from previous studies, infected pure microglial, astrocyte and neuron cultures were found to actively produce some cytokines. The infected cultures showed significant increase in cytokine release compared to uninfected cultures. The neurological isolate induced a backward dose effect with lower cytokine levels produced at higher concentrations of the organism. This effect was not seen with the orophanyngeal isolate and could be reversed by heat inactivating the infecting inoculum.
The findings demonstrate a time and dose dependant profile of cytokine production in the CNS in response to pneumococcal infection and may be important in understanding the neurological pathogenesis of pneumococcal infection.
12 -Differentiation of primary adult microglia alters their response to TLR8-mediated activation but not their capacity as APC We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein and produced larger amounts of pro-inflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia.
While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses.
13 -Immunoregulatory role for perforin in an animal model of CD8+ T cell-mediated demyelinating disease Accumulating evidence indicate that CD8+ T cells may contribute significantly to disease pathogenesis in multiple sclerosis and other autoimmune diseases of the nervous tissue. To gain insights into the potential mechanisms by which this T cell subset may induce nervous tissue injury, we study an animal model (herein referred to as L31 mice) that spontaneously develops a CD8+ T cell-mediated demyelinating disease associated with axonal damage primarily due to the transgenic expression of the B7.2 (CD86) T cell costimulatory ligand on microglia. We previously reported that the CD8+ T cells that accumulate in the central nervous system (CNS) of L31 mice are potent IFN-gamma producers and that responsiveness to this cytokine is a prerequisite for disease development. Since IFN-gamma is a powerful inducer of MHC-I expression and a common mechanism by which CD8+ T cells mediate destruction of MHC I-expressing cells is through the perforin pathway, we hypothesized that IFN-gamma production by CNS CD8+ T cells promotes upregulation of MHC-I expression on neurons and/or oligodendrocytes, which could become targets of a MHC-I-restricted perforin-dependent CD8+ T cellmediated cytotoxic attack.
MHC-I expression was detected on oligodendrocytes but not on neurons in pre-clinical L31 animals and CD8+ T cells were observed in close contact to MHC I-expressing oligodendrocytes. L31 mice deficient in perforin expression (Perf−/−) exhibited the same clinical and pathological features as perforin competent animals but with an accelerated onset and hastened expansion/accumulation of CD8+ T cells in the CNS that couldn't be attributed to disparities in the peripheral CD8+ T cell population.
Our results indicate that oligodendrocytes have the potential to interact with CNS CD8+ T cells in a MHC-I-dependent fashion before clinical manifestations of disease but demyelination does not occur through a perforin-dependent mechanism. Perforin appears rather to regulate the expansion of CD8+ T cells within the CNS.
14 -Diabetic ketoacidosis in mice elicits a unique cytokine response between serum and brain Diabetic keotacidosis (DKA) is a complication of type 1 diabetes requiring immediate recognition and treatment. Despite careful treatment, children are at high risk for intracranial complications during DKA therapy, such as brain swelling, stroke and bleeding. The causes of these complications are unknown. However, DKA elicits a systemic inflammatory response associated with elevated cytokines. Therefore, systemic or local production of cytokines in the CNS might contribute to neuroinflammation through a variety of mechanisms. We sought to determine levels of cytokines in serum and brain tissue during DKA.
We used our juvenile mouse model of DKA that develops DKA 72 h after injection of streptozocin and alloxan. Serum and cortex samples obtained from both control and DKA mice were quantitatively analysed for the cytokines interleukin (IL)-1a, IL-1b, IL-2, IL-6, IL-9, IL-10, IL-18, keratinocyte-derived chemokine (KC; mouse ortholog of human IL-8), Interferon (IFN)-g and tumor necrosis factor (TNF)-a using multiplexed immunoassays. Transforming growth factor (TGF)-b1 levels were analysed by ELISA. Mice were confirmed to be in DKA by having significantly higher serum glucose, beta-OH-butyrate levels and weight loss compared to controls (p b 0.01, n = 10). Analysis of serum revealed DKA mice had significantly elevated levels of proinflammatory cytokines IL-6 and KC (p b 0.05, n = 9-10) and antiinflammatory cytokines IL-10 (p b 0.01, n = 7-9) and TGF-b1 (p b 0.05, n = 9-10) versus controls. In contrast, only anti-inflammatory cytokines were significantly altered in brain, IL-10 decreased (p b 0.05, n = 9-10) in DKA mice, and TGF-β1 increased (p b 0.01, n = 10). There was no significant difference in the levels of proinflammatory cytokines, IL-6 (p = 0.198, n = 10) and KC (p = 0.791, n = 10), in DKA mice versus controls. We also found that IL-6 (r^2 = −0.661) and KC (r^2 = −0.727) levels in serum correlate with weight lost in DKA mice (p b 0.05, n = 10) and blood glucose levels (r^2 = 0.923; r^2 = 0.914, p b 0.001, n = 10).
These results are novel since the inflammatory response that occurs in the brain is unique from the systemic response. These differential changes in inflammation between serum and brain might be a contributing factor to intracranial complications during DKA. This also indicates that there maybe a relationship between levels of proinflammatory cytokines and severity of DKA. Panatecs, Tuebingen, Germany Autoimmune central nervous system (CNS) inflammation such as found in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) requires activation of myelin reactive T cell in the periphery and inside the target organ by antigen presenting cells (APC). Microglia are CNS resident APC and have been shown to present a cryptic epitope from myelin basic protein (MBP) inside CNS lesions in MS patients. In order to better understand the events leading to generation and display of this epitope, we investigated the antigen processing machinery and MBP processing during acute EAE.
Different APC populations were isolated from susceptible DA and resistant ACI rats immunized with MBP85-99 at the onset of disease in DA rats. Cathepsin activities were determined using active site directed probes. In addition, human recombinant MBP was digested with lysosomal contents from microglia, splenic B cells and splenic DC and fragments analysed. We found that MBP processing in microglia from both rat strains was dominated by cathepsin D which destroyed the main immunogenic epitope MBP85-99 and that processing in both strains was similar both in the periphery and the CNS.
MBP was efficiently destroyed in all cell types investigated, with no obvious differences between the two strains, suggesting that differences in processing cannot account for the differences in susceptibility. While in mouse microglia cultures, CatG is the protease dominating MBP processing, in rat primary microglia, CatD limits presentation of the MBP85-99 epitope during acute EAE, highlighting the need for caution in translating knowledge about antigen processing from one species to another.
Oriane Cedile⁎ 1 , Natalia Popa 1 , Lucile Gioda 1 , Jose Boucraut 1
We studied, in the model of olfactory axotomy, the recruitment of NK cells and the expression of MHC-Ia, b and NKG2D ligands, including Rae-1. The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway. The MOB receives the afferences from the olfactory mucosa and is the target of the neural progenitor cells generated in the subventricular zone (SVZ) and which migrate along the rostral migratory stream (RMS). Sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons induces retrograde neuronal cell death in the mucosa, an increase of apoptotic cell death in the SVZ and along the rostrocaudal extent of the RMS. In parallel, an increase in cell proliferation in the SVZ and in the extension of the RMS located in the MOB preceding full neuronal repair appears.
Using qPCR, we observed the expression in the olfactory bulbs of NKG2D and the NK specific receptor NKp46. The level of expression was similar than those observed in the hepatic tissue. The recruitment of immune cells after axotomy was confirmed with other techniques such as flow cytometry and immuno-labeling with anti-NKp46 polyclonal antisera. In the same experiment, we did not detect recruitment of immune cells in the SVZ. A weak expression of Rae-1 was detected by qPCR solely in some part of normal brain and its expression was strongly induced in the MOB after axotomy.
We showed for the first time the recruitment of immune cells including NK cells in the model of olfactory axotomy. Deciphering the roles in vivo of NK cells and Rae-1 in neuronal death and neuronal repair is now our main objective. mice have shown that PD-L1 or PD-L2 blockade leads to an earlier onset and increased severity of the disease. Although, PD-1 and its ligands were observed on infiltrating immune and CNS cells during EAE, which cell types express these molecules have not been completely resolved, and no data are available on human CNS. Our goal is to assess the expression of PD-Ls by human CNS cells and to establish whether these molecules could locally contribute to modulate immune responses.
Human primary CNS cultures were either kept in basal conditions or stimulated with pro-inflammatory cytokines respectively mimicking healthy vs. MS CNS environment. PD-L1 and PD-L2 expression were analyzed by real-time PCR and flow cytometry. Human primary cultures of astrocytes, oligodendrocytes, and brain endothelial cells expressed barely detectable PD-L1 and PD-L2 levels, at the mRNA and protein levels. Following pro-inflammatory cytokine (IFN-gamma, TNF-alpha, IL-1beta) treatments, we observed an increased expression of both PD-L1 and PD-L2 on these cells, especially PD-L1 on astrocytes and oligodendrocytes, while brain endothelial cells up-regulated preferentially PD-L2. Combinations of cytokines were more potent to enhance PD-L1 and PD-L2 expression on all cell type tested than either cytokine alone.
Our results underline the enhancing effects of a pro-inflammatory environment on PD-L1 and PD-L2 basal expression by human brain cells. We are currently evaluating whether PD-Ls expressed by human CNS cells immuno-modulate human T cells.
Research funded by a grant from Multiple Sclerosis Society of Canada. Neurocysticercosis (NCC) is the most common parasitic disease of the central nervous system (CNS) caused by the helminth Taenia solium. The disease is associated with a hyper inflammatory response which is thought to be detrimental to the host. Our primary goal is to understand the mechanisms involved in the induction of the inflammatory response in NCC. Toll-like receptors play important role in the induction of host inflammatory responses. We propose to study the role of TLRs in CNS immunopathogenesis in murine NCC.
During murine NCC expression of Toll like receptors 1 through 13 was analyzed by real time PCR, and IF microscopy. All the TLRs, except TLR5, were expressed and up regulated differentially among different cell types. Among them, TLR2 was the first to be upregulated after parasite infection and was most abundantly expressed, particularly in astrocytes and their foot processes that terminate on blood vessels present in the periventricular and meningeal areas. Next, the role of TLR2 was analyzed in vivo. The tlr2−/− mice were highly susceptible to NCC as compared to their wild type counterparts. Histopathological analysis revealed severe pathology characterized by lack of perivascular lymphocytic cuffing, increased microgliosis, and more tissue damage when compared to the wild type animals. The tlr2−/− mice were observed to elicit a weaker immune response as determined by the reduced reactivity of the sera to the soluble parasitic antigens. Both the total number of infiltrating immune cells and individual cell types such as CD11b+ myeloid cells, alphabetaT cells, gammadeltaT cells, and B cells were significantly reduced both early (1 wk) and late (3 wk) during infections. Accordingly the levels of several of the pro-inflammatory cytokines and chemokines such as IL6, TNF-alpha, IFN-gamma, MCP1, RANTES, MIP1alpha and MIP1beta were reduced in CNS of TLR2-deficient mice during NCC. In TLR2-deficient mice the expression of inflammatory cytokines was reduced in astrocytes present in leptomeningeal and periventricular areas which are thought to be important early during CNS infection.
TLR2−/− mice exhibit reduced immune responses during murine NCC. The death of TLR2-deficient mice during experimental NCC along with increased pathology, and parasite burden suggests an important role for TLR2 in the immunopathogenesis in murine NCC. Here, we report on the isolation of subpopulations of murine CNS-DC on the basis of different surface markers. The CD11c+ DC subsets differ in their capacity to stimulate T cell proliferation. In fact, one DC population inhibited the proliferation of T cells. The DC types also differ in their population dynamics during the course of EAE, which may indicate that they play different roles in the evolution of the disease. In order to investigate how EAE-CNS derived DC exert their different effector mechanisms, we compared regulatory and stimulatory DC by performing gene expression arrays. Pathway analysis showed that cellular growth and movement were the main biological processes distinguishing the three DC subsets. Genes involved in growth and cellular proliferation were investigated, particularly the expression of genes involved in the recognition, capture and presentation of antigens was validated. Several genes involved in chemotaxis and homing were differentially expressed in the DC subsets, suggesting that the DC subsets may attract different types of cells to the CNS.
The differences observed between the three DC subsets may indicate distinct roles in EAE pathogenesis. The identification of specific markers as well as the phenotypical characterization of the DC subsets may help to specifically manipulate DC subsets in order to attenuate CNS autoimmune inflammation. 20 -MYD88-deficiency is associated with reduced disease severity and decreased TH1 inflammatory response in the brain during murine neurocysticercosis The symptomatic phase of neurocysticercosis (NCC) is associated with a TH1 type inflammatory cytokine response in the central nervous system (CNS), which is thought to be responsible for the severe neuropathology and mortality associated with the disease. Brain infection with Mesocestoides corti, a murine model for neurocysticercosis, induces a massive influx of immune cell types and a TH1 cytokine expression. To understand the mechanism involved in the development of the inflammatory response, the role of toll like receptors (TLRs) was analyzed. In murine NCC, all the TLRs (TLRs 1-13) , except TLR5 were upregulated and differentially expressed among various nervous tissue and infiltrating immune cells. Mice lacking MyD88 (MyD88−/−), the central regulator of TLR induced response, displayed enhanced ability to survive the infection as compared to the wild-type mice, although the numbers of parasites in brain remained similar. The CNS of MyD88−/− mice was more quiescent with an absence of perivascular lymphocytic cuffing, decreased microgliosis and tissue damage. The numbers of CD11b+ve myeloid cells, aβT cells, gdT cells, and B cells were present in the brain. Real time PCR and ELISA analysis showed a decreased level of several of the inflammatory cytokines and chemokines such as IL-6, TNF-a, IFN-g, and MCP-1 during the course of infection. IF microscopy analysis revealed that in MyD88−/− mice the expression of inflammatory cytokines was reduced in leptomeningeal and periventricular areas of brain which are known to influence leukocytes infiltration in NCC. MyD88 signaling also appeared to influence the humoral immunity as infection in the MyD88-deficient mice was associated with a lower parasite soluble antigen-specific IgG2a Ab titers.
In patients infected with Taenia solium, the destruction of larvae by therapeutic treatment or during parasite life cycle associates with strong inflammatory response and disease manifestation. The better containment of the disease along with the absence of inflammatory response and lack of neuropathology in MyD88-deficient mice may help in formulating therapeutic immune intervention strategy for neurocysticercosis.
Matsumoto Yoh⁎ 1 , Kohyama Kuniko 1 , Hiraki Keiko 1 1 Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan
Accumulating evidence strongly suggests that T cells and autoantibodies reactive with myelin oligodendrocyte glycoprotein (MOG) play a critical role in the pathogenesis of multiple sclerosis (MS). In the present study, we have tried to elucidate the pathomechanisms of lesion formation and progression of MS by analyzing the role of T and B cells in MOG-induced experimental autoimmune encephalomyelitis (EAE) that exhibits various clinical subtypes mimicking MS. Special attention was paid to examine the presence or absence of autoantibodies against conformational epitope(s) of the MOG molecule.
Relapsing remitting (RR) or secondary progressive (SP) EAE was induced in LEW.1AV1 rats by immunization with recombinant rat MOG or synthetic peptides covering the entire extracellular domain of MOG. The responses of T cells from immunized rats to MOG and MOG peptides were determined by T cell proliferation assay. Autoantibodies against linear and conformational epitopes were assayed with conventional ELISA and flow cytometry using native MOG-expressing cells, respectively.
Immunization with synthetic peptides revealed that encephalitogenic epitopes resided in Peptide 7 (P7, residue 91-108) and P8 (residue 103-125) and induced RR or SP EAE. However, the disease severity of MOG peptide-EAE was generally milder than that of MOG-EAE. T cells taken from MOG-immunized and MOGP7-immunized rats responded to MOG and MOGP7 and sera from MOG-immunized rats reacted to MOG and MOGP1 throughout the observation period. Interestingly, sera from MOGP7-immunized rats with RR-EAE did not react to MOG protein and peptides during the observation period. There was no epitope spreading at both T cell and antibody levels. Analysis of conformational epitopes (cme) in sera from MOG-immunized rats revealed that the titers of anticme antibodies paralleled the disease status.
These findings demonstrate that the development and relapse of MOG peptide-EAE occurs without autoantibody involvement but that autoantibodies play a role in the progression of the disease as found in MOG-EAE. Collectively, the disease processes of EAE, and probably of MS, are regulated by several different mechanisms and immunotherapy should be performed properly on the basis of such information. Tissue transglutaminase (TG2) is a multifunctional enzyme, involved in post-translational modification of proteins. During various inflammation-related processes, including cell adhesion and migration, TG2 has been suggested to play a major role. Therefore, we propose that TG2 can contribute to some of the pathological processes underlying MS.
Since we observed that TG2 immunoreactivity is elevated in MS lesions, we questioned whether TG2 is present in the CNS of rats suffering from chronic relapsing EAE (cr-EAE). To study the expression of TG2 during cr-EAE, animals were sacrificed at different phases of clinical disease and we found an increased level of TG2 mRNA and protein in various cell types including infiltrating cells, mainly in the upper parts of the spinal cord during clinical disease of cr-EAE. Subsequently, we studied whether TG2 contributes to clinical disease during cr-EAE. KCC009, a specific irreversible inhibitor of TG2 was injected i.p. daily in cr-EAE animals from first clinical signs onwards. All animals were sacrificed when the PBS-treated controls reached the relapse phase of cr-EAE. Our data showed that inhibition of TG2 activity reduced clinical symptoms during cr-EAE dramatically and has an effect on the neuropathological outcome of cr-EAE. There was no significant effect on the mRNA levels of different genes which are normally higher expressed during cr-EAE such as CD68, inflammatory mediators, and genes involved in adhesion and migration processes. Immunohistochemical analysis with OX6, a MHC class II marker detecting monocytes, activated macrophages and microglia showed that KCC009 treatment attenuated monocyte infiltration in the spinal cord of cr-EAE animals whereas microglia remained activated in KCC009 treated animals.
These data suggest that KCC009 mainly has a peripheral effect reducing TG2 activity in infiltrating cells and thereby attenuate cell adhesion and migration, an important pathological process underlying MS. Therefore, TG2 can be a novel therapeutic target in MS. This protocol induced an EAE-like disease characterized by massive infiltration of CD8+ T-cells to the brain and spinal cord as analysed by flow cytometry and histological analysis. Preliminary FACS analysis showed that 80% of the CNS infiltrating T-cells were CD8-positive in symptomatic mice. Non-symptomatic mice also showed T cell infiltration to the spinal cord, and 50% of the infiltrating T-cells were CD8-positive. These CD8 proportions were substantially greater than in our previous studies of conventionally-immunized MOG-or MOG peptide-induced EAE where proportions of CD8+ T-cells in the CNS of symptomatic mice were in the 10-30% range.
Those studies indicated that CD8+ T-cells play a regulatory role despite displaying an activated memory-effecter phenotype. Results of this study suggest that the route of antigen presentation may determine whether activated CD8+ T-cells play a pathogenic role and that presentation through viral recognition pathways results in an encephalitogenic CD8+ T-cell response. This has implications for our understanding of MS. In an attempt to observe the effect of blocking angiogenesis in neuroinflammation, we synthesize 10 new prodrugs of thalidomide. A series of molecular or macromolecular amphiphilic derivatives of thalidomide were prepared starting from 3-N-(3-aminopropyl)aminothalidomide and exhibiting a modular hydrophilic-lipophilic balance. Their biological activities were examined using myelin oligodendrocyte glycoprotein 35-55 peptide-induced acute EAE in the C57BL6 mouse. When mice were treated on the second day when clinical signs were observed for a total of 7 days, two of these thalidomide derivatives (having molecular weights 7986 (AP05/09) and 14,244 (AP06/06) showed clear efficacy in reversing the clinical signs of EAE at 100 mg/ kg equivalent dose of thalidomide.
These studies provide additional evidence that VEGF and angiogenesis are components of an acute lesion and this could provide an interesting target for limiting the extent of the acute MS attack. Experimental autoimmune encephalomyelitis (EAE) is a central nervous system (CNS) specific disease and a primary animal model of multiple sclerosis (MS). The common marmoset (Callitrix jacchus), which is used in this study, is a small New World monkey. The marmoset provides a highly useful model to study the pathogenesis of MS, due the outbred nature and immunological proximity to human.
Immunization of this non-human primate with a synthetic peptide representing amino acids 34 to 56 of the human myelin/oligodendrocyte (MOG) protein in mineral oil (incomplete Freund's adjuvant; IFA) induces full-blown clinical EAE. Animals sensitized at 4 weeks interval all developed clinical EAE with body weight loss. Magnetic resonance brain images of presymptomatic monkeys were recorded on a 4.7 T spectrometer showing large hyperintense areas in the white matter. Immunohistology showed that the MRI-detectable abnormalities were large demyelinated lesions in the brain, and that lesions were also present in spinal cord and optic nerve.
Positive proliferating T-cell responses to the MOG peptide 34 to 56 (MOG34-56) were observed and T-cell lines generated from the spleen and lymph nodes against MOG34-56 expressed phenotypes of NK-CTL like cells; CD3+, CD4+ and/or CD8+, CD56+ and CD16−. Specific cytotoxicity of the specific T-cells lines was confirmed using peptidepulsed EBV-transformed autologous and allogenous B-cells by chromium release assay. Target cell exchange studies show that cytolysis is peptide specific but target cell independent. This pleads for peptide presentation by an invariant MHC class I allele, being Caja-E.
We conclude that the minimal requirement for induction of EAE in marmosets is prime-boost with MOG34-56 peptide in IFA. EAE is caused by the autoreactive cytotoxic T-cells which display specific cytolytic activity towards cells presenting peptide via non-classical MHC class I Caja-E alleles. IL-15 (KO) mice were recently available from Taconic where they were backcrossed 5 times onto C57BL/6NTac mice and intracrossed to make them homozygous for the IL15 (KO). We immunized the IL-15 (KO) (n =10) and C57BL/6NTac mice (background, n= 10) with myelin oligodendrocyte glycoprotein 35-55 peptide (200 μg) in complete Freund's adjuvent. Bordetella pertussis toxin (200 ng) was also administered i.p. on day 0 and 2 post immunization (p.i.). IL-15 (KO) mice had the same clinical signs as control mice until day 15-21 p.i. when they displayed more severe clinical signs (pb 0.05). Pathological changes were also significantly worse in spinal cord sections stained for inflammation (p = 0.024) and demyelination (p = 0.014). Unexpectedly, extensive demyelination (N20% of the sections) was observed in 4/10 of the IL-15 (KO) mice.
Mice lacking IL-15 showed more severe acute EAE. We propose that there is a compensation by other inflammatory mediators which exacerbates the disease.
27 -Discrepant oxyradical production by phagocytes from marmosets and rhesus monkeys with differential encephalitis pathology Experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS), can be induced in the non-human primate species rhesus macaques and common marmosets with recombinant human myelin oligodendrocyte glycoprotein (rhMOG). While EAE induction in rhesus macaques leads to acute ADEM-like neurological disease with highly destructive lesions, rhMOG immunization of marmosets induces MS-like progressive neurological disease with primary demyelination and conservation of axons. Since axonal damage may be caused by reactive oxygen species (ROS) that are produced by macrophages and microglia during phagocytosis of myelin debris, we have investigated the profile of ROS produced by activated phagocytes from both species.
The production of superoxide anion (O 2 −) and hydrogen peroxide (H 2 O 2 ) was analyzed in PMA stimulated peripheral blood mononuclear cells (PBMC) and granulocytes. Superoxide dismutase inhibitable superoxide anion production was determined by lucigenin luminescence. The production of superoxide anion is much higher in rhesus monkey cells (1000-5000 cpm) than in marmoset cells (200-300 cpm). Production of hydrogen peroxide was measured by the horseradish peroxidase-catalyzed oxidation of amplex red to resorufin that can be measured at 570 nm. Hydrogen peroxide production by marmoset cells is hardly detectable (OD b 0.1), in contrast to the production by rhesus monkey cells (OD 0.6).
In conclusion, we have found differences in the oxidative burst between both species. Sialic acid residues on gangliosides expressed in the axonal membranes and myelin are prominent scavengers of H2O2. We hypothesize that reaction of sialic acids with H 2 O 2 will affect the specific binding of neurons to myelin-associated glycoprotein (MAG) contributing to disruption of compact myelin and enhanced binding of autoantibodies. Since rhesus monkey cells produce more hydrogen peroxide than marmoset cells this may lead to more and faster disruption of compact myelin explaining the acute disease and destructive lesions in rhesus monkey EAE. We will discuss the expression of sialic acids and MAG in the brains of rhesus and marmosets with EAE. Moreover, data on the damage to DNA and proteins caused by ROS will be discussed. CD40 is a co-stimulatory molecule functioning at the interface of antigen presenting cell (APC) in the immune system. In the central nervous system (CNS), it is also expressed in reactive microglia and plays a role in neuroinflammatory disorders such as Alzheimer's disease, Parkinson's disease and multiple sclerosis by inducing various inflammatory molecules such as inducible nitric oxide synthase (iNOS). Therefore, we investigated a role of Class4 Semaphorin Sema4D/CD100, which could modulate CD40 signaling.
Since the receptors of Sema4D are identified to be Plexin-B1 in the CNS while CD72 plays a major role in the immune system, we prepared primary microglia from wild-type, CD72-deficient and Plexin-B1-deficient newborn mice (C57 BL/6 background). When microglia are incubated with recombinant Sema4D, CD40-induced iNOS expression was enhanced. In addition, immunohistochemical studies revealed that Sema4D was expressed in the infiltrating cells in the spinal cord of experimental autoimmune encephalomyelitis (EAE) and Plexin-B1 was up-regulated in microglia/macrophage. When EAE was induced by adoptive transfer of wild-type MOG-reactive T cells to wild-type or Plexin-B1-deficient mice, Plexin-B1-deficient recipient mice exhibited a milder phenotype than wild-type.
Collectively, these results suggest that Sema4D participates in the neuroinflammatory process by enhancing CD40-related inflammatory process via Plexin-B1. Interaction between an antigen-presenting cell (APC) and a T-cell leads to the rapid formation of an immunological synapse. Upon recognition of the appropriate antigen, a swift rise of intracellular calcium is triggered within the T-cell which can be used to monitor the activation status of the lymphocyte. While this approach is successful in cell culture, in vivo calcium imaging in T-cells still remains a major challenge as synthetic calcium indicators do not persist intracellularly for a sustained period. To overcome this problem we wanted to create transgenic T-cells stably expressing a fluorescent calcium sensor for long-term tracking and calcium measurement studies.
By retroviral transduction we generated T-cells transgenically expressing the novel fluorescence resonance energy transfer (FRET)-based calcium sensor TNXXL. This sensor features spectrally optimized derivatives of the fluorescent proteins CFP and YFP as FRET pair linked by the calcium-sensitive domain Troponin C. Originally designed for calcium imaging in neurons, this sensor is also capable of responding to the more modest alterations in lymphocyte intracellular calcium levels.
These transgenic T-cells will be used for in vivo imaging studies in experimental autoimmune encephalomyelitis (EAE). We plan to track the migration and activation of T-cells upon the encounter of APCs, e.g. during or after passing the blood-brain barrier and while infiltrating the CNS. For this purpose various inducible as well as spontaneous models of EAE are available in our lab thus providing the opportunity to investigate T-cell activation under different disease conditions. Interleukin-23 (IL-23) is a crucial mediator of tissue inflammation during EAE. As heterodimeric cytokine consisting of the IL12-B (p40) and the IL23-A (p19) subunits, it is produced by antigen-presenting cells as macrophages and dendritic cells and acts primarily on T cells. In the MOG35-55 model of EAE, IL-23 was shown to be vital for the development of disease. It is clear, that EAE induction upon peptide immunization will trigger a CD4 mediated response and that IL-23 has some influence on the polarization of TH17 cells. Nevertheless, we found, that IL-17 production is not only restricted to CD4+ T cells, but even a greater number of CD8+ T cells. Due to the CD4 bias of peptide induced EAE, CD8+ T cells have long been neglected in experimental models of CNS inflammatory disease. CD8+ T cells have been implicated in mediating axonal damage in both mice and men. Nevertheless, the interplay between IL-23 and CD8+ T cells in autoimmune CNS inflammation remains ill defined.
Our mouse model relies on the expression of OVA under the control of a modified oligodendrocyte-specific myelin basic protein (MBP) promoter. Upon active immunization with the MHC class I OVA peptide or adoptive transfer of OT-1 T cells the mouse shows severe EAE symptoms. To address the impact of IL-23 on CD8+ T cells, we crossed these ODC-OVA mice onto the p40−/− and p19−/− background, the two subunits of IL-23. Preliminary data indicate that loss of IL-23 leads to a change in the disease pattern and that the IL-23/IL-17 axis applies not only to TH cells, but also to encephalitogenic CTLs.
The primary impact of IL-23 appears to be on the cytotoxic capacity of CTLs rather than the local (CNS) tissue response. Further experiments will reveal the interaction of IL-23 with encephalitogenic CD8 T cells and the role of IL-17-secreting CD8 T cells during EAE.
Bacterial cell wall peptidoglycan (PG) is a strong pro-inflammatory agent promoting development of Th17 cells and potentially stimulating multiple sclerosis (MS) lesion activity. PG is present in innate immune cells infiltrating MS brain tissue, and Staphylococcus aureus PG stimulates EAE (experimental autoimmune encephalomyelitis) in wildtype mice when admixed with autoantigen in incomplete Freund adjuvant (IFA). PG stimulates cells via innate immune receptors like membrane-associated Toll-like receptor 2 (TLR2) and cytosolic Nod1/ CARD4 and Nod2/CARD15. We hypothesized that the stimulatory effect of S. aureus PG on EAE is independent of TLR2 action in vivo. Hence we asked whether S. aureus PG as the single bacterial component admixed with autoantigen in IFA can induce EAE in animals genetically deficient for TLR2.
Indeed, TLR2 knockout mice still developed EAE after induction with this well-characterized highly purified soluble PG from S. aureus and MOG35-55. TLR2 knockouts had similar disease incidence and severity compared to wildtype mice. TLR2-dependent and -independent activity of this S. aureus PG preparation in vitro was assessed using bone-marrow derived dendritic cells (DC). When stimulated with PG, wildtype DC upregulated the expression of CD40, and secreted the proinflammatory cytokines TNF-a, IL-6 and IL-12p70, as well as the anti-inflammatory cytokine IL-10. As expected, these parameters were reduced in DC from TLR2 knockout mice. However, the expression of TNF-a, IL-12p70 and IL-10 was not fully ablated in DC lacking TLR2, suggesting only a partial TLR2-dependent effect of PG.
[Ongoing: Importantly, S. aureus PG induced production of IL-23, which is essential for sustaining Th17 polarization, by (human) antigen presenting cells].
Our data provide proof of principle that this highly purified soluble S. aureus PG can effectively induce EAE in the absence of TLR2 engagement. This suggests that the Nod2-mediated action of S. aureus PG is reasonable sufficient to promote EAE.
32 -Loss of non-canonical NFkB signalling in antigen-presenting cells aborts adaptive immunity Hofmann Janin⁎ 1 , Greter Melanie 1 , Becher Burkhard 1 1 Neuroimmunology, University Zürich, Switzerland
The inflammatory canonical NFkB pathway is critically involved in virtually all aspects of inflammation in general. On the other hand, the role of the alternative, non-canonical NFkB pathway in inflammation and adaptive immunity remains elusive. The alternative pathway is primarily mediated through the NFkappa-B inducing kinase (NIK) which in turn leads to the phosphorylation and the cleavage of p100 to p52. Among the receptors engaging NIK is the LTbR, which is also required to form the anlage for secondary lymphoid tissues (SLTs). Due to a point mutation within NIK, alymphoplasia (aly) mice do not develop SLTs and are highly immunodeficient. However, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of NIK itself could potentially lesion immunity.
To verify this notion, we generated a series of bone marrow chimeric mice (BMC) in which the absence of SLTs was disconnected from the hematopoietic loss of NIK function. We generated mice, which lack all SLTs, but are equipped with a normal systemic immune system (wt→aly), and conversely, mice with normal SLTs, but lacking NIK in all leukocytes (aly→wt). Surprisingly, we discovered that NIK is vital for the development of autoimmune disease, while SLTs (ie. LNs, spleen etc.) are essentially dispensable for T cell priming. We discovered that NIK is required for the polarization of effector T cells and that TH17 and TH1 cells cannot be generated in the absence of NIK. However, the NIK lesion does not cause an intrinsic T cell defect but leads to the inability of APCs to drive T cell polarization. Diphtheria toxin-mediated cell depletion suggested a crucial role of NIK in dendritic cells (DCs) for the development of EAE. This data is strongly supported by the finding that aly DCs show functional deficits in maturation and pro-inflammatory cytokine production.
Taken together, loss of non-canonical NFkB signalling in DCs ultimately leads to abortive T cell priming. Phosphodiesterases (PDEs) are enzymes degrading cyclic nucleotides. Inhibitors of some PDEs have been reported as therapeutic agents for experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis (MS), by inhibiting antigenmediated T cell proliferation and down-regulating the production of Th1 cytokines. Cilostazol is known as a specific inhibitor of PDE3, which is used for treatment of ischemic disease by acting on antiplatelet and vasodilator properties. Besides, cilostazol suppresses the expression of such adhesion molecules. In this study, we investigated possible therapeutic effect of cilostazol for EAE. EAE was induced in B6 mice by inoculation with MOG35-55 as usual protocol. Mice were fed with blended solid diet with cilostazol (n = 28) or vehicle control (n = 28). Cilostazol suppressed the maximum EAE score and the average EAE scores on days 15, 16 and 17, compared with the control group. In the recall responses with MOG35-55, the proliferation and IFN-g production of lymphocytes from cilostazol-fed mice were significantly reduced in comparison with those from control mice. In contrast, no significant differences were detected in the level of TNF-a, IL-17 and IL-10 productions between these groups. Serum level of sICAM-1 was significantly lower in cilostazol-fed mice than in control mice.
Cilostazol may have repressive effects on EAE by reducing the antigen specific T cell response and decreasing the expression level of adhesion molecule, ICAM-1. Although further investigation is needed to clarify the detailed mechanisms of therapeutic effects in EAE, cilostazol is a hopeful choice for the treatment of MS. IL-17 has recently emerged as a critical cytokine in T cell-mediated autoimmune diseases. IL-17 is produced by a T cell subset distinct from Th1 and Th2 cells, and induces IL-1, TNF-alpha and IL-6 expression in inflamed tissues. IL-6 and TGF-beta can induce naïve T cells to differentiate into IL-17-secreting CD4+ T cells in vitro and we have shown that IL-1 and IL-23 promote IL-17 production from T cells in the absence of TCR engagement.
In this study we examined the infiltration of IL-17-producing T cells into the CNS during the development of experimental autoimmune encephalomyelitis (EAE). Mononuclear cells were isolated from peripheral immune and CNS tissue 7, 10, 14, 21 and 35 days post immunization and analyzed for cytokine expression by flow cytometry. The kinetics of changes in mRNA expression of a number of associated cytokines was also examined by quantitative PCR.
We found significant infiltration of CD4+ and CD8+ T cells into the brain and spinal cord of mice immediately prior to the onset of clinical signs of EAE. Approximately, 6% of total CD3+ T cells in the CNS of mice produced IL-17 on day 14 after induction of EAE. CD4+ T cells constitute up to 40% of this CD3+ population, and 10-20% of these expressed IL-17. Furthermore, gamma delta T cells were detected in the CNS. Whilst these constitute only around 2% of the total CD3+ population, we show that 70% of these produced IL-17, but not IFNgamma. These findings correlated with increased expression of IL-1beta, IL-23, IL-6, TGF-beta, IFN-gamma, IL-17A and TNF-alpha mRNA in the cerebral cortex, cerebellum and spinal cord of mice on day 14. In contrast, IL-17 mRNA expression, as well as that of IL-1beta, IL-6 and IL-23, was significantly increased in the spleens of mice on day 7 of EAE, but subsided at the peak of disease. In addition, IL-17 producing CD3+ T cells were significantly increased in the spleen on day 7. NK cells and CD8+ T cells in the CNS did not express significant IL-17 over the course of disease.
We hypothesise that IL-17-producing T cells are activated in the periphery early after immunisation and subsequently migrate to the CNS, where they stimulate local production of proinflammatory cytokines from glial cells. We found that gamma delta T cells infiltrated the CNS during the development of EAE and, at the onset of clinical symptoms, a very high percentage of these cells express IL-17 and may have the potential to induce local inflammatory response during EAE. Proteolipid Protein (PLP)-inoculation in SJL/J mice generates a form of Experimental Autoimmune Encephalomyelitis (EAE) that exhibits a relapsing-remitting course and is used to investigate mechanisms of disease relating to Multiple Sclerosis (MS). Hitherto, most studies have concentrated solely on the central aspects of MS pathology. However, it is becoming increasingly evident that systemic events also contribute to MS pathogenesis. We have observed histological changes in the livers of individuals with MS including the recruitment of neutrophils. We were interested to discover whether our model of EAE also exhibits cellular and molecular changes that might contribute to systemic pathology. We have previously shown that neutrophil recruitment to the liver after brain injury is associated with hepatic chemokine production and the expression of classical Acute Phase Proteins (APPs). We examined the liver from animals with EAE to discover whether this was also true for immune-mediated Central Nervous System (CNS) diseases.
To induce EAE, female 8-12 week old SJL/J mice were injected subcutaneously with 25 μg of PLP emulsified in 50 μl Complete Freund's Adjuvant (CFA) or with Phosphate Buffered Saline (PBS) in 50 μl CFA for our control group. On the day of the immunization and 48 h later, both treatment groups received an intravenous injection of 200 ng pertussis toxin in 0.2 ml PBS. Using RT-PCR, we were able to establish the presence of an extended expression of APPs and chemokines in the liver of EAE mice prior to the development of overt clinical signs. We show an elevated expression of Serum Amyloid A1 (SAA1), Serum Amyloid A2 (SAA2), Serum Amyloid P (SAP), CCL 2 and CXCL 10 just before the onset of clinical symptoms at day 8 which in some cases persisted into the clinical phase of disease. Most of the APPs returned to naïve levels by day 18, during the period of remission. Furthermore, the production of these APPs and chemokines was associated with the recruitment of leukocytes to the liver.
Thus systemic pathology accompanies EAE and MS and is likely to impinge on clinical signs and contribute to disease progression. The identification of the liver in particular as a preclinical component of MS defines an important time window between chemokine formation, lesion formation and an MS relapse which may be exploited for therapeutic purposes. An individual's risk to multiple sclerosis (MS) is determined by the triad of genes, environment and infection. Studies showing that the concordance of MS is higher in non-identical twins than in siblings from independent births suggest an influence of the simultaneous in utero exposure to maternal factors. We have tested in marmoset monkeys whether corticosteroid hormones leaking through the placenta influence the severity and course of MS-like disease in later life.
In the context of a large multinational experiment funded by the European Commission (Grant QLRI-CT-2002-02758, EUPEAH) led by the German Primate Center pregnant female marmosets were treated with dexamethasone (DEX) in the form of a daily 5 mg/kg oral dose for 7 days. The offspring developed moderate metabolic changes reminiscent to those observed in children from mothers who received dexamethasone to limit complications of preterm delivery. We received five female monkeys that were siblings from mothers treated daily with DEX in the first trimester of pregnancy (day 42-48) and four females from mothers receiving DEX late in the second trimester (day 90-96) of the total 144 days gestation period. Four normal age and sex-matched control monkeys were added to the experiment. EAE was induced in all 14 monkeys by repeated immunization at 28 days interval with a mix of two MOG peptides (34-56 and 74-96) in adjuvant.
All 14 monkeys developed overt clinical EAE. However, EAE in the monkeys that were prenatally exposed to DEX developed more rapidly and with a higher lesion load than in the control monkeys. The most remarkable immunological effect was that T-cells from the control monkeys proliferated against the two immunizing peptides, but not against recombinant human MOG protein (rhMOG), while T-cells from both DEX exposed groups proliferated also against rhMOG suggestive of epitope diversification beyond MOG peptides 34-56 and 74-96.
These data suggest an influence of antenatal exposure to dexamethasone on the severity of EAE induced at adult age. To observe the change of phosphoprotein 53 (P53), tumor necrosis factor-a (TNF-a) and transforming growth factor-b (TGF-b) expression, as well as to study the immunosuppressive effects of Simvastatin in a dosage of 1 mg/kg intraperitoneally in Wistar rats with experimental autoimmune encephalomyelitis (EAE).
Guinea pig spinal cord homogenate, in Complete Freund's adjuvant, mycobacterium bovis bacillus calmette-guerin and pertussis toxin were used to immunize Wistar rats. They were divided into two groups for the immunosuppressive effects of the medicine. Simvastatin group was intraperitoneally injected with Simvastatin once a day from 0 to 10 p.i. The clinical scores, body weight were recorded every other day. The brains and spinal cords were removed on days from 11 to 18 p.i. for HE staining and immunohistochemistry staining in different parts of nervous system.
When compared with that of EAE group (2.33 ± 1.75), the clinical symptom of Simvastatin group (0.83 ± 1.28) was statistically significantly decreased (p b 0.05). The loss of body weight and the incidence in Simvastatin group was statistically significantly decreased when compared with those of EAE group (12.27 ± 4.95 vs 18.13 ± 5.00, p b 0.05; 40% vs 80%, p b 0.05, respectively). So was the differences of morbidity and number of infectious foci and onset date between Simvastatin group and EAE group (p b 0.05). On days 11 to 18 p.i., the expression of P53 and TGF-b in Simvastatin group was statistically significantly increased when compared with those of EAE group Simvastatin is a potential agent to inhibit EAE effectively. The inhibiting mechanism of Simvastatin is related to up-regulation of P53 and TGF-b and down-regulation of TNF-a, which may promote the transdifferentiation of Th1 cells to Th2 cells. P53 may exert protective function by suppressing expression of TNF-a and promoting expression of TGF-b in rats with EAE reported here in this paper. Multiple sclerosis is associated with some cognitive deficits, from the disturbances of memory, language fluency to full developed dementia. These deficits start, in some cases, very early during first years of the disease and develop independently from motor deficits. Animal studies showed that cognitive deficits in autoimmune encephalomyelitis (EAE), model of MS, also exist and may depend on degeneration of the cholinergic pathways.
In this study we showed that such memory deficits existed in EAE may be also depended on pyramidal neurons loss in hippocampus. Lewis 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. T cells infiltrated all the white matter in the brain and also hippocampus CA1 and CA4 areas 5 days after EAE induction, than disappeared during recovery period. The number of pyramidal CA1 and CA4 neurons was decreased by 10-15% on day 21 and 60 after EAE induction. No cortical (frontal cortex) degeneration was visible on these time points. Microglial and astrocytes activation increased from day 5 to 21 in CA1 ad CA4 regions, and NG2+ cells reaction was also increased on day 30.
In conclusion, anti-MBP CD4+ T cells are capable not only to evoke inflammatory reaction in spinal cord and brain leading to motor deficit, but also they may directly injure hippocampal pyramidal neurons.
Tischner Denise⁎ 1 , van den Brandt Jens 1 , Herold Marco 2 , Reichardt Holger 1 1 Institute for Cellular and Molecular Immunology, Goettingen, Germany 2 Institute for Viroloty and Immunobiology, Wuerzburg, Germany
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system of presumed autoimmune origin. Acute relapses are most commonly treated with high doses of glucocorticoids (GCs), necessitating a better understanding of the mechanisms that underlie GC actions in MS. By using different cell-specific glucocorticoid receptor (GR) knockout mouse strains we could show that GCs mainly act on T cells. Nevertheless, the differential effects of GCs on effector versus bystander T cells remain elusive.
Therefore we inactivated GR expression by retrovirally expressed miRNAs in fully differentiated encephalitogenic T cells (Tenc) and generated two cells lines with a constitutive knockdown of the GR as well as one control cell line. In various apoptosis assays the GRdeficient Tenc cells displayed a significantly increased survival rate after GC treatment, indicating a successful knockdown. In addition, repression of the effector cytokines IFN-gamma and IL-17 by GCs was impaired in those cells, suggesting that also the transrepressive activity of the receptor was affected.
Collectively, we envisage that inactivation of the GR in differentiated Tenc cells by RNAi will allow for investigating the influence of GCs on pathogenic effector T cells during Experimental Autoimmune Encephalomyelitis (EAE) in vivo and thereby help to further elucidate the mechanisms underlying GC therapy of MS. Variations in severity and duration of experimental autoimmune encephalomyelitis (EAE) depend on the species and the method of induction. Previous studies have shown that astrocytic connexin43 (Cx43) expression is decreased in demyelinating lesions and increased in remyelinating lesions in the guinea pig chronic model. The purpose of this study was to determine whether Cx43 was also involved in chronic and recovering mouse EAE models and used RNA microarray studies to investigate the modulation of other gap junction activity and other potential therapeutic targets.
In this study, we compared two methods of immunization with myelin oligodendrocyte glycoprotein (MOG 35-55) peptide or human recombinant MOG protein (rMOG), in female C57BL/6J mice. Animals used in this study include: MOG peptide chronic mice, n= 15, chronic controls, n =8, rMOG, n = 11, and acute controls, n=4. MOG peptide caused a chronic mouse model with continued demyelination and immune cell infiltration for at least 55 days post-immunization. rMOG immunization produced a less severe disease course with many animals recovering clinically with spinal cord remyelination Cx43 RNA levels were increased in the rMOG EAE model, as well as the oligodendrocyte connexin, Cx47, which shows that intercellular communication via connexin proteins in astrocytes and oligodendrocytes. The other connexin proteins expressed in the CNS were not altered significantly. Other, non-connexin RNA transcripts, and potential therapeutic targets, altered by at least 3-fold, were related to lymphocyte activation and immune regulation. Other molecules of interest with a 6-fold increase compared to healthy controls were: proteoglycan 4; neutrophilic granule protein; Hox10 (involved in CNS development), Clec7a (a calcium binding protein) and CD84 (found on activated T helper cells).
These are the first studies to compare spontaneously recovering and non-recovering chronic mouse models of EAE. Each model has useful characteristics: the rMOG recovering model, using microarrays, in order to identify possible therapeutic targets, and the MOG peptide chronic model, to use for intervention testing. Cell communication via connexin gap junctions is potentially a significant factor required for recovery, since increased expression of Cx43, and potentially Cx47, has been observed in recovering mice, compared to chronic EAE mice and healthy controls. Recent studies have suggested that Th17 cells may play an important role in the development of autoimmune diseases, including EAE. However, the encephalitogenicity of Th17 cells has not been well studied.
Using adoptive transfer, we demonstrated that MBP Ac1-11 specific Th17 cells differentiated in vitro with IL-6+TGFβ+/−IL-23 from naïve T cell receptor transgenic T cells do not transfer disease, whereas Th1 cells do. After secondary stimulation of these MBP-specific Th17 cells with MBP Ac1-11, three populations of Th cells are observed which include IL-17 producing cells, IFN-g producing cells and cells that produce both IL-17 and IFN-g. This mixed population of T cells transfer disease as efficiently as Th1 cells. Since IL-1β has also been implicated in Th17 differentiation, we have also investigated the possibility that an alternative differentiation pathway may result in Th17 cells that are encephalitogenic. We found that induction of T-bet was necessary for encephalitogenicity, regardless of differentiation pathway.
Together, our findings suggest that Th1 cells are encephalitogenic and that IL-6+TGFβ induces Th17 cells that cannot transfer disease alone. The actual biological function of IL-23 in the context of inflammation of the central nervous system (CNS) remains elusive. Initial reports point towards a major impact of IL-23 on TH17 cell polarization and survival (the population of auto-aggressive cells thought to mediate autoimmune effector function). Loss of IL-23 results in the complete resistance to tissue-directed autoimmunity but surprisingly in the absence of IL-17A producing cells experimental autoimmune encephalitis (EAE) is only slightly ameliorated contradicting to the previous observation. However, it becomes increasingly evident that IL-23 has a profound impact on accessory cells and may not just be required for Th17 generation in vivo. Our goal is to decipher the precise mechanism by which IL-23 drives autoimmune inflammation and to systematically assess the effect it has on the biology of T cells as well as macrophages and dendritic cells (DCs).
We have shown that in the absence of functional T and B cells, in vitro differentiated bone-marrow derived macrophages after IL-23 stimulation produce high amounts of pro-inflammatory cytokines such as MIP-1 alpha and TNF-alpha. Peritoneal injection of IL-23 into RAG animals upregulates the expression of its receptor and induces the production of IL-23, IL-6, IL-17F among others in a T cell free environment. To restrict the functional impact of IL-23 to accessory but not T cells, we generated mixed BM chimeras using the bone-marrow of RAG deficient and IL-12Rb1 knock-out animals. After the recovery period, EAE was induced and for our surprise these animals showed strong signs of EAE and we could observe the same number and pattern of infiltrating cells to the CNS as in wild-type animals indicating the crucial impact of IL-23 on antigen presenting cells(APCs).
To further elucidate the function of IL-23R on a variety of cell types, we have generated a conditionally targeted allele of the IL-23R in C57Bl/6 mice. The gene-targeting strategy and its impact on immunity will be discussed. T helper 17 (Th17) cells have been identified to play a pivotal role in the development of experimental autoimmune encephalomyelitis (EAE) through the production of inflammatory IL-17. Although alltrans retinoic acid (ATRA) has been reported to suppress the development of Th17 cells, the application of ATRA for therapeutic treatment is limited due to its instability, poor bioavailability, and side effects. In this study, we examined whether Am80, a synthetic RARagonist with superior pharmacological properties, could control EAE by attenuating the inflammatory properties of pathogenic T cells.
Splenic T cells stimulated under Th17-priming conditions (TGFb + IL-6) in the presence of ATRA, Am80, or LE540 (RAR-antagonist) were assessed for their expression of inflammatory cytokines and lineage-specific transcription factors. Suppressive effects of those retinoids were assessed for in vitro differentiated Th17 cells as well. B6 mice immunized with MOG35-55 peptide to induce EAE were treated orally with Am80 or LE540 every other day. CNSinfiltrating T cells were analyzed for cytokine production and gene expression. Draining lymph node cells from MOG immunized B6 mice with or without oral retinoid treatment were analyzed for their proliferation and cytokine production after restimulation with MOG peptide.
Am80 exerted more potent suppression of Th17 differentiation and reciprocal induction of regulatory T cells than ATRA did in a dose dependent manner. Both compounds suppressed the IL-17 secretion and RORgt expression of already differentiated Th17 cells as well. Oral administration of Am80 ameliorated severity of EAE, whereas LE540 exacerbated the disease. Am80 treatment showed 90% reduction of IL-17 or IFN-g production and 60% reduction of TNF-a production by CNS-infiltrating T cells upon stimulation, without affecting their accumulative properties into CNS. Intriguingly, infiltrated T cells in brain or spinal cord were characterized by their diverse production of inflammatory cytokines and unique response after Am80 treatment. Oral administration of Am80 to B6 mice immunized with MOG peptide led to a significant suppression of IL-17 production by draining lymph node T cells upon restimulation with MOG peptide, without affecting their proliferative response.
Synthetic retinoid Am80 successfully inhibits inflammatory cytokine production by pathogenic T cells and could provide a novel therapeutic approach on multiple sclerosis. The goal of this study is to test the hypothesis that inhibiting glycogen synthase kinase-3 (GSK3) provides protection from multiple sclerosis (MS) and other inflammatory autoimmune diseases affecting the central nervous system (CNS), using the mouse model experimental autoimmune encephalomyelitis (EAE).
Growing evidence indicate that the serine/threonine protein kinase GSK3 is a major regulator of the inflammatory response in the periphery, but the extent to which GSK3 regulates inflammation in the CNS is unresolved. We therefore investigated if lithium, an inhibitor of GSK3, can ameliorate EAE in mice.
The GSK3 inhibitor lithium was therapeutic in EAE using three different treatment protocols: (1) treatment before induction of disease prevented EAE, (2) treatment initiated at the onset of clinical symptoms suppressed exacerbation of disease, and (3) treatment initiated at the peak of disease reduced clinical symptoms and induced partial recovery. The amelioration of EAE was associated with reduced demyelination, microglia activation, and leukocyte infiltration in the spinal cord. Conversely EAE was more severe in mice expressing constitutively active GSK3. Spleen and lymph node cells obtained from lithium-treated MOG35-55-immunized mice proliferated poorly to in vitro restimulation with antigen and also secreted much lower amounts of interferongamma, interleukin-6 and interleukin-17 as compared to mononuclear cells obtained from untreated MOG35-55-immunized mice. However, the ability of T-cells from lithium-treated mice to respond to anti-CD3 stimulation was normal indicating that lithium does not intrinsically affect T-cell activation. These results suggest that lithium therapy suppresses the generation, differentiation and/or expansion of MOG35-55-reactive effector T cells by acting on cells of the innate immune system.
A major form of clinical MS is a relapsing/remitting disease, which is modeled in female SJL mice immunized with proteolipid protein peptide (PLP139-151) . The initiation of lithium treatment in PLPimmunized SJL mice after the first clinical episode of EAE suppressed relapses for over 70 days; however the disease relapsed after lithium withdrawal. Remarkably, restoration of lithium therapy led to partial recovery.
Our results demonstrate that lithium suppresses EAE and identify GSK3 as a new target for inhibition that may be useful for therapeutic intervention of MS and other autoimmune and inflammatory diseases afflicting the CNS. Here we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigenspecific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE accompanied with an early T cell infiltration and a decreased proportion of regulatory FoxP3expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor (TNF-alpha) induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL 17 production in the CNS.
Our findings suggest DCs as a rate limiting factor for neuroinflammation and imply DC as a target for therapeutic strategies for CNS diseases. The clear association of the TH17 phenotype with autoimmune pathogenicity implicates TH17 cytokines as critical mediators of chronic autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE).
To study the actual impact of IL-17 on CNS inflammation, we generated transgenic mice in which the expression of high levels of IL-17A can be initiated by cre-recombination. While the general overexpression of IL-17A leads to skin inflammation and granulocytosis, T cell driven IL-17A expression does not impact visibly on the development and health of the mouse. In the context of EAE, here we demonstrate that neither the T cell-driven overexpression of IL-17A nor its complete loss has a significant impact on the development of clinical disease. Since TH17 cells also produce IL-17F, which evidently acts through the same receptor complex as IL-17A, it is feasible that it could compensate for the loss of IL17A in IL-17Anull mice. To determine the actual function of IL-17F in autoimmune inflammation of the CNS, we next generated IL-17Fnull mice. They not only lack IL-17F, but also produce significantly less IL-17A. Nevertheless, they too are fully EAE susceptible and display unaltered emergence, expansion, and polarization of auto-reactive T cells during EAE.
We conclude that IL-17A and IL-17F, while prominently expressed by encephalitogenic T cells, do not or only marginally contribute to the development of autoimmune CNS-disease. Experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), is an ideal disease paradigm for dissecting the multiple functions of transcription factor NF-kB during autoimmune demyelination and neurodegeneration. In this study we applied conditional gene targeting in mice to delete the inhibitor of NF-kB kinase b (IKKb), a catalytic molecule in canonical NF-kB activation, in selected CNS cell lineages and to study its cell-specific functions during the development of EAE.
Cell-specific IKKb deletion was achieved by mating mice carrying a loxP-flanked (F) IKKb gene (exon 3) (IKKbF/F) with transgenic mice expressing Cre recombinase under promoters specific for endothelial cells (Tie2-Cre mice), macrophages/microglia (CD11b-Cre) and neurons (CamkII-Cre). IKK-b deletion in endothelial cells (endIKKb-KO mice) did not alter the clinical course of EAE while deletion in CD11b-positive macrophages/microglia (mIKKb-KO mice) delayed the onset and ameliorated clinical symptoms. The deletion of IKK-b in CamkII-expressing neurons showed dual and opposite effects in EAE. Mice lacking neuronal IKK-b (nIKKb-KO) showed significant delay in disease initiation but subsequently developed a more severe, non-resolving disease compared to control mice. The increased pathology in nIKKb-KO spinal cord was associated with reduced expression of neuroprotective molecules and enhanced expression of immune mediators, including Th1-and TH17-related cytokines and chemokines that are known to be important for EAE pathogenesis.
Our results show that neuronal IKK-b is important for the correct regulation of neuroprotective mechanisms and CNS immune responses during CNS autoimmune disease and further emphasize the importance of neuroprotective strategies in the treatment of multiple sclerosis. The growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) is a well known cytokine affecting the differentiation of dendritic cells (DCs). DCs play a crucial role in regulating beneficial immune responses and in the development of autoimmune diseases such as multiple sclerosis (MS) or its animal model experimental autoimmune encephalomyelitis (EAE). GM-CSF preferentially induces the expansion of CD11b+ myeloid DCs (CD45hi CD11c+). This DC subset can be further differentiated into a F4/80-negative and a F4/80positive fraction. We previously found that, in contrast to F4/80-DCs, the F4/80+ DCs isolated from the EAE-diseased central nervous system (CNS) inhibited T cell proliferation and EAE occurrence. Thus we aimed at defining the differential role of GM-CSF in the development and remission of EAE.
GM-CSF producing cells were injected intracerebrally into mice which were immunized with myelin oligodendrocyte glycoprotein (MOG). Depending on the time point of GM-CSF treatment, mice developed more severe EAE early after immunization. In order to analyze the origin of GM-CSF-induced DCs, we generated congenic bone marrow-chimeric animals. The treatment with GM-CSF resulted in a preferential induction of F4/80-blood-derived DCs. In contrast, CNSderived DCs were predominantly of the F4/80+ phenotype. Furthermore, the GM-CSF-recruited F4/80-DCs stimulated T cell proliferation whereas the F4/80+ DC subset inhibited T cell proliferation. Finally, GM-CSF receptor (Csf2r)-deficient mice did not develop EAE and did not contain any infiltrates within the CNS during disease development despite unaltered priming compared to wild type controls.
In summary, the data demonstrate that both the DC recruitment and the disease severity of EAE are enhanced in the presence of GM-CSF. The chimera experiment further suggests that the effect of GM-CSF on stimulatory F4/80-blood-derived DCs dominates over the GM-CSF-mediated induction of inhibitory F4/80+ DCs. In addition, the results with the Csf2r-deficient mice indicate that GM-CSF plays a crucial role in the effector phase of EAE. Experimental Autoimmune Encephalomyelitis (EAE) in Lewis rats was induced by transfer of myelin basic protein-reactive T cells genetically engineered to express green fluorescent protein (TMBP-GFP cells). After a disease free period of 3 days the autoreactive T cells induced CNS inflammation and severe paralytic disease. Using 2photon microscopy we visualized the infiltration process of TMBP-GFP cells into their target organ during incipient EAE. Intravital imaging of spinal cord in situ combined with imaging of spinal cord explants enabled us to track TMBP-GFP cells throughout the entire CNS parenchyma. Imaging was complemented by gene expression profiling of the autoaggressive TMBP-GFP cells isolated from different CNS locations by cytofluorometric cell sorting.
The first TMBP-GFP cells arrived at the CNS within 24 h after T cell transfer (p.t.). These T cells were located in close contact to leptomeningeal blood vessels. The number of these vessel-associated TMBP-GFP cells increased during the next 24 h. The vast majority of T cells moved continuously at the vessel surface. A small proportion of the T cells (5%), however, were arrested in contact with perivascular phagocytes. On day 2.5 p.t. the TMBP-GFP cells started to distribute throughout the entire meningeal areas and to immigrate into the CNS parenchyma along the perivascular Virchow-Robin spaces. An increasing part of the T cells (25%) were now forming long lasting contacts to perivascular/meningeal phagocytes. From day 3 p.t. on, the T cells invaded the parenchyma adjacent to the meninges and CNS vessels and within the next 24 h they diffusely distributed throughout the entire CNS tissue. Within the CNS parenchyma 65% of the T cells were constantly moving whereas 35% were stationary.
2-Photon microscopy was complemented by studies of the gene expression profiles of TMBP-GFP cells on their way into the CNS tissue. TMBP-GFP cells residing in the spleen and circulating in blood displayed a "migratory" phenotype with low activation markers and upregulation of genes relevant for cell migration. Importantly, upon entry into the meningeal, TMBP-GFP cells were strongly activated producing high amounts of pro-inflammatory cytokines. Further activation occurred following entry into the parenchyma.
We present data indicating that autoaggressive T cells enter their target organ via the meninges where they recognize endogenous autoantigen presented by resident meningeal/perivascular phagocytes.
Multiple Sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). We aim to find mechanisms by which toll like receptors (TLRs) exacerbate or suppress CNS inflammatory demyelination. Both effects have been observed in experimental autoimmune encephalomyelitis (EAE) an animal model of MS-TLRs 2 and 9 in exacerbating disease, TLR4 in delaying onset and TLR3 in reducing severity. Naturally occurring CD4+CD25+ regulatory T cells (Tregs) can suppress autoimmune reactions and were recently found to express TLRs.
We are in the process of studying the effect of TLR3 on Treg suppression in the mouse by co-culturing CD4+CD25-responder cells with different ratios of Tregs with or without poly(I:C). Preliminary studies indicate that TLR3 ligand poly(I:C) increases the suppressive ability of Tregs in C57BL/6 mice and this is a potential mechanism by which EAE disease severity is reduced after poly(I:C) administration. We also determined which cytokines and chemokines are induced by TLR stimuli in murine splenocytes. The inflammatory chemokine MCP-1 was unregulated by TLR stimuli-Pam3CSK (TLR1/2), HKLM (TLR2), LPS (TLR4), Flagellin (TLR5), follistatin-like 1 (TLR6) and ssRNA40 (TLR7). The anti-inflammatory cytokine IL-10 was unregulated by the same stimuli and also by poly(I:C) (TLR3) and CpG ODN1826 (TLR9). TLR9 also induced increased production of IL12p70. All TLR stimuli also induced increased T cell proliferation. The response of human monocyte derived dendritic cells (MDDCs) to TLR stimuli in terms of maturation and cytokine and chemokine production was also studied. MCP-1 was unregulated by TLR stimuli poly(I:C) and LPS. The inflammatory cytokines IL-6 and TNF-a were unregulated by these two stimuli and also by Pam3CSK4 and HKLM. The effects of TLR stimuli on MDDCs in MS patients are in progress. We are also in the process of determining the effect of a range of TLR stimuli on the clinical and pathological outcome of EAE.
TLR stimuli alter the regulatory ability of Tregs and this may contribute to disease exacerbation or suppression in EAE. TLR stimuli lead to the production of cytokines and chemokines that may regulate autoimmune diseases and there is potential to aid immunotherapy by targeting these TLRs and the cytokines produced.
Rigolio Roberta⁎ 1 , Biffi Alessandro 1 , Oggioni Norberto 1 , Cavaletti Guido 1 1 Università degli Studi Milano-Bicocca, Monza, Italy Actively induced Experimental Autoimmune Encephalomyelitis (EAE) is a well reproducible model for portraying the acute phase of multiple sclerosis as well as good model to develop new promising treatments. Both lymph node and spinal cord (s.c.) have been widely described by means of molecular biology and immunohistochemistry. The aim of this study was to characterize both spleen and s.c. lymphocyte populations and to highlight possible useful markers to be monitored during new therapeutic treatments. Thus at the onset (day 10 after EAE induction) and at the peak of the disease (day 14 after EAE induction) we investigated CD4, CD8 and encephalitogenic TCRVb8.2+ T cells prevalence as well as the expression of adhesion molecules CD49d (VLA-4) and CD11a (LFA-1).
EAE was actively induced by subcutaneously inoculation of 50 μg guinea pig Myelin Basic Protein (gifted by P. Riccio) in complete Freund's adjuvant with inactivated Mycobacterium tuberculosis. The spleen and s.c. were dissected from both healthy (controls) and EAE-induced animals, cells were collected, stained with different combinations of conjugated antibodies and acquired using a flow cytometer.
In the spleen of EAE animal we reported a significant decrease in the mean absolute cell number on day 14, together with the presence of a CD45+ population other than lymphocytes as soon as day 10. Furthermore we observed an increase in CD4 and decrease in CD8 T cells compared to the controls on day 14, while no changes were reported on day 10. Moreover no alteration in the TCRVb8.2+ lymphocytes percentage as well as in CD49d (VLA-4) and CD11a (LFA-1) expression on CD4 and CD8 T cells was never observed. In the spinal cord no infiltrating lymphocytes were detected until day 14. Therefore the results on s.c. were compared to those obtained in the spleen on day 14. Thus CD4 lymphocytes percentage was higher in the s.c., while CD8 lymphocytes prevalence decreased. We also observed an increase in CD4 TCRVb8.2+ T cells percentage together with a significant increase in the expression of CD49d (VLA-4) which was more accentuated for CD4 than for CD8 T cells.
Our results confirm that EAE in Lewis rats is a CD4 T cell mediated disease in which the expression of TCR Vb8.2 and adhesion molecules is probably regulated mainly in the target organ. We show that multiparametric flow cytometry can be an informative method to describe immune system cells during the EAE ongoing.
While the elevation of several members of the matrix metalloproteinase (MMP) family is thought to promote the pathophysiology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), the rise of particular MMP members may have contrary beneficial functions during disease processes. MMP-12 mRNA levels rise significantly in the spinal cord in EAE-afflicted mice (Weaver et al., FASEB J 19:1668-1670, 2005) and may have a beneficial role in EAE pathogenesis. We examined the role of MMP-12 in EAE and its potential mechanism(s) of action.
129/SvEv MMP-12 −/− mice were induced with myelin oligodendrocyte glycoprotein for EAE. Mice were followed for 60 days to determine EAE disease profiles. The spinal cords were isolated and analyzed using flow cytometry, protein array, western blot and histology during EAE.
While MMP-12 −/− mice had similar day of onset of clinical signs as wildtype controls, their subsequent relapses in relapsing-remitting EAE were more severe. Residual disability of MMP-12 −/− mice at remission was also higher compared to wildtype controls. The worsened relapses and remissions in MMP-12 −/− mice occurred despite a deficiency of the antigen recall capacity of lymph nodederived cells, and a reduction in the proportion of macrophages in the spinal cord during the chronic phase of EAE. Significantly, large increases of levels of chemokines, pro-inflammatory and antiinflammatory cytokines, but low G-CSF levels, were found in the spinal cord of MMP-12 −/− mice during chronic EAE. We discovered as well that MMP-12 processes the multi-functional protein, osteopontin, into smaller fragments in vitro and in EAE; significantly, the higher severity of EAE in MMP-12 −/− mice returned to wildtype EAE scores in osteopontin and MMP-12 double −/− mice.
These results highlight MMP-12 as a beneficial MMP in EAE through cytokine and chemokine regulation, as well as through an interaction with osteopontin. Therapeutic interventions in MS that are aimed at reducing MMP activity should avoid MMP-12 as a target. The cytostatic drug Edelfosine is a synthetic analog of Lysophosphatidylcholin. It was originally regarded as an antitumor agent in cancer treatment. Edelfosine is incorporated by cells and it selectively activates the cell death receptor Fas/CD95. In contrast to other cytostatic drugs, Edelfosine does not intercalate into DNA. In vitro treatment of cells with Edelfosine showed that it 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 encephalitis (EAE).
In order to analyze the anti-inflammatory properties of Edelfosine we have used two models, C57BL/6 mice immunized with the peptide 35-55 of the myelin oligodendrocyte glycoprotein (MOG 35-55) and SJL mice immunized with the peptide 139-151 of the proteolipid protein (PLP 139-151) . The effect of Edelfosine on EAE disease course was evaluated by clinical, histological and flow cytometric analysis. We found that daily treatment of C57BL/6 mice with 10 mg/kg Edelfosine by gavage from the day of immunization led to a reduction of the maximum mean EAE score of 2.8 in control animals (gavage of PBS) to 0.7 in a 0 to 5 scale. In the control group all animals developed full blown EAE whereas only one mouse in the Edelfosine treatment cohort showed severe disease, while the others demonstrated weak signs of EAE or stayed healthy. No side effects due to drug administration were observed. Flow cytometry studies at day 29 after immunization in the C57BL/6 model revealed a reduction of the activated CD4+ lymphocyte fraction in secondary lymphoid organs of treated mice when compared to controls.
These results point to a beneficial effect of Edelfosine on EAE disease severity and suggest that it interferes with immune cell infiltration into the central nervous system. Further analysis of the therapeutic value of Edelfosine is currently underway in order to examine further the potential of this drug Edelfosine for the treatment of multiple sclerosis.
To identify the most critical factors of inflammation during animal model of multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE), we have used microarrays for the molecular analysis of the transcriptional profile of the central nervous system (CNS) infiltrate. Heat shock proteins (hsp), especially hsp70 were among the most prominent protein groups that genes expression was upregulated during EAE. Hsp, major inflammatory-induced proteins, were demonstrated to play both protective (e.g. anti-apoptotic), and proinflammatory (e.g. activation of TLRs) roles. In the recent study we aim to analyze the in vivo role of hsp expression in the development of autoimmune demyelination.
To obtain a definite proof for the role in vivo of hsp70 in myelin antigen recognition we used mouse deficient for the major gene encoding inducible hsp70, hsp70.1. Immunization of hsp70.1−/− mice with myelin/ oligodendrocyte glycoprotein (MOG) peptide 35-55 showed their almost complete resistance to induction of experimental autoimmune encephalitis (EAE). The EAE resistance correlated with lost of MOG35-55 proliferative T cell responses as well as significantly downregulated secretion of proinflammatory cytokines like interferon gamma (IFNg) or interleukin 17 (IL17) by CD4+ T cell in hsp70.1−/− mice. Interestingly hsp70 deficiency resulted in CD4 T cell activation dysfunction demonstrated by antigen presentation assays which showed that MOG35-55 reactivity was abolished when T cells from hsp70.1−/− mice were cocultured with wild type APC. Hsp70 deficient CD4+ T cell also failed to transfer disease in passive model of EAE. The mechanism of CD4+ T cell failure in hsp70.1−/− mice in responding to MOG35-55 was TCR dependent and involved activation-induced apoptosis.
These results provide a direct proof for the in vivo role of hsp, especially hsp70 in recognition of self myelin antigen MOG and strengthen the hypothesis that stress conditions leading to hsp70 induction may enhance autoimmune reactions. The present study was aimed to characterize the individual functions of myelin specific Th1 and Th17 T cells in EAE. We established polarized Th1 and Th17 populations, derived from MOG specific TCR transgenic mice with C57BL/6 genetic background. These cells were adoptively transferred, individually or as combination, to Rag2 deficient animals to test their encephalitogenic potential.
We found that both Th1 and Th17 CD4+ subsets alone are capable to induce EAE. Notably, however, co-transfer of Th1 and Th17 cells induced more severe EAE with earlier onset. EAE differed in clinical presentation: Th1 alone and Th1+Th17 co-transfer induced more than 90% classical EAE, while around 50% of Th17 transferred mice developed an ataxic phenotype.
Histological analyses indicated that lesion intensity largely correlated with astrogliosis and microglia activation. In Th1+Th17 transferred mice this was evident both in spinal cord and brain parenchyma, especially cerebellum. On the other hand, Th1 transferred mice showed more infiltration of spinal cord and perivascular spaces in brain, while Th17 transferred mice with classical EAE presented T cell infiltration both in spinal cord and brain parenchyma.
We preformed cytotoxicity assays of Th1 and Th17 cells on astrocytes in order to elucidate in detail the pathogenic mechanisms of these two T cell subsets in CNS resident cells. Preliminary data show that, in contrast to Th17, Th1 cells are cytotoxic to astrocytes and that this cytotoxicity may be independent on the presence IFNg, as blocking MAbs added during co-culture did not affect cytotoxicity. However, we found a correlation between extent of cytotoxicity and the percentage of IFNg+ cells in the total CD4 Th1 population applied to the astrocytes culture.
We conclude that both T helper cell subsets alone have the potential to induce EAE, probably by different mechanism, and that their interaction potentiates the autoimmune response. Both myelin-specific Th1 and Th17 cells have been implicated as the pathogenic T cells in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Recent studies have shown that IL-23-deficiency and systemic administration of anti-IL-23 monoclonal antibodies significantly reduces the incidence and severity of EAE, suggesting a critical role for IL-23 in the pathogenicity of EAE. IL-23 has been implicated as a critical factor in the differentiation, expansion, and survival of Th17 cells, thus speculating that IL-23 is critical for the production autoreactive Th17 cells and the subsequent induction of EAE. However, IL-23 receptor was originally described as a memory Th1 cell marker.
We have confirmed that myelin-specific T cells differentiated into Th1 and Th17 cells express similar levels of IL-23 receptor, suggesting that in addition to Th17 differentiation, IL-23 may play a role in the effector functions of Th1 cells. To determine if IL-23 is playing a critical role in the differentiation or effector function of myelin-specific Th1 cells, we differentiated naive T cells specific for MBP Ac1-11 in vitro with IL-12 or IL-12 + anti-IL-23 antibody, and transferred these cells into naive recipient mice. Although both populations made equivalent amounts of IFN-gamma, the incidence of EAE in the mice that received Th1 cells differentiated in the presence of anti-IL-23 was significantly reduced compared to the mice that received Th1 cells differentiated with IL-12 only.
This data suggest that IL-23 contributes to the encephalitogenic capacity of Th1 cells in immune-mediated demyelinating disease.
57 -Both blood-derived and CNS-derived APC contribute to antigen presentation during the effector phase of EAE
During experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, MHCII expression is upregulated on a variety of different types of antigen presenting cells (APC) in the CNS. Defining the respective roles of antigen presentation by these APC during EAE remains a major challenge. To address this question we have generated mice exhibiting altered patterns of MHCII expression by exploiting the fact that the class II transactivator (CIITA) gene, which encodes the master regulatory factor of MHCII genes, is controlled by three cell-type specific promoters called pI, pIII and pIV. We recently generated a strain of mice lacking pI driven CIITA expression.
The analysis of MHCII expression in pI−/− mice demonstrated that pI is essential for driving CIITA and MHCII expression in interferongamma activated macrophages and microglia. It is also required to a variable degree in various dendritic cell (DC) subsets. In contrast, MHCII expression is affected only marginally in thymic epithelial cells and B cells. pI is thus critical for driving CIITA and MHCII expression in the APC (microglia, macrophages and DC) that are most likely to be implicated in the effector phase of EAE. We therefore studied the susceptibility of pI−/− mice to EAE induced by active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, as well as by the adoptive transfer of MOGreactive encephalitogenic CD4+ T cells. In both models, pI−/− mice were almost completely protected. In the active immunization model, MOG-specific T cell priming was essentially normal despite the fact that EAE did not ensue. Taken together these results demonstrate that the block in EAE development in pI−/− mice lies at the level of antigen presentation during the effector phase in the CNS. To determine whether the absence of MHCII expression by CNS resident microglia or blood-derived APC is responsible for the resistance of pI−/− mice to EAE, we generated reciprocal bone marrow-chimeras between wild type and pI−/− animals. Interestingly, both types of chimeric mice (pI−/− into wt and wt into pI−/−) developed EAE, whereas pI−/− into pI−/− control chimeras remained strongly protected.
These results show that MHCII-restricted antigen presentation by both blood-derived and CNS-derived APC is involved during the effector phase of EAE, and that these two APC compartments can compensate for each other's absence. Experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis, is mediated by autoreactive CD4+ T cells targeted against CNS myelin. Phosphoinositide 3-kinases (PI3Ks) are intracellular signaling molecules that have been implicated in susceptibility to autoimmune disease. Existing evidence suggests that PI3K-gamma is important for migration of antigen presenting cells (APCs).
We found that PI3K-gamma−/− (ko) mice, when immunized with myelin proteins, show a profound suppression of EAE clinical signs and a marked reduction in demyelination and inflammatory cytokine production relative to wildtype (wt) controls. We hypothesize that the primary mechanism of protection observed in ko mice is due to impaired myeloid cell migration to sites of inflammation, and not an inherent defect in the ability of APCs to activate T cells. Co-culture of ko APCs with competent T cells induced T cell proliferation, suggesting that PI3K-gamma is not critical for processing and presenting antigen. We developed ko and wt chimeras to test this hypothesis and found that wt animals reconstituted with ko bone marrow had less severe EAE than wt chimeric controls. This suggests that the protective phenotype observed in ko mice is mediated by immune cells located in the periphery.
Taken together, these results demonstrate a key role for PI3Kgamma in the activation of peripheral immune compartments, and suggest that PI3K-gamma could be an important target for the treatment of autoimmune diseases. (Supported by NIH grants GM 068412, AI 064320 and National MS Society grant RG3272).
59 -The dominancy of encephalitogenic peptide correlates to its ability to induce potent regulatory T cells Lin Youwei⁎ 1 , Miyake Sachiko 1 , Yamamura Takashi 1 1 National Institute of Neuroscience, NCNP, Tokyo, Japan
Like human counterpart MS, animal MS models can show a great diversity in clinical courses. The most famous model is EAE and its susceptibility is believed to be determined by genetic factors. SL/J mice are well known to develop relapsing-remitting EAE (RR-EAE) by immunization of PLP139-151. However, we found that priming with PLP136-150, overlapping peptide, induces monophasic EAE (M-EAE) resistant to relapse and also to reinduction of EAE with any peptides, unlike RR-EAE. We have already discovered that such resistance originated from a continuously high and selective induction of Foxp3+CD4+CD25+ regulatory T cells (Foxp3+Treg) co-expressing CD103 and CD69 in the LN cells during remission phase of M-EAE. We furthermore clarified the underlying mechanism.
First, we compared T cell response or EAE primed with several peptides concerning with PLP136-150 and PLP139-151. Contrary to conventional understanding, truncation of N-terminal residues in PLP136-150 or elongation of C-terminal residue in PLP139-151 exacerbated relapse and re-induction of EAE. As such, dangerous self-peptide can differ from a less dangerous one only at flanking residues through prohibiting induction of potent Treg. We next checked the dominancy of PLP peptides in SJL/J mice by immunization with spinal cord homogenate. Surprisingly, PLP136-150 was more dominant than PLP139-151 among immuno-dominant epitope in SJL/J mice, and PLP136-150-primed LN cells could react effectively than PLP139-151-primed ones. By the experiments of priming with low-dose PLP peptides, we disclosed that the more dominant the encephalitogenic peptide is, the more resistant to reinduction EAE the peptide induced showed, indicating that TCRpeptide affinity determined the ability to induce potent Treg. Finally, we immunized the mice with peptide emulsified in IFA (not CFA) to check whether EAE induction is necessary or not and found that such phenomenon depends on peptide itself independent of EAE induction.
These findings suggest that the variety of clinical prognosis in MS might depend on the different kinetics and potency of Treg induced by the different 'regulatogenicity' in the individuals, controlled by the dominancy of self-peptide. This would confirm the idea 'immunological homunculus'. Through exploring this, a new therapy into full recovery and resistance to relapse for a long period would be available. Pregnancy affects the clinical course of autoimmune disease such as multiple sclerosis (MS), rheumatoid arthritis and lupus. In this study, we determined the immunoregulatory role of pregnancy on the effector phase of murine experimental autoimmune encephalomyelitis (EAE), a model for studying MS.
We used an adoptive transfer system with myelin basic protein (MBP) peptide NAc1-11 activated MBP T cell receptor (TCR) transgenic (Tg) donor cells. Donor cells are Th1 (Tg cells+Ag+/-IL-12) and Th17 (Tg cells+Ag+IL-6+TGFbeta+IL-23) cells. Transfer of Ag-specific activated Th1 cells into non-pregnant or virgin recipients resulted in EAE. However, transfer of Ag-specific activated Th17 cells to non-pregnant recipients resulted in severe EAE with 100% mortality. Our results show 1) transfer of activated Ag-specific Th1 cells to recipients during pregnancy resulted in EAE with a delayed onset and reduced clinical severity; 2) transfer of activated Ag-specific Th17 cells to recipients during pregnancy resulted in EAE with reduced cumulative scores but no reduction in mortality. We did observe the termination of pregnancy in some recipients of Th1 or Th17 cells. We investigated the mechanisms of pregnancy suppression of adoptively transferred EAE induced by Th1 cells. We explored three alternatives: 1) deletion of Ag-specific pathogenic T cells, 2) anergy of Ag-specific pathogenic T cells and 3) deviation from Ag-specific pathogenic T cells to Th2 and regulatory T cells. Our results show that pregnancy suppresses Agspecific proliferation and Th1 cytokine production. However, the number of donor Tg CD4+ cells increased in the pregnant environment compared with non-pregnant control mice.
These results suggest that pregnancy provides an immunoregulatory environment including anergy and deviation that suppresses the activity of encephalitogenic T cells. Although several studies clearly demonstrate the ability of Foxp3+ T cells to prevent EAE in mice, not many address their phenotypic characteristics and their mechanism of action.
We have performed a kinetic and cytokine analysis of the cellular composition of secondary lymphoid organs and of the central nervous system (CNS) at different time points after relapsing-remitting EAE induction in SJL by immunization with PLP139-151. We describe the kinetic and possible migration features of different subpopulation of CD4+Foxp3+ T cells during the course of the disease. We also, surprisingly, found that, approximately 10-12% of Foxp3+ T cells were positive for IL-2, IL-17, or IFNγ by intracellular cytokine staining. Cytokine positive CD4+Foxp3+ T cells were enriched in the CD25+, CD25-, CD103+ subpopulations.
Our results, showing a small but consistent percentage of Tcells double positive for Foxp3 and effector cytokines, question the concept of Foxp3 as a marker tout court associated to suppressive functions also in mice.
62 -Liver X receptor agonist T0901317 ameliorates experimental autoimmune encephalomyelitis through suppressing IL-17 production Xu Jihong⁎ 1 , Drew Paul 1 1 University of Arkansas for Medical Sciences, Little rock, United States Liver X receptors (LXRs) are part of the nuclear receptor family and are believed to regulate cholesterol and lipid homeostasis. We have recently shown that liver X receptor agonist T0901317 has antiinflammatory effects on activated microglia and astrocytes, and it also has been demonstrated that T0901317 decreased the clinic severity of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). MS is a T-cell initiated autoimmune disease, and recent data have established that IL-17-producing T cells, driven by IL-23 and referred to as Th17 cells, play a pivotal role in the pathogenesis of EAE. The aim of this work was to determine the effects of T0901317 on IL-17 production and IL-23R expression.
Our studies demonstrated that LXR agonist T0901317 inhibited the production of IL-17 and IFN-gamma by activated T-cells from both MOG primed C57BL/6 mice and Va2.3/Vβ8.2 TCR-transgenic mice. T0901317 also inhibited IL-23R expression in T-cells. In addition, we assessed the effects of T0901317 on mRNA and protein expression of IL-17, IL-17R and IL-23R from spinal cord of EAE mice, and found that T0901317 significantly suppressed these molecules.
These data suggest that LXR agonists may mediate autoimmune diseases by suppressing IL-17 production. Proliferation studies showed that naïve lymphocytes responded better in the presence of Non-PDC compared to PDC. Increasing the ratio PDC/T cells did not alter the T cell proliferation in contrast to non-PDCs.
Since IL-17 is known to amplify effector immune responses the interplay of IL-17 with PDC is of great interest. Yet, the differentiation/ maturation of PDC from BM cells from IL-17ko and wt mice was comparable. Furthermore, the proliferation responses of naïve lymphocytes using PDC from IL-17ko versus wt mice showed no difference. Of specific note, adding anti-ICOS to the culture revealed inhibition only with wt-PDC not IL-17ko-PDC. When maturation of PDC was studied in mice lacking CD28 this resulted in less PDCA-1+ PDC among the BM cell population compared with wt animals. Nevertheless, no differences in stimulating autoantigen (MOG)specific lymphocytes using PDC from wt or CD28ko animals were observed. Interestingly, when using PDC from CD28ko mice MOGspecific secretion of IL-10 (anti-inflammatory) was decreased whereas the secretion of TNFa (pro-inflammatory) was increased.
Our data help to better understand the maturation conditions of murine PDC, and their role in immunoregulation.
Supported by BMBF 01 GZ 0708. This recombinant protein, produced in house in CHO cells, showed over a 90% homology to human OPN-A. Surprisingly, therapeutic daily administration of the OPN-like protein at 0.3 mg/ kg s.c. resulted in a significant decrease of the clinical score as well as in a reduction of duration of the paralysis during the acute phase. Histological analyses performed either after the peak of the primary attack or at the end of the treatment showed a reduction of leukocyte infiltration and number of macrophages in the CNS. In addition, detection of endogenous OPN plasma levels in EAE rats showed an increased quantity compared to that observed in healthy rats mimicking what is already known in MS. Administration of the OPN-like protein provided stable circulating levels during the course of the disease reaching around 100 ng/ml. This level did not induce changes in the endogenous OPN levels. However, it corresponds to over a 10-fold lower level than the one used in the previously published study where an exacerbating activity of rOPN was observed in mouse EAE.
These findings suggest that OPN-like protein under certain treatment regimen may play an anti-inflammatory activity by modulating the infiltration of leukocytes within the CNS thus suggesting a possible action through its binding to CD44 and/or alpha-v-beta integrin receptors. To determine the role of TGF-B signaling cells in EAE pathogenesis, we used a transgenic TGF-B signaling reporter and ablative mouse model (SBE-lucRT). In the SBE-lucRT mouse, upon ligation of TGF-B1 with its receptors, Smad2/3 are phosphorylated, dimerized and then translocated into the nucleus subsequently activating the Smad binding element (SBE) promoter. Activation of the SBE promoter then drives the simultaneous expression of luciferase (luc), RFP (R) and herpes simplex virus-thymidine kinase (T).
We show that upon initial EAE progression, microglia and astrocytes activation and TGF-B1 production precedes the onset of clinical disease. While, astrocytes and microglia are the main producers of TGF-B1, neurons and infiltrated T cells were identified as the primary TGF-B signaling cells. Blockade of TGF-B signaling by the systemic administration of a pharmacological drug reduced EAE disease, the accumulation of T cells and expression of IL-6 in the CNS. Concurrently, treatment of EAE in SBE-lucRT mice with Ganciclovir (GCV) led to the ablation of TGF-B signaling and dividing cells, as well as the alleviation of EAE disease. Upon further cellular analysis of systemic GCV treatment, there was little effect on the peripheral immune cell populations while the composition of CNS-infiltrating CD4 T cells were altered. Together, these results suggest a pathogenic role of TGF-B signaling cells, which broadens the view of potential pathogenic cells during EAE progression.
Zuiderwijk-Sick Ella A. 1
Activated microglia are prominent in Multiple Sclerosis lesions, but it is still unclear whether their role is beneficial or detrimental. In contrast to other organs, where terminally differentiated populations of resident antigen presenting cells (APC) outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated, immature state. Whereas the effects of differentiation on the activation process of monocyte-and bone marrow-derived APC have been extensively studied, much less is known about the effects on microglia activation. Recently we have demonstrated that differentiation can also affect microglia activation. Differentiation of primary microglia from adult rhesus monkeys on M-CSF or GM-CSF did not alter their APC capacities, but did change their responses to Toll-like receptor (TLR)8-mediated activation.
New data from our lab show that exposure of microglia to the prototypic DC differentiation regime of GM-CSF + IL-4 indeed induces a cell surface molecule expression profile reminiscent of a DC-like phenotype. However, by marked contrast with conventional DC generated from the same donors, these cells no longer produce proinflammatory cytokines in response to TLR-mediated activation, irrespective of the TLR ligand used. We will here present data on the mechanism behind this block in TLR-induced pro-inflammatory cytokine production.
The apparent paradox between cellular phenotype and the absence of a pro-inflammatory response to activating stimuli was unique to microglia, and is likely to impact on the strength and the type of immune response generated by microglia-T cell encounters. We will therefore also present data on the functional consequences of interactions between GM-CSF + IL-4-differentiated microglia and T cells.
The anti-inflammatory or tolerogenic potential of these microglia might lead to new therapeutic avenues to halt ongoing immune responses and possibly even re-educate infiltrating T cells. The aim of the present study was to identify basic mechanisms how infections may induce a neuron-specific autoimmune response.
We generated mice expressing OVA as neuronal autoantigen under control of the neuron-specific enolase promoter (NSE-OVA mice). Intracerebral, but not systemic infection with attenuated Listeria monocytogenes secreting OVA (ΔactA LMova) induced an atacticparetic neurological syndrome in NSE-OVA mice after bacterial clearance from the brain, whereas WT mice remained healthy. Immunization with ΔactA LMova prior to intracerebral infection strongly increased the number of intracerebral OVA-specific CD8 T cells aggravating neurological disease. T cell depletion and adoptive transfer experiments identified CD8 T cells as decisive mediators of the autoimmune disease. Importantly, NSE-OVA mice having received OVA-specific TCR transgenic CD8 T cells developed an accelerated, more severe and extended neurological disease. Adoptively transferred pathogenic CD8 T cells specifically homed to OVA-expressing MHC class I+ neurons and, corresponding to the clinical symptoms, ∼30% of neurons in the anterior horn of the spinal cord became apoptotic.
From these studies it is concluded that molecular mimicry between a pathogen and neurons can induce a CD8 T cell-mediated neurological disease with its severity being influenced by the frequency of specific CD8 T cells and its induction, but not its symptomatic phase requiring the intracerebral presence of the pathogen. expression in lymphocytes Torres Karen⁎ 1 , Souza Bruno 1 , Sampaio Andre 1 , Barros Alexandre 1 , Gollob Kenneth 1 , Dutra Walderez 1 , Romano-Silva Marco Aurélio 1
NCS-1 is a protein involved in the Ca2+ signaling. Its expression was found in neural and neuroendocrine cells and it is involved in several functions as exocytosis and neurotransmitter release enhancement and dopamine D2 receptor desensitization inhibition. Currently, it has been demonstrated that NCS-1 is important in mast cell functions as stimulates Fc epsilon RI-triggered exocytosis and the release of arachidonic acid metabolites.
Then we sought to investigate whether NCS-1 could be expressed in lymphocytes and monocytes and if so evaluate which intracellular signaling pathways were compromised with NCS-1 expression.
With PKB and MEK (PD0956 and UO126) inhibitors we have investigated the participation of MEK/ERK, PI3K/PKB intracellular signaling pathways in the NCS-1 expression from CD4+ T lymphocytes stimulated in vitro, using flow cytometry.
Results: Data showed a downregulation of NCS-1 expression, dosedependent, in lymphocytes, in the presence of both MEK and PKB inhibitors, when cells were stimulated. The activation of cells was accompanied by CD69 increased expression.
NCS-1 expression in CD4+ T lymphocytes depends on the PI3K/PKB and MEK/ERK commitment and these cells could be a good model for NCS-1 studies. Financial support: CNPq, FAPEMIG. To assess the relevance of the neuroinflammatory response, behavioral, neurochemical, neuropathological, cerebrospinal fluid (CSF) and peripheral blood analyses were performed in male adult C57Bl/6 mice exposed to systemic administration of MPTP. The data collected were correlated with longitudinal [at both early (3-24 h) and late (1, 2, 3, 4, 5 and 6 weeks) time points upon MPTP administration] wide gene expression analysis of mRNA species involved in inflammation, immunity, stemness, self-renewal, neural differentiation, migration, DA metabolism and signaling, using TaqMan® Low-Density-based Array (TLDA) in ventral mid brain (VM) and striata (Str). Here we describe that MPTP-induced DA neurodegeneration and self-repair is likely to be associated to a localized biphasic astroglial response, a spatiotemporal recovery of tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunoreactivity (IR) paralleling the spatiotemporal return of synaptosmial DA uptake capacity. Temporal reconstitution of damaged DA cell bodies and axon terminals appeared also paralleled by a switch of nigrostriatal astroglial phenotype. Among a total of 93 mRNA species analysed, we have identified a major time-and region-dependent up-regulation of inflammatory chemokines, such as CCL3/MIP-1a, CXCL10/IP-10, CXCL11-I-TAC and key transcripts involved in DA neurodevelopment, especially the morphogen wingless-type MMTV integration site (Wnt) 1. Activated astrocytes of the VM were identified as potential elements of the Wnt signalling pathway sustaining nigrostriatal self-repair. This work suggests a surprising self-repair ability of the nigrostriatal DA system following MPTP neurotoxicity. It further implicates the MPTP-reactive "in situ" inflammation as key event in the molecular cascade linking tissue injury to repair. A deeper understanding (and manipulation) of the neurodevelopmental cues undergoing recapitulation upon CNS tissue injury is likely to have important implications for the treatment neurodenegerative diseases including PD. Increasing evidence suggests that spinal microglia could be an important player in the pathogenesis of neuropathic pain. We reported previously that peripheral nerve injury induced an early and transient spinal microglial cell proliferation which is in close correlation with spinal microglial activation and the development of neuropathic pain. To further test our hypothesis on the role of new microglia to the genesis of neuropathic pain symptoms, in this study, we investigated the effects of TGF-α1, a potent antiproliferation and anti-inflammatory cytokine on spinal microglia reaction following peripheral nerve injury and the impact on nerve injury induced-hypersensitivity. Rats having a partial ligation on the left sciatic nerve (Seltzer model) had a sharp decrease in paw withdrawal thresholds bilaterally in response to mechanical Von Frey Hair stimulation. Withdrawal latency to heat insults (Hargreaves) was significantly lower unilaterally on the injured side. Correspondingly, following nerve lesion, microgliosis was induced on the spinal cord. A significant increase on the number of BrdU+ cells and the intensity of iba-1 (marker for microglia) immunoreactivity (ir) was observed on the ipsilateral side dorsal horn, with a slight increase on the contralateral side as well. Intrathecal infusion of rat recombinant TGF-β (2 μg and 5 μg) for 14 days, starting on the day of nerve ligation, inhibited spinal microgliosis on the ipsilateral side dorsal horn by a reduction of BrdU+ cells from 22.71 in saline treated rats to 7.6 (2 μg), 10.5(5 μg) in TGF-β1 treated rats, and a reduction on the iba-1 ir intensity (83.21/saline vs. 61.23/2 μg TGF-β and 49.81/ 5 μg TGF-β ). Intrathecal treatment of TGF-β1 reduced partially nerve injury induced mechanical allodynia at both injured and non-injured sides, and completely abolished thermal hyperalgesia at the ipsilateral side. Together, our results suggest that peripheral nerve injury triggered a spinal microglial cell proliferation and these newly generated spinal microglia are involved in the genesis of hypersensitive pain behaviour. Inhibition of spinal glial response with a central TGF-β1 treatment, which is effective in limiting spinal microgliosis, could relieve partially mechanical allodynia, however completely prevented the development of thermal hyperalgesia. which deductively are cytotoxic T lymphocytes, show similarities concerning V-beta/J-beta usage, length of the complementarity determining region 3 (CDR3), and some biochemical characteristics of the CDR3 amino acids suggesting common driving antigen(s) without shared clones. Additionally, these CD8+ T cells persist in the periphery hinting towards either an autoantigen or a persisting viral infection as the antigenic cause of the immune reaction.
Taken together, our TCR analysis provides strong evidence for an antigen-driven MHC class-I restricted, CD8+ T cell mediated attack against CNS structures dominating the pathogenesis in RE. Neural progenitor cells (NPC) possess the ability to regenerate new neurons and could be used as a potential therapy for neurodegenerative disorders. In diseases such as multiple sclerosis (MS) and HIVassociated dementia (HAD) this approach may be impacted by activated T cells. In this study, we investigated the effect of activated T cells and granzyme B on NPC proliferation and differentiation.
Primary human fetal NPC were treated with supernatants from activated T cells or GB (1-4 nM) in either maintaining medium or differentiating medium. Cell proliferation was studied with BrdU incorporation assay after 24 h and neuronal differentiation by using beta-III-tubulin immunostaining after 4-7 days. The NPC lysates were used detecting the intracellular cyclic AMP (cAMP) by ELISA and the gene expression of Kv1.3, a voltage dependent potassium channel, by real-time PCR. The NPC cultures were also pretreated with pertussis toxin (PTX, 100 ng/ml) or Kv1.3 blocker margatoxin (MgTX, 10 nM) or Kv1.3 siRNA to study mechanisms. Supernatants from activated T cells decreased NPC proliferation and differentiation significantly which were attenuated by immunodepleting GB (P b 0.05). Recombinant GB treatment also decreased both NPC proliferation and differentiation (P b 0.05). GB also decreased cAMP level in the NPC after 20 min of treatment in a dose-dependent manner (P b 0.05) and increased Kv1.3 gene expression after 3 h (P b 0.05). Pretreatment with PTX reversed the GB-induced effect on cAMP and Kv1.3 expression and increased NPC proliferation (P b 0.05). Kv1.3 specific blocker MgTX or siRNA pretreatment also protected NPC neurogenesis against GB treatment as determined by the beta-III-tubulin positive neuron counting (P b 0.05). Kv1.3 siRNA also protected primary human fetal neurons from GB-caused neurite damage as determined by measuring the neurite length (P b 0.05).
GB released from activated T cells inhibits NPC proliferation and differentiation. The underlying mechanisms involve a PTX-sensitive Gi-protein-coupled receptor and the subsequent decrease in intracellular level of cAMP and the activation of Kv1.3. Blockade of Kv1.3 provides protection to NPC and neurons and could be used as a potential therapy for T cell-related neurodegenerative diseases such as MS and HAD.
Minocycline has been reported to have neuroprotective activity in various animal models of neurological diseases, where a major mechanism for its effectiveness is attributed to the inhibition of microglia inflammation. Much less has been described on the possibility that minocycline could be acting directly on neurons independent of microglia activity. In this study, we tested the protective role of minocycline against H 2 O 2 toxicity in enriched human fetal neurons (HFNs) in the absence of microglia and identified new targets of minocycline using microarray analyses. Furthermore, we demonstrate that an induced enzyme in response to stress, heme oxygenase-1 (HO-1), may be important in the protection of minocycline both in vitro and in vivo.
HFNs treated with minocycline were exposed to H 2 O 2 . Relative intensity of neuronal markers was then measured with In-Cell western analysis. Gene expression profiles of the treated HFNs were generated using microarrays. Real-time PCR (RT-PCR) was conducted to validate the microarray results.
Using HFNs which contain 85 to 90% neurons and 10-15% astrocytes, but devoid of microglia, we found that H 2 O 2 , kills neurons which was documented by obvious cell loss, and by decreased intensity of neuronal markers and increased active caspase-3 in In-Cell western analyses. Minocycline significantly attenuated these effects of H 2 O 2 . As neurons could therefore be protected by minocycline in the absence of microglia, we subjected HFNs to DNA microarrays using a 14,000 gene chip. Across 14 experiments involving 7 different HFN preparations, we found that many genes were up-regulated by H 2 O 2 and that a minority of these were reduced in expression levels when neurons were protected by minocycline. Of the latter targets, real time PCR and In-Cell western investigations confirmed that HO-1 was up-regulated by H 2 O 2 and reduced by minocycline. Immunohistochemistry for HO-1 showed that neurons and astrocytes were both reactive for this enzyme following H 2 O 2 exposure. The necessity of HO-1 in minocycline protection against H 2 O 2 is currently under investigation using both in vitro and in vivo experiments.
We conclude that minocycline confers protective effect of HFNs against H 2 O 2 without the need of microglia activity and that this may involve a hitherto unknown mechanism of minocycline, that of modulating HO-1 levels in neurons and/or astrocytes. The neuroprotective action of estrogen (Es) against 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown in various reports in both female and male mice, however the exact mechanisms of that phenomenon remain obscured. One potential mechanism by which Es may exert this action is by modulating the inflammatory response in nigrostriatal system, which occurs as an early consequence of injury this system.
We studied the chronic effects of 17b-estradiol -E2 (0.25 mg per pellet, 21-days release) administered prior (7 days -Experiment 1) to or after (3 days -Experiment 2) MPTP treatment in C57BL male mice (12 months old) on dopaminergic neurons degeneration and inflammatory reaction in nigrostriatal pathway. C57BL mice received 40 mg/kg of MPTP intraperitoneally. We estimated striatal: tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) content (Western blotting method), cytokines (TNFa, TGFb1, IFNg) and trophic factor (GDNF) gene expression (RT-PCR method), and CD4+ and CD8+ cells influx to nigrostriatal pathway (immunohistochemistry) at 1,7 and 21 (Exp.1) and 7 and 21 (Exp.2) days post intoxication. MPTP treatment reduced striatal TH within 1-21 days following intoxication. We showed that E2 exerted a neuroprotective effect upon nigrostriatal system when administered 7 days prior intoxication. We also observed that E2 protected striatum from MPTP insult when administered on the 3rd day after intoxication. The implantation of E2 pellets after intoxication attenuated the MPTP-induced loss of striatal TH at 7-and 21-day time-points. E2 also decreased the striatal GFAP content, when administered prior and after intoxication. MPTP caused a rapid increase of striatal TGFb1, TNFa and IFNg gene expression. Pre-treatment with E2 decreased the early expression of TGFb1 and IFNg mRNA but failed to suppress the MPTP-induced increase of striatal TNFa mRNA level. E2 pre-treatment also induced an increase of the striatal GDNF gene expression and CD4+ cells influx to the injured brain areas. In contrast, E2 decreased the CD8+ cells infiltration when administered prior and after MPTP intoxication.
Our studies suggest that the neuroprotective effects of E2 indicated in MPTP model might mediate through a modulation of both molecular and cellular factors in neuroinflammatory reaction.
10 -Characterization of lysolecithin-induced demyelination, with a focus on the early inflammatory responses and their possible roles Döring Axinia⁎ 1 , Yong V. Wee 1 1 Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
Demyelinating diseases of the central nervous system (CNS) such as multiple sclerosis (MS) feature oligodendrocyte (OL) and myelin loss but it is the concordant loss of axons and neurons that is thought to drive the progression of disability in patients. In several studies of MS brain specimens, it has been shown that there is good correspondence of regions of axonal injury with areas containing leukocyte infiltration or microglia activation. In tissue culture studies, products of microglia/macrophage or lymphocytes produce axonal dysfunction and kill neurons. More recently there is an appreciation that some aspects of neuroinflammation can be beneficial, as highlighted by experiments where the significant depletion of microglia/ macrophages in demyelinating models retarded remyelination. The interplay between inflammatory cell subsets, axonal injury and demyelination thus require a closer examination, particularly with the prospect of providing neuroprotection, preventing demyelination, or enhancing repair.
We have used a toxin-mediated demyelination of the CNS to characterize the early inflammatory and neural responses. Following the deposition of 1.5% lysolecithin solution into the dorsal column of T3/T4 region of the spinal cord, mice were killed and analysed from 1-7 days after injection using a variety of markers. We find axonal injury (accumulation of β-APP and non-phosphorylated neurofilament) and demyelination (myelin basic protein) to be already evident by 1 day of lysolecithin administration. This time frame corresponded with upregulation of Iba1 immunoreactivity, a marker of microglia/ macrophage activation. We were not able to detect an increase of OLs at the lesion side at these time points. Flow cytometry shows that the macrophage (CD11bCD45high) infiltration is most marked at day 3 of injury. Current experiments are directed at increasing or decreasing the extent and period of microglia/macrophage reactivity and to address the subsequent impact on axonal injury and demyelination.
These experiments seek to clarify the beneficial or detrimental roles of immunity, particularly that of microglia/macrophage activation, on the exacerbation of injury, or conversely, the protection and repair of axons and myelin. Preservation of axons in Wallerian degeneration slow (Wld^S) mice has been shown to be beneficial in experimental autoimmune encephalomyelitis (EAE), an autoimmune model for MS. In Wld^S mice, axonal degeneration is delayed due to an increased nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme Nmnat activity. SIRT1 is the downstream effector of the increased Nmnat activity. Resveratrol is a natural polyphenol compound of red wine and has received considerable attention as a chemical responsible for the health benefits of red wine and is currently being tested in clinical trials as a potential therapy for cancer. Resveratrol can enhance SIRT1 activity, potentially resulting in axonal preservation, and exhibit anti-inflammatory and anti-viral activities. We tested whether resveratrol could be therapeutic, possibly by limiting axonal damage in a viral model for MS, Theiler's murine encephalomyelitis virus (TMEV) infection.
SJL/J mice were infected intracerebrally with the DA strain of TMEV. Groups of infected mice were fed a diet containing 0.04% resveratrol (20 mg/kg/day) during the acute stage of infection (days 0 to 14) or during the chronic stage (days 21 to 35), or a control diet. Clinically, the mice treated with resveratrol during the acute stage showed more weight gain than control mice at 2 weeks post infection (P b 0.001, by ANOVA). At 5 weeks after infection, central nervous system (CNS) tissues and spleen mononuclear cells (MNCs) were harvested. Conversely, mice treated during the chronic stage tended to have higher clinical and pathological scores than control mice, although they did not reach statistical significance. There were no significant differences in numbers of cells expressing viral antigen in the CNS or MNC proliferative responses to virus among the experimental groups.
We speculate that significant weight gain during the acute stage in the early resveratrol treatment group could be due to a neuroprotective property of resveratrol. The potential axonal sparing activity by resveratrol could be of great benefit, since permanent clinical disability in MS results form degenerated axons that do not regenerate in the CNS.
12 -17b-estradiol administration protects against dopamine depletion, down-regulates astroglial activation and modulates cytokine production in the nigro-striatal system in female mice In neurodegenerative disorders an immune system involvement in the pathological process is postulated. The potential role for estrogen (Es) acting as a neuroprotectant of dopaminergic system may be supported by the epidemiological reports showing a gender differences in Parkinson's disease (PD). The aim of the present study was to examine the chronic effects of Es (17b-estradiol, 0.25 mg per pellet, 21-days release) on dopaminergic system and cytokines production in female C57Bl mice (12 months old) in a murine model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).
Estrogen was administered 7 days prior to (exp.1) or 3 days after MPTP intoxication (exp.2). MPTP (40 mg/kg) was injected to the animals in four intraperitoneal (i.p.) injections at 1-h intervals. Animals were sacrificed by spinal cords dislocation at the different time intervals: 1, 7 and 21 (exp.1) or 7 and 21 days (exp.2) following MPTP intoxication. Striatal dopamine (DA) was quantified by highperformance liquid chromatography (HPLC), glial fibrillary acidic protein (GFAP) expression was measured by Western Blot method, TNFa, IFNg and TGFb mRNA expression was examined by RT-PCR. MPTP treatment decreased DA concentration within 1-21 days, enhanced GFAP expression within 7-21 days, increased TNFa and TGFb mRNA expression at 1 day, and IFNg mRNA at 7 days following MPTP injection. Estradiol exerted a neuroprotective effect upon dopaminergic system (less pronounced MPTP-induced decrease of DA) when administered 7 days prior to MPTP, but not when Es was implanted 3 days after MPTP. Estrogen inhibited GFAP expression at 7 day time point when implanted prior to intoxication and within 7-21 days time points when administered post MPTP injection. Estradiol decreased TNFa and IFNg mRNA expression at 7 day and TGFb mRNA expression at 1 day time point (exp.1), as well as TGFb mRNA expression at 7 day after intoxication (exp.2).
Our findings suggest that the neuroprotective effects of Es in MPTP mouse model of PD might be mediated by modulation of an inflammatory reaction. 17b-estradiol may inhibit the activity of astroglial cells and decrease production of inflammatory mediators, including cytokines. The contribution of macrophages to spontaneous recovery from spinal cord injury (SCI) is currently under debate. Macrophages have long been perceived as inhibitory to CNS recovery, but under different conditions these cells can acquire distinct activities whose effect on the tissue varies from destructive to beneficial. We wanted to define the specific contribution of infiltrating monocytes to spinal cord recovery.
To that aim we used a unique model that allowed us to identify and subsequently ablate the active blood-derived myeloid cells but sparing the resident microglia. This ablation of monocyte-derived cells found in close proximity to the injured site, resulted in impaired recovery of motor function. Restoration of the monocyte pool restored the healing process. Immunization, which was previously shown to augment recovery from severe spinal cord injury, resulted in enhanced recruitment of these myeloid cells. Importantly, these cells exhibited characteristics of myeloid derived suppressor cells (MDSC) and restricted the local immune response via, at least in part, the anti-inflammatory cytokine interleukin 10 (IL-10).
We argue that the discrepancy regarding macrophages contribution to recovery is due, in part, to the heterogeneity of these cells and the need for their timely-regulated response following the insult. This study identifies an essential novel role, in recovery from spinal cord injury, for infiltrating blood-borne monocytes, which can not be provided by resident microglia.
14 -Oral fingolimod (FTY720) suppresses established EAE and allows remyelination Fingolimod (FTY720) is a novel oral immunomodulatory agent that significantly reduced the rate of inflammatory disease activity compared to placebo in patients with relapsing MS in a Phase II clinical trial. Fingolimod is converted in vivo to its active phosphate by sphingosine kinase and then binds to S1P1,3,4 and 5 receptors (S1PRs) with high affinity. Binding of fingolimod to S1P1 on lymphocytes causes their sequestration within peripheral lymph nodes, thus reducing lymphocytic infiltration into the CNS, which is considered an important part of the mechanism of action of fingolimod. In addition, fingolimod readily crosses the blood brain barrier and CNS cells also express S1P receptors, suggesting further direct effects within the CNS. In the current experiments we determined if fingolimod can protect from experimental autoimmune encephalomyelitis (EAE) after the onset of demyelination.
DA rats were immunized with MOG plus CFA. Fingolimod was administered to rats daily p.o. (0.3 mg/kg) for 9 days beginning at 29 days post immunization or when the 3rd phase of EAE started. The effect of fingolimod on clinical scores, demyelination, inflammation and axonal degeneration was determined by histology and MRI (MTC). Using MTC we established that demyelination was present at the initiation of treatment. Oral administration of fingolimod (2 experiments) to rats with well-established EAE significantly reduced clinical EAE severity compared to vehicle. MTC analysis revealed that white matter loss continued to increase in the vehicle treated animals, but was stabilized by treatment with fingolimod. In some individual lesions we observed a normalization of MTC signals suggestive of remyelination. Histopathological analysis showed a reduction in macrophage and T cell infiltrates in the spinal cord of fingolimodtreated rats, and enhanced myelin and axonal content.
These studies demonstrate that fingolimod can reduce EAE severity even if administered in late stage EAE and after the onset of demyelination. This supports the possibility that fingolimod could also have protective effects in the progressive stages of MS.
Slaets Helena Stefanie Elisabeth⁎ 1 , Hendriks Jerome 1 , Carmans Sofie 1 , Stinissen Pieter 1 , Hellings Niels 1
Leukemia inhibitory factor (LIF) is produced by infiltrating immune cells and glial cells during auto-immune and injury responses in the central nervous system (CNS). During these responses, LIF promotes survival of glial cells and neurons and has immunomodulatory potential, but data on the underlying events are lacking. The goal of this study was to study the mechanisms of LIF-mediated protection of mature oligodendrocytes (OLG) and to determine the effects of LIF on macrophage function.
LIF mediated effects were studied in primary rat oligodendrocyte and macrophage cultures. Cell viability was measured by AnnexinV/PI staining. Activation of signalling pathways was analyzed by western blotting and RNAse protection assays. Myelin phagocytosis was determined using DiI-labelled myelin and flow cytometry.
We show that LIF protects OLG selectively against the synergistic insult of the proinflammatory cytokines IFNgamma and TNFalpha, while it does not protect against oxidative stress nor against staurosporine induced apoptosis. We further demonstrate that LIF protection is independent of suppressors of cytokine signalling and Bcl-2 mRNA expression levels. Our results indicate that LIF induces a shift in the cellular machinery towards a pro-survival execution program, illustrated by an enhanced expression of isoforms of the anti-apoptotic molecule 14-3-3 and an activation of the Jak/STAT3 and the phosphatidylinositol 3 kinase/Akt pathways. We further demonstrate that LIF modulates macrophage function by inhibiting the production of oxygen radicals and TNFalpha, both mediators of CNS injury in neuroinflammatory diseases such as multiple sclerosis (MS). We show that LIF plays a role in myelin phagocytosis as it stimulates myelin uptake by macrophages.
These results demonstrate that LIF has both neuroprotective and anti-inflammatory properties and enhances myelin clearance, implicating it is an important factor in lesion development and limitation in immune mediated demyelinating diseases such as MS.
Harada Yohei⁎ 1 , Yawata Izumi 1 , Takeuchi Hideyuki 1 , Sonobe Yoshifumi 1 , Jin Shijie 1 , Doi Yukiko 1 , Liang Jianfeng 1 , Kawanokuchi Jun 1 , Mizuno Tetsuya 1 , Suzumura Akio 1 1 Nagoya University, Nagoya, Japan
We have shown previously that the most neurotoxic factor from activated microglia is glutamate that is produced by glutaminase utilizing extracellular glutamine as a substrate and released through gap junction. In this study, to elucidate whether or not a similar mechanism is operating in macrophages infiltrating into the central nervous system during inflammatory and demyelinating diseases, we examined the neurotoxicity induced by macrophages in comparison with microglia and experimental allergic encephalomyelitis (EAE), the model of multiple sclerosis, treated with glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) and gap junction inhibitor carbenoxolone (CBX). Microglia were isolated from primary mixed glial cell cultures prepared from neonatal C57BL/6J mice. Peritoneal macrophages elicited with 2 ml of 4% thioglycolate injected intraperitoneally into C57BL/6J mice were collected. Neuronal cultures were prepared from the mice at embryonic day 17. Microglia and macrophages were cultured with 1 micro g/ml LPS for 24 h in the graded concentrations of CBX or DON. Glutamate concentration of the conditioned media was determined with the Glutamate Assay Kit colorimetric method. Inflammatory cytokines and chemokines were detected by RT-PCR. Neurotoxicity in the conditioned media of microglia and macrophages treated with CBX or DON was determined by dye exclusion method with propidium iodide. EAE in C57BL/6J mice were induced by myelin oligodendrocyte glycoprotein peptide. DON and CBX were injected intraperitoneally into the mice every other day from day-2. Microglia and macrophages stimulated with LPS produced glutamate. LPS-stimulated macrophage conditioned media induced robust neurotoxicity, which was completely inhibited by the NMDA receptor antagonist MK801. Both the DON and CBX effectively suppressed glutamate production and subsequent neurotoxicity by activated microglia and macrophages. EAE severity was significantly reduced in 2.0 mg/kg CBX-treated mice, and EAE onset was delayed and EAE severity was significantly reduced in 1.6 mg/kg DON-treated mice compared with PBS-treated control.
This study demonstrated that a similar machinery is operating in macrophages as well, and DON and CBX that prevent microgliamediated neurotoxicity should be effective for preventing macrophage-mediated neurotoxicity. Thus, these drugs may be effective therapeutic reagents for inflammatory and demyelinating diseases. We reported that transgenic mice constitutively expressing the B7.2 (CD86) T cell costimulatory ligand on microglia spontaneously develop a T cell-mediated demyelinating disease. We have shown that CD8+ T cells are the primary effector T cell-subset that initiates the pathological process and TCR clonotype mapping demonstrated clonotypic CD8+ T cell expansions within the CNS. To further understand the pathological process that occurs in this animal model, we characterized the phenotypic and functional status of the CD8+ T cells that accumulate in the CNS.
The vast majority of the CNS CD8+ T cells displayed phenotypic features of effector-memory cells (CD44hi, CD62-Llo, CD127hi, Ly6Chi, CCR7-) in an activated state (CD43hi, CD69+, CD25+). However, unlike conventional effector cells, they expressed low levels of CD122. A similar memory CD8+ T cell subset (CD122lo, CD62-Llo, CD44hi, CD127hi) was present in increased number in peripheral lymphoid organs. CNS CD8+ T cells exhibited potent cytotoxicity directly ex vivo and were capable of co-producing high levels of IFN-g, TNF-a and IL-2 upon restimulation in vitro. The activating receptor NKG2D was also expressed by a high proportion of CNS CD8+ T cells. High levels of granzyme B were detected directly ex vivo and a significant number of CNS CD8+ T cells also showed surface mobilization of CD107a without restimulation, consistent with active engagement in cytolytic activity in vivo.
Our data suggest that the CD8+ T cells that are reactivated in the CNS by B7.2-expressing microglia may originate from the CD122low memory CD8+ T cell subset that has been reported to be maintained through interactions with commensal or self-antigens. The CNS CD8+ T cells are potent effector cells, well equipped to induce tissue injury through various pathways. Transplantation of multipotent neural stem/precursor cells (NPCs) protects the central nervous system (CNS) from inflammatory damage via a number of bystander mechanism(s) alternative to cell replacement, including immune modulation.
Here we show that subcutaneous injection of NPCs significantly protects mice from chronic-recurrent autoimmune CNS inflammation via a novel immune-regulatory mechanism occurring within secondary lymphoid organs. Injected NPCs accumulate and survive within draining lymph nodes, where they stably modify the perivascular (micro)environment. Within this context, NPCs restrain dendritic cell function via a bone morphogenetic protein (BMP)-4-dependent mechanism, andin turnprevent the expansion of antigen-specific encephalitogenic T cells. This effect is completely reverted by the BMP-4 antagonist Noggin.
Our results suggest a novel tolerogenic mechanism exerted by immune regulatory NPCs in vivo, which may be further exploited to modulate aberrant peripheral immune responses leading to chronic tissue-specific autoimmunity. Stroke is the leading cause of long-term disability in industrialized countries.
Here we have investigated the molecular and cellular mechanisms regulating functional recovery of mice subjected to transient (45′) focal cerebral ischemia [e.g., middle cerebral artery occlusion (MCAO)] and transplanted intravenously (i.v.) in the early post-acute phase (namely 72 h after MCAO) with syngenic neural stem/precursor cells (NPCs). Transplanted NPCs selectively accumulated in the periischemic area corresponding to the lateral striatum between 3 and 10 days post-transplantation (dpt) and survived up to 45 dpt. Histopathology revealed that about 0.3% of i.v.-injected NPCs accumulated into the brain, while lower numbers of NPCs were found in peripheral organs (e.g., lung, liver, spleen, and kidney). The great majority of transplanted NPCs accumulating in the brain displayed undifferentiated or only intermediate-differentiated features at 10 dpt, with no NPCs expressing markers of terminal neural differentiation. Starting from 3 dpt, i.v.-injected NPCs induced a gradualthough slow -amelioration of neurological deficits: the clinical improvement in NPC-transplanted MCAO mice compared to Sham-treated controls reached statistical significance from day 15 dpt on and persisted up to 60 dpt. Further neuronal survival, astroglial glial reaction, (neo)angiogenesis and inflammatory reaction were then analysed in the perischemic area at both protein and mRNA level. Neuropathological analysis showed that the i.v. delivery of NPCs is associated to an increase of the number of surviving endogenous neurons, to a significant reduction of the axonal loss as well as to a diminished gliotic scar tissue reaction. Gene expression analysis confirmed the down-regulation of a number of key genes associated with glial proliferation as well as up-regulation of genes controlling the neuronal survival.
These preliminary data provide evidence that the post-acute systemic injection of NPCs promotes significant clinico/pathological recovery in mice with experimental MCAO by bystander protection of endogenous hypoxia-injured neurons and inhibition of the astrocytic glial scar formation.
Bonnamain Virginie⁎ 1 , Michel Delphine 1 , Nerrière Daguin Véronique 1 , Thinard Reynald 1 , Dugast Anne-Sophie 1 , Brachet Philippe 1 , Anegon Ignacio 1 , Vanhove Bernard 1 , Neveu Isabelle 1 , Naveilhan Philippe 1
Porcine neural precursor cells (pNPCs) from fetal brain have been proposed as an alternative to the use of human fetal neuroblasts (NBs) in neurodegenerative disorders. Initially thought as a useful source in cell replacement therapy because of their ability to be expanded in vitro, pNPCs are of a great interest also because of particular immunomodulatory and/or immunogenic properties. Indeed, we recently observed that 40% of the rats transplanted with pNPCs exhibited a healthy graft 63 days post-grafting whereas pNBs grafts were all rejected at this time point. Interestingly, the surviving grafts did not exhibit any sign of inflammation. Immunostaining of activated macrophages/microglial cells with ED1 was negative. In addition, we did not observe any premise of T lymphocyte infiltration as indicated by the absence of R7.3+ cells.
To determine potential immunomodulatory properties of NPCs, rat T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies were mixed with increasing number of pNPCs. Thymidine assays clearly showed that NPCs inhibited T cell proliferation. This inhibitory effect was closely dependent of the cell ratio. We did not find major inhibitory effect at a ratio of 1 pNPC for 4 T cells, but a 50% decrease in the proliferating rate was observed at a ratio of 1 pNPC for 2 T cells. Interestingly, a 90% decrease in the number of proliferating T lymphocytes was observed at a ratio of 2 NPCs for 1 T cell. The same result was obtained with NPCs derived from fetal rat tissues. To determine whether contacts between T cells and NPCs were necessary or if soluble factors were sufficient, NPCs were plated in the upper part of a transwell whereas T cells were cultured in the lower part.
Thymidine assays were performed three days later. Inhibitory effect similar to the one observed when the cells were in direct contact was observed indicating that soluble factors released by NPCs are sufficient to inhibit the proliferation of activated T cells.
The next step was to search for soluble factors known to interfere with T cell proliferation. RT-PCR and immunocytochemistry analyses revealed the expression of several immunoregulatory molecules by virtually all NPCs, as indicated by their co-localisation with nestin, a marker of neural stem cells. The molecules critical for the immunoregulatory effects of NPCs are currently under investigation using specific inhibitors.
Recent characterizations of human mesenchymal stem cells (MSCs) and their role in hematopoiesis and immune modulation suggest that their potential for cell therapy extends beyond their traditional accessory function in HSC engraftment. MSCs, easy to obtain and able to differentiate into multiple lineages such as osteoblasts, adipose tissue, cartilage and stromal cells, contribute significantly to tissue restructuring and immune functioning, in addition to facilitating durable, long-lasting stem cell engraftment.
We focus our analysis on immune synapse formation and cytoskeleton reorganization.
To investigate cell-cell contact we analysed by confocal microscopy immune synapse formation in the presence of MSCs. Furthermore, we study the nature of MSC/DC cell-cell contact by electron microscopy. We found that MSCs impair active immune synapse formation as shown by confocal analysis and we evidenced at electron microscopy two types of intimate contact between MSCs and DCs: gap and adherent junctions. In the same experiments we show that MSC contacts induce a reorganization of DC cytoskeleton by the formation of actin podosomes, these structures are typical of an immature state, and therefore tolerogenic, of DCs.
These results suggest that MSCs exert a tolerogenic effect on DCs by mechanism not only mediated by soluble factors, that should be further investigated, but also mediated by cell-cell contact, contact that induces DC cytoskeleton reorganization with formation of actin podosomes. So far, several hypothesis have been made on the mechanisms underlying the tolerogenic effect due to coculturing MSCs and DCs, here we show that short term contacts may induce a tolerogenic phenotype by actin redistribution.
We recently reported that mesenchymal stem cells (MSCs) exert a profound inhibitory effect on T cell proliferation both in vivo and in vitro. Subsequently, MSCs were shown to exert similar effects also on B cells, dendritic cells, and NK cells. In addition, MSCs support the recruitment of local precursor cells and provide trophic factors supporting survival and repair of injured cells. These data suggested that MSCs could be used to ameliorate immunomediated diseases. Although it is accepted that MSCs play an important role in maintaining the hematopoietic stem cell in an undifferentiated state, the exact mechanism by which this happens is still unknown. To start addressing that question, we recently characterized the transcriptional profile of purified MSCs and compared it to that of 13 other cell types. We showed that this profile is enriched in transcription factors and component of Wnt signaling pathway.
We expand our transcriptional analysis and characterize the signature of MSCs either alone or in co-culture with T cells under different culture conditions. At the end of the period of culture T cells were gently removed from the well leaving adherent MSCs untouched. Pure MSCs exposed to stimuli provided by T cells either proliferating will be analyzed for gene expression upon RNA extraction, amplification and hybridization onto Affymetrix Mouse Genome 430 2.0 arrays according to the manufacturer's instructions (Affymetrix, Inc. Santa Clara, CA).As control groups we utilized resting MSCs, MSCs stimulated by anti-CD3 and anti-CD28 in the absence of T cells as well as T cells stimulated by anti-CD3 and anti-CD28 in the absence of MSCs.
For in vivo study, we i.v. injected e-GFP labeled MSCs in C57B/6J mice at the peak of EAE induced with MOG 35-55; animals were followed for 40 days and then brain and spinal cord were removed and stained for immunofluorescence examination. Upon identification of eGFP+ cells under an optical microscope, each cell of interest was captured by laser micro-dissection with the Laser Capture Microdissection. eGFP+ cells were pooled and subjected to RNA extraction, amplification and hybridization onto Affymetrix Mouse Genome 430 2.0 arrays as described above.
Emphasis is given to the expression of population-defining transcription factors and differentiation signals under each condition. This analysis resulted in the identification of key molecules with potential therapeutic implications. DCs stimulated with LPS in the presence of MSCs, failed to up-regulate MHC classes I and II and co-stimulatory molecules thus resulting in an impaired capacity of priming CD4+ antigen specific cells. mMSC-treated DCs showed an impaired secretion of the Th1 orienting cytokine IL-12. Furthermore, mMSC significantly decreased DCs surface expression of CCR7, a key receptor for homing into lymph nodes. mMSC-treated DCs down-regulated intracellular components of the antigen processing machinery (APM). In order to dissect the impact of MSCs on LPSmediated activation of DCs through Toll-like Receptor 4 (TLR4) we analyzed by real-time PCR the expression profile of TLRs genes of DCs activated by LPS. We observed a significant down-modulation of key molecules involved in TLR signaling including NF-kB, MAPK and IP-10.
These results suggest that mMSC affect adaptive immunity not only through a direct effect on T and B cells but also through an impaired capacity of DCs to properly instruct T cells. Although the pathogenic mechanisms involved in experimental autoimmune encephalomyelitis/multiple sclerosis (EAE/MS) are not well understood, accumulating data suggest that oxidative stress plays a major role in lesion development. Metallothioneins (MTs) are a family of low-molecular weight, cysteine-rich proteins that act in protection against oxidative stress. In the central nervous system (CNS), two neuroprotective antioxidant MT isoforms (MT-I and MT-II), and a brainspecific isoform (MT-III), acting as an inhibitor of neuronal growth, have been identified. The abundance of MT isoforms induced in CNS by tissue injury suggests a role for MTs in several neuropathologies. The present study was aimed at evaluating the expression profiles of the three brain MT isoforms during EAE progression and following murine mesenchymal stem cells (MSCs) treatment. In parallel, oxidative stress conditions were monitored by assessing the activity of the main antioxidant enzymes.
Expression of all MT isoforms was significantly increased in EAE mice with respect to controls. A significant up-regulation of MT-I, MT-II and MT-III was observed already at day 11 from the onset of the disease (about a 2-fold induction for each MT with respect to the healthy mice). A further increase in MT transcription was observed at day 40. In mice treated with MSCs, up-regulation of MTs was partially reversed. At day 40 of disease the up-regulation of all MT isoforms decreased to values lower than those observed at day 11 in EAE animals. Changes in CAT and SOD activity parallel those observed in MT expression pattern.
The results demonstrate that EAE progression is clearly associated with the development of oxidative stress conditions in the brain, as indicated by increased expression of all MT isoforms and activities of CAT and SOD. Treatment with MSCs significantly reduces both MT upregulation and increased antioxidant enzyme activities accompanying the progression of the disease. Human mesenchymal stem cells (MSC) are endowed with remarkable immunomodulatory properties that resulted in their exploitation for the treatment of immune-mediated diseases such as multiple sclerosis (MS). In this study we addressed their effect on unconventional T cells, namely NKT and gammadelta T cells, which are likely to play a role both in control and pathogenesis of autoimmune diseases.
We first studied the effect of MSC on in vitro expansion of both populations from PBMC stimulated with a-Galactosyl Ceramide (for NKT) and Isopentenyl Pyrophosphate (for gammadeltaT cells) respectively. MSC inhibited NKT and gammadelta T cells expansions both in cell-to-cell contact and in trans-well experimental systems without the induction of apoptosis. We observed that such inhibition was partially counteracted by indometacin, an inhibitor of PGE2 synthesis. When we specifically blocked other molecules reported to be involved in the modulation of the immune response by MSC, such as indoleamine2,3-deoxigenase (IDO) and TGFBeta we could not detect any effect on NKT+ and gammadelta+ cells expansion.
These data indicate that PGE2 represents the soluble factor preferentially involved in the MSC-mediated inhibition of VAlpha24+ VBeta11+ and gammadelta+ cell expansion. We also studied the effects of MSC on OKT3-mediated proliferation, cytokine production and cytotoxic activity of in vitro expanded NKT and gammadelta T cells. MSC moderately inhibited TNFAlpha and IFNgamma production only by NKT while they impaired CD3-mediated proliferation without significantly interfering with the cytotoxic potential of both NKT and gammadelta T cells. In contrast, MSC were lysed by activated gammadelta T cells though TCR recognition. This information is relevant especially for clinical applications of MSC-based therapy like treatment of autoimmune diseases as multiple sclerosis. Hydrogen sulfite (H2S) is generated endogenously in mammalian tissues by the normal processing of sulphur-containing amino acids. Some of its known actions are carried out in the cardiovascular, nervous and immune systems. Besides this endogenous source of H2S, it is known that thermal waters rich in sulphur are precursors of this molecule, and they have been traditionally used in bath therapies because of their beneficial effects on human health. Up to now, all the still scarce scientific studies on this subject have focused on the effects of H2S on the different physiological systems separately. In the present work we have investigated the role of sulfurous thermal waters in the neuroimmune communication.
Twelve-month old female ICR-CD1 mice were used. They were divided in two groups: the first (called "sulfurous water group", SG) was submitted to a daily bath of 15 min in sulfurous water, and the second ("control water group", CG) in normal water. The treatments were performed during 4 weeks, 5 days per week. Animal motor condition and neuromuscular vigour were assessed through the tightrope test (% of animals that reach one of the side poles of the rope and % of animals that fall down). In addition, peritoneal leukocytes were obtained and total glutathione levels (nmol/10^6 cells), a key antioxidant of the organism, and natural killer activity (NK, % lysis of tumoural cells) were determined.
As regards the tightrope test, mice of the SG group showed higher percentages of reaching one of the side poles situated the end of the rope (50% for SG and 16% for CG) and lower percentages of falling downs (25% for SG and 66% for CG). With respect to total glutathione, SG showed increased levels with respect to the CG group (0.61 ± 0.15 nmol/10^6 cells and 0.41 ± 0.09, respectively p b 0.01). The same tendency was observed in NK activity, since mice of the SG group show a higher percentage of lysis (29 ± 9% for SG and 20 ± 6% for CG, p b 0.05) with respect to those of the CG group.
Bath sessions in thermal waters rich in sulphur compounds modulate the neuroimmune relationship by improving in mature mice their neuromuscular coordination and one relevant immune function such as the NK activity in peritoneal leukocytes. In these cells that water also increase the total glutathione levels. These results show a better preservation of homeostasis and health of the treated animals.
Financial support: Foundation for Research and Innovation in Medical Hydrology and Spas (Bilbilis Foundation) grant. RETICEF (RD06/0013/0003). We have previously shown that triple-transgenic mice for Alzheimer's disease (3xTgAD; harboring PS1M146V, APPSwe, tauP301L transgenes), which develop plaques and tangles and show genderspecific cognitive and behavioral impairments, also suffer detrimental changes in some key lymphocyte functions, namely proliferation in response to the mitogenes concanavalin A and lipopolysaccharide, natural killer cytotoxic activity of YAC-1 tumoural cells and chemotaxis towards the formylated peptide, in both spleen and thymus, with males showing worse function than females. The regulatory cytokines tumour necrosis factor-α and interleukin-2 in the supernatants of lymphocyte cultures were also altered in 3xTgAD mice. Environmental enrichment (EE) was found to be efficient in improving those changes.
In the present work, 3xTgAD and wild type male and female nonand environmentally enriched mice were used. EE began in the adulthood (6 months) and lasted for 5.5 months (house and wheel were maintained permanently, whereas other objects of different shapes and colors were changed every 2 days). The animals were sacrificed at the age of 15 months, and thymus and spleen were removed. Lymphocytes were obtained and total glutathione content was measured by enzymatic kinetic spectrophotometric procedure. Plasma corticosterone was determined by radioimmunoassay.
The results showed that gender exerted an overall effect on plasma corticosterone, which was higher in females (males 35.83 ± 5.72, females 72.88 ± 4.85 ng/ml, P b 0.001). Further analysis revealed that AD increased corticosterone in males only (non-3xTgAD males 21.90 ± 4.75, 3xTgAD males 79.60 ± 5.07 ng/ml, P b 0.001), and the beneficial effects of the EE decreasing those levels were shown especially in 3xTgAD males (non-environmentally enriched 3xTgAD males 79.60 ± 5.07, environmentally enriched 3xTgAD males 27.85 ± 3.36 ng/ml, P b 0.001). As regards glutathione content, no differences were observed in thymus lymphocytes, whereas gender showed an overall effect on spleen lymphocytes, being higher in females (males 2.37 ± 0.71, females 8.24 ± 0.64 nmol/mg prot, P b 0.001). EE exerted a general effect decreasing the levels of glutathione in spleen lymphocytes (non-EE 6.95 ± 0.68, EE 3.65 ± 0.67 nmol/mg prot, P b 0.001).
In conclusion, corticosterone seems to play an important role in the gender-specific immune function and behavioral changes exerted by both AD and EE. The gender differences could contribute to differential development of the disease and therapeutic effects of the EE. Oxidative stress in lymphocytes from old 3xTgAD mice is similar to that of the wild type animals of the same age. However, females show higher glutathione levels in spleen lymphocytes, which could indicate a leveling off mechanism aiming to control the increased oxidative stress that occurs with ageing. EE would be efficient in decreasing the oxidative stress in spleen lymphocytes from old subjects, which would be reflected in lower glutathione levels needed. Therefore, EE stands out as a key strategy to decrease the oxidation and improve the function of the immune system in both healthy and non-healthy elderly subjects.
UAB140267, MEC (BFU2005-06777), Research Group UCM-CM (910379), RETICEF (RD06/0013/0003).
3 -Complement inhibition abrogates nerve sodium channel disruption in a rabbit model of Guillain-Barre syndrome-Yuki Nobuhiro⁎ 1
Guillain-Barre syndrome is divided into two subtypes, acute inflammatory demyelinating polyneuropathy and acute motor axonal neuropathy (AMAN). Autopsy results indicate complement activation followed by membrane attack formation is an important mechanism for both subtypes. Complement-mediated disruption of voltage-gated sodium channel clusters at the node of Ranvier plays a role in the pathogenesis of AMAN. To examine whether nafamostat mesilate (NM), a serine protease inhibitor exhibiting anti-complement activity, could inhibit complement-mediated Nav disruption in a rabbit model of AMAN.
Each AMAN rabbit was intravenously implanted with two osmotic pumps containing NM in 50% dimethyl sulfoxide (treatment group, four rabbits) or 50% dimethyl sulfoxide alone (control group, five rabbits) at a delivery rate of 0.8 mg/kg/h for 7 days. Immunohistochemical analysis was performed to examine the frequency (%) of deposited complement products and disrupted sodium channel clusters at the nodes of Ranvier in the spinal anterior roots. Complement deposition and sodium channel disruption were significantly less frequent in NM-treated rabbits compared to control rabbits. The clinical signs in two rabbits rapidly improved after NM treatment, although there was no significant difference for the total clinical scores between NM-treated and control groups.
NM could inhibit complement deposition and consequently prevent complement-mediated sodium channel disruption in the AMAN model. The results provide a rationale for a clinical trial using NM. Microglia, the resident macrophages of the CNS, are implicated in many acute and chronic neurological diseases, including multiple sclerosis, trauma and stroke. While many laboratories are studying the role of activated microglia in CNS pathophysiology, the molecular mechanisms controlling the activation process of microglial are less understood.
Recent progress in genetics has identified a new class of RNA molecules called microRNAs (miRNA) involved in gene regulation. miRNAs are single-stranded RNA molecules of ∼ 22 nucleotides complementary to a site in the 3′ untranslated region (UTR) of mRNAs. The annealing of the miRNA to the mRNA either inhibits protein translation or facilitates cleavage of the mRNA. This regulation adds an unexpected layer of complexity to the classic "linear" concept of DNA→mRNA→protein.
The functional role of miRNAs in microglial activation is currently unknown. Using miRNA arrays and qRT-PCR for miRNAs we have found a limited number of miRNAs constitutively expressed at high levels, which are most likely involved in the maintenance of the microglia phenotype. Furthermore, we identified several miRNAs which are regulated during microglial activation by LPS/IFN-gamma stimulation. We have correlated the miRNA regulation profiles with parallel mRNA gene array data for target evaluation. To gain insight into the effects of specific miRNAs we used transfection of miRNA mimics to validate the predicted miRNA targets by qRT-PCR and Western blot.
miRNAs are an integral part of the microglial activation process. Identification of miRNA specific pathways may constitute new targets for therapeutic intervention in CNS injuries associated with microglial activation such as stroke, multiple sclerosis or Alzheimer's disease. In a study of eleven MS patients and eight healthy controls, Tregs were co-cultured with CD4+CD25-(responder) T cells at 0:1, 1:1, 1:2 and 1:4 ratios in the presence or absence of BLP. We found that BLP reversed Treg suppression at low Treg/responder ratios. In the absence of BLP, Tregs from patients had lower suppressive activity than Tregs from healthy controls at the 1:4 ratio, in agreement with a previously reported defective Treg function in MS (Viglietta et al., J. Exp Med 2004). Preincubation of Tregs or responder cells with BLP before co-culture demonstrated a predominant effect of TLR2 stimulation on Tregs, which were rendered less suppressive. To clarify the dose effect of TLR2 stimulation by BLP on human Treg activity, Tregs were cocultured with responder T cells at 0:1, 1:4, 1:8 and 1:16 ratios in the presence or absence of BLP (0.1, 1 and 5 μg/ml). Suppression of responder cell proliferation by Tregs was significantly reduced by 5 μg/ml BLP compared with 0.1 and 1 μg BLP concentrations at lower Treg/responder cell ratios. By contrast, lower concentrations of BLP did not reduce suppression of responder cell proliferation by Tregs.
This study demonstrates the previously uncharacterised nature of Treg responses to different doses of TLR2 stimulation and suggests that infections may facilitate MS exacerbations by suppressing Treg functions. Seventy-four MG patients participated in this study, and they were treated with either FK506 (N = 65) or CyA (N = 22). We measured blood drug concentrations of trough level (C0). The MG-ADL scores were examined in relation to the blood drug concentrations (C0).
The mean FK506 C0 for MG patients (n = 38) having the CYP3A5⁎3/⁎3 genotype was significantly higher than those (n=20) having CYP3A5⁎1/⁎3 genotypes (7.05 ng/mL versus 2.87 ng/mL, P = 0.00002) or those ( n = 7) having CYP3A⁎1/⁎1(7.05 ng/mL versus 1.30 ng/mL, P b 0.00035). The improvements of mean MG-ADL scores (beginning→4 months after FK506 administration in MG patients having either CYP3A5⁎3/⁎3 (5.4→3.4 or CYP3A5⁎1/⁎3 (6.2→4.3) genotypes tended to be better than those having CYP3A5⁎1/⁎1 (4.0→3.7 genotypes. But in the CyA concentrations, the comparisons of the CYP3A5 genotypes revealed no significant differences.
With MG treatment, comparisons of the CYP3A5 genotypes revealed the significant differences in the FK506 concentrations with ADL improvements. Multiple Sclerosis (MS) is considered an autoimmune disease (AD) and susceptibility to this condition is controlled by multiple genes and environmental factors. Association between MS and other autoimmune disorders has been suggested. Recently, the protein tyrosine phosphatase (PTPN22) 1858CNT gene polymorphism has been proposed as a common susceptibility factor for several autoimmune diseases, but association with MS couldn't be demonstrated.
1) To identify coexisting systemic autoimmune features in MS patients and in their relatives. 2) To investigate whether PTPN22 polymorphism was associated with autoimmune features in these patients.
A total of 266 patients with definitive MS, according to McDonald criteria, were included in the study. Patients were characterized for the presence of positive family history of AD (group 1); for the coexistence of other autoimmune diseases (group 2) and for the presence of positive serum autoantibody (group 3). Genotyping of the 1858C N T polymorphism was performed using a TaqMan® assay.
Twenty nine patients (11%) reported at least one relative with AD such as MS (15), Hashimoto thyroiditis (3), psoriasis (2) and other AD (9). Thirteen patients (5%) reported at least one coexisting AD. Sixty nine patients (26%) had at least one positive serum autoantibody but no other AD. No significant differences in PTPN22 allele frequencies among the different groups were observed.
Disregarding the known familiar aggregation of MS, these results do not show an increased frequency of ADs in MS patients and their relatives, compared with the Portuguese general population. The lack of association of AD features with the PTPN22 polymorphism in these patients may be due to lack of statistical power given the small number of cases identified. This study provides no support for the concept that MS might arise on a background of increased susceptibility to autoimmunity, in agreement with the results from a larger Canadian study [ Although specific autoimmune disorders (ADs) are associated with unique clinical and serological features, some overlapping and a tendency towards family aggregation may be explained by the presence of common genetic susceptibility polymorphisms in immune system related genes (HLA-DRB1, PTPN22). It has been hypothesized that these could influence susceptibility, disease course or both in different ADs. We previously showed, in our population, that HLA-DRB1⁎15 positive MS patients had better clinical outcome, and also a higher frequency of the PTPN22 1858T allele.
To assess the prevalence of coexisting ADs in Portuguese MS patients and their families, and to relate the PTPN22 1858T polymorphism to disease aggressiveness.
A total of 160 definitive MS patients, with at least 10 years of disease, were evaluated. Patients were subdivided into 2 groups: patients who maintained an EDSS score of = 3 at 10 years were considered to have 'benign' MS (n = 69); patients with EDSS score N3 were defined as having 'non-benign' MS (n = 81). Genotyping of the PTPN22 1858CNT polymorphism was performed using a TaqMan® assay.
Families: eleven 'benign' patients reported at least one first degree relative with an AD, compared to five 'non-benign' patients (16% vs. 6.2%, p = 0.06). These were mostly due to MS familiar aggregation. Coexisting AD: four 'benign' and four 'non-benign' patients had coexisting AD (5.8% vs. 4.9%). The PTPN22 1858T phenotype frequency was higher in 'benign' than in 'non-benign' patients (18.1% vs. 8%, p = 0.054, OR = 2.5).
The PTPN22 1858T variant, previously associated with increased susceptibility to autoimmunity (but not with MS), seems to be associated with a good prognosis in MS patients. This, together with the observed higher frequency of autoimmune diseases in the relatives of patients with benign course, suggests that different pathogenic pathways are implicated in this subgroup of patients, or alternatively, that genetic factors related to autoimmunity modulate the course of the disease. Murine experimental autoimmune encephalomyelitis (EAE) is an induced, autoimmune, demyelinating disease model for human multiple sclerosis (MS). Copaxone is an approved immunomodulatory therapy for MS that was discovered through studies in EAE. It is thought that the main mode of action of Copaxone is the induction of a Th1 to Th2 shift in CD4+ T-cells. We have shown that, in human MS, Copaxone therapy induces an upregulation of cytotoxic, suppressor CD8+ T-cell responses, suggesting that these cells are integrally involved in mediating the immune effects of this drug.
In this study, we show that Copaxone induces robust CD8+ T-cell responses even in mice. Whereas Copaxone immunization ameliorates the clinical and histologic severity of EAE in wild type C57/BL6 mice, such protection is not observed in CD8(−/−) mice, indicating that CD8+ T cells are required in mediating the effects of Copaxone. Moreover, adoptive transfer of CD8+ T-cells from Copaxone-immunized (but not OVA-immunized) mice results in amelioration of EAE in CD8(−/−) and wildtype mice, suggesting therapeutic potential for Copaxone-specific CD8+ T-cells. These cells appear to mediate their effects through a cytotoxic/suppressor mechanism, similar to our findings in human MS.
In conclusion, our studies provide strong evidence that the in vivo induction of immune modulatory CD8+ T-cells is an essential step in mediating therapeutic protection during autoimmune demyelination.
In the past decade, several new radioligands have been developed for positron emission tomography for potential clinical use. Since microglia play important roles in health and disease, studying their activation states with such radioligands may prove useful for assessing disease prognosis and progression. Radioligands that bind to the translocator protein of 18 kDa (TSPO), formerly known as the peripheral benzodiazepine receptor, are currently being scrutinized, but the protein expression of TSPO in human brain has not been well-studied. In addition, the cell types that express TSPO in vivo has been debated. Autoradiography used in combination with immunohistochemistry (IHC) for cell-specific markers on autopsy tissue or in animal models has shown that microglia are the main cell type that upregulate the TSPO, but astrocytes are a cell type of interest for some conditions. The purpose of this study is to detect the protein expression of TSPO in normal and diseased human brain via IHC and to delineate the cells that express TSPO.
We have assessed the expression of TSPO via IHC using two antibodies, one polyclonal and one monoclonal, developed against TSPO. The two antibodies have good agreement and show mild staining in normal brain, but elevated staining in pathologic conditions such as Alzheimer's disease, multiple sclerosis, ischemic stroke and HIV encephalitis. Microglia and macrophages appear to be the main cell types expressing TSPO, although limited astrocyte expression was also observed.
The IHC of TSPO will be useful in animal models of inflammation and will shed light on the conditions that govern its upregulation. Future studies will look into regional expression of TSPO and may probe the timing of upregulation of TSPO with in vitro studies. Tongue muscle atrophy is relatively common in MuSK-MG patients. However, these are not clear whether tongue muscle atrophy can be reversed or not. We observed retrospectively a change of the tongue atrophy before and after immunosuppressive treatment by MRI.
We observed retrospectively a change of the tongue atrophy before and after immunosuppressive treatment by MRI from 2002 to 2008. In retrospective evaluation with mid-sagittal MRI, numbers of cross sectional areas of tongue are; 2088 mm 2 in 2002, 1401 mm 2 in 2005, and 1736 mm 2 in 2008.
In MuSK-MG, immune-modulating treatments can result in dramatic clinical recovery, even after long course. Tongue muscle atrophy can be reversed, too.
12 -Meningeal myeloid cells as middlemen between T cell deficiency and depressive behavior - Peripheral immune status is tightly linked to central nervous system (CNS) function. Original studies showed that T cells have a beneficial effect on neuronal survival following CNS injury. More recently, the role of the immune system has been extended to normal brain function. T cells have been implicated in cognitive function and adult neurogenesis, two major aspects of adult brain plasticity. Severe combined immune deficient (SCID) mice exhibit reduced neurogenesis and cognitive impairment as examined on spatial learning and memory tasks, (e.g. Morris Water maze). Moreover, depletion of the immune system in wild type (WT) mice followed by bone marrow transplantation from SCID mice also impairs cognitive function. T cell transfusion restores cognitive function and neurogenesis in both groups. Thus, the immune system affects aspects of brain plasticity and cognition.
This led us to examine the effect immunodeprivation might have on the ability of the brain to withstand stressful conditions. We assayed SCID and WT mice using the forced swim test (FST), in which increased immobility is indicative of a "depressed" phenotype. Here we show that SCID mice are strikingly more immobile than WT in the FST. We assayed SCID and WT mice in an open field assay and found no differences in baseline activity. We then examined the population of meningeal cells in both groups. Immunohistochemical observations revealed that although the numbers of meningeal macrophages/monocytes (CD11b+CD11c−) were comparable in both groups, the numbers of MHCII positive cells were significantly lower in SCID mice. When meningeal isolations were examined by flow cytometry, the reduced MHCII expression was substantiated. Moreover, we found that meningeal macrophages/ monocytes from SCID mice show an increased pro-inflammatory phenotype, characterized by expression of cytokines such as IFNγ, TNFa, and IL1β.
We propose that the lack of mature and active T cells leads to a skewed pro-inflammatory phenotype of myeloid cells. As a consequence, this leads to accumulation of pro-inflammatory cytokines in meninges and finally to depressive behavior. These results may shed new light on the "etiology" of depressive behavior in chemotherapy patients, the elderly, HIV-dementia, and other conditions, all tightly associated with T cell dysfunction. If found true in the abovementioned conditions, our study might lead to development of new therapeutic approaches for depression, based on modulation of adaptive immunity.
13 -The pathway of CX3CR1 and fractalkine interaction is involved in the leukocytes infiltration on experimental autoimmune encephalomyelitis in mice -Muramoto Kenzo⁎ 2 , Kuboi Yoshikazu 2 , Ogasawara Hideaki 1 , Rikitsu Etsuko 1 , Mizuno Keiko 1 , Nishimura Miyuki 1 , Imai Toshio 1 1 Kan research institute, Kobe, Japan 2 Eisai Co., LTD., Tsukuba-shi, Japan
Leukocytes migration to central nervous system is involved in severity of the demyelinating diseases multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Fractalkine (FKN, CX3CL1) is a unique chemokine with unique chemokine/mucin hybrid structure. CX3CR1, the solo receptor for CX3CL1, is expressed on microglia, monocyte/macrophage, dendritic cells and cytotoxic/effector T cells. It was reported that Fractalkine and CX3CR1 were constitutively expressed in several region of the CNS and this interaction was involved in EAE. To clarify the role of CX3CR1/FKN pathway on EAE model, the effect of anti-FKN monoclonal antibody (mAb) on an EAE model was investigated in mice.
Anti-FKN mAb was administered every other day from 6 days after the sensitization with MOG (myeline-oligodendrocyte-glycoprotein). The administration of anti-FKN mAb at the dose of 0.5 mg/mice significantly inhibited the development of EAE symptoms. The infiltrated cells into the spinal cord were analyzed by FACS. These infiltrated cells including T cell, dendritic cells and so on was decreased by administration of anti-FKN mAb. The mRNA levels of inflammatory and immune genes in spinal cord were analyzed by real-time PCR analysis. The elevation of mRNA such as leukocytes markers (CD45, CD4, CD8, CD11c), cytokines and chemokines were observed in an EAE model and suppressed by administration of anti-FKN mAb. The immunohistochemical analysis also showed the inhibitory effect of anti-FKN mAb on infiltration of leukocytes into spinal cords and activation of microglia. This anti-FKN mAb also showed therapeutic effect when it was administered after the induction of disease in PLP-induced EAE model.
These results suggested CX3CR1/FKN pathway facilitates the development EAE through leukocyte migration into spinal cord on both of acute and chronic phase.
14 -Modulation of the HIV-1 infected microglial proteome by astrocytes - Astrocytes are brain homeostatic regulators that modulate a broad range of neural functions including regulating extracellular glutamate and neuronal networks, controlling neuronal activities and neurogenesis and secretory a broad range of growth factors including neurotrophins and cytokines. In neurological diseases such as those caused by human immunodeficiency virus (HIV-1) infection astrocytes affect mononuclear phagocyte (MP; perivascular and tissue macrophages and microglia) function and the secretion of neurotoxic factors. Nonetheless, little is known about MP-astrocytes communicate during infection.
To examine these questions we employed a proteomics approach. Our primary method of profiling was 2-dimensional gel electrophoresis with DIGE technology. Protein identification was accomplished using nano-LC-MS/MS (LTQ Orbitrap, Thermo Scientific) sequencing of tryptic digest peptides. The primary method of validation was Western blot analysis. We examined differential regulation of HIV-1 infected MP proteins during co-cultivation with astrocytes and confirmed by Western blotting. These revealed the differential regulation of redox and structural proteins. Preliminary results demonstrated that gelsolin, leukotriene A4 hydrolase, and adenylyl cyclase-associated proteins were decreased in MP by astrocytes. Increases in annexin, carbonyl reductase and calreticulin precursor proteins were observed.
We conclude astrocytes significantly affect the MP proteome following HIV-1 infection and as such can affect disease onset and progression.
15 -Anti-leukocyte adhesion therapy for the treatment of seizures and 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% of the general world population. Both experimental and clinical data indicate that repeated seizure activity can lead to the chronic recurrent seizures of epilepsy, but the mechanisms responsible for this transition remain unclear. Our GOAL was to study the role of leukocyte trafficking mechanisms in the pathogenesis of seizures and epilepsy.
Epilepsy was induced by injection of C57Bl mice with pilocarpine. Intravital microscopy and in vivo staining were performed in cortical brain vessels postseizures. Magnetic resonance imaging, confocal microscopy, immunohistochemistry, telemetry, power spectrum analysis, behavioral evaluation and cognitive tests were also performed.
In mouse models of epilepsy, seizures caused elevated expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-and P-selectin on brain vessels and recruitment of leukocytes to the brain. Intravital microscopy revealed enhanced granulocyte and Th1 lymphocyte rolling and arrest in brain vessels after seizures and these leukocyte-vascular interactions were inhibited by blockade of the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) or integrins alpha4beta1 and alphaLbeta2. Treatment of mice with anti-alpha4-integrin or anti-alphaL integrin mAb dramatically reduced acute and chronic seizure activity induced by pilocarpine. Genetic deficiency of PSGL-1 or of the alpha1-3-fucosyltransferases, enzymes involved in the glycosylation of PSGL-1, also inhibited seizure activity. Neutrophil depletion reduced seizure induction and spontaneous recurrent seizures as well. Blood-brain barrier (BBB) leakage was induced by seizures, but was drastically reduced in anti-alpha4 treated or PSGL-1 or FucT deficient animals, establishing a pathogenetic link between leukocyte adhesion, BBB damage and seizure generation.
Our results suggest leukocyte recruitment mechanisms as potential targets for the prevention and treatment of epilepsy. Monocytes human immunodeficiency virus type 1 (HIV-1) infection is highly restricted. Such virus-cell exposure is thought to have limited consequence on the physiology of the monocyte. With the advent of functional proteomics the tools are available to investigate the consequences to cell function following HIV-1 exposure. To this end we investigated changes to the proteome of the plasma membrane (PM) to monocytes and monocyte-derived macrophages (MDM) 7 days after virus exposure.
Viable human monocyte PM was labeled with CyDye fluorophores and lysed the subjected to two dimensional differential ingel electrophoresis (2D DIGE). This was used to compare PM proteomes of unaffected and HIV-1ADA exposed monocytes. Tandem mass spectrometry (LC/MS-MS) assay showed that 45% of the PM proteome was changed after HIV-1 exposure with differentially expressed proteins showing higher than 2 fold changes. Of these proteins 20% were redox proteins linked to PM protection from respiratory burst. Heat shock protein 70 (HSP70), leukotriene hydrolase A4 (LTHA4) and biliverdin reductase (BVR) were representative upregulated PM proteins that are known to protect against free radicals. Confocal images, flow cytometry analyses of HIVexposed and control permeabilized and control cells showed these proteins were upregulated and translocated to the PM in virusexposed monocytes. Western blot and flow cytometry assays of PM and cytozolic fractions stained against HSP70, LTHA4 and BVR confirmed the 2D DIGE results. We next investigated whether the translocation of the three proteins to the plasma membrane was specific to HIV-1 and whether use of PM protecting antioxidants such as tocopherol acetate and sanguinarine attenuate the oxidative burst in monocytes and alter levels of the proteins under investigation.
The alterations in the metabolic and redox systems provide an insight into cell-pathogen interactions and of compensatory events between engagement of pathogen killing host defenses and modulation of a respiratory burst. Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). MS is thought to be a T-cellmediated disease, although the major focus of prior research has been on CD4+ T-cells. We have shown previously that MS patients and healthy subjects harbor CNS-specific CD8+ T-cells with a mixed functional profile. The roles of these CD8+ T-cells in the pathogenesis and/or regulation of MS are still unclear. We have also shown that untreated MS patients show an overall deficit in the immune suppressive ability of CD8+ T-cells, which is corrected following glatiramer acetate (Copaxone) therapy.
In the current study, we addressed the role of CNS-targeted CD8+ T-cells in MS patients by developing a novel, flow cytometry-based in vitro immune suppression assay. First, we confirmed the validity of this approach by replicating the suppression by CD4+CD25+ regulatory T-cells ("T-regs") from healthy subjects. We observed a deficit of CD4+CD25+ T-reg-mediated suppression in untreated MS patients. Moreover, MS patients also had an overall deficit in CD8+ T-cellmediated suppression in Copaxone-or anti-CD3-stimulated cultures. Interestingly, CD8+ T-cells from MS patients and healthy controls subjects that were targeted against CNS autoantigens, but not those specific for control antigens, exhibited regulatory ability, in that they suppressed the proliferation of CD4+CD25− T-cells.
In conclusion, our results not only confirm that MS is a disease of perturbed immune regulation but also provide the first evidence that CNS-specific CD8+ T-cells may have a regulatory role in this disease.
18 -MicroRNAs regulate brain to body anti-inflammatory signaling - MicroRNAs (miRs) contribute to both neuronal and immune cell fate, but their involvement in inter-tissue communication remained unexplored. Here, we report a role for the acetylcholinesterase (AChE) mRNA-targeting miRs 132, 182⁎ in regulating brain-to-body control of inflammation.
We show increases in miRs 132, 182⁎ and decreases in AChE in human leukocytes exposed to bacterial endotoxin, reduced AChE and inflammation following treatment with mimics of miRs 132, 182⁎, and impaired cholinergic anti-inflammatory regulation in leukocytes from mice over-expressing 3′-UTR-null AChE devoid of miR-responding elements.
Our findings identify AChE mRNA-targeting miRs as functional regulators of brain to body signaling, opening avenues for studying the neuro-immune dialogue and for post-transcriptional therapeutic interventions in inflammatory reactions.
19 -TLR3 and TLR4 are innate antiviral immune receptors in human microglia -Suh Hyeon-Sook⁎ 1 , Zhao Meng-Liang 1 , Belbin Thomas J. 1 More than 80% of patients with typical generalized myasthenia gravis (MG) have auto-antibodies against the acetylcholine receptor (AChR) at the postsynaptic membrane of the neuromuscular junction. Early-onset MG is frequently associated with thymic abnormalities and the myasthenia often ameliorates after thymectomy. To characterize the autoreactive antibodies that are produced in the thymus, memory B lymphocytes were isolated from the thymus of MG patients and immortalized.
Immortalization of B cells by Epstein Barr Virus (EBV) transformation provides a tool to characterize the auto-antibodies in MG patients. To overcome the low efficiency of EBV-based immortalization, the polyclonal B cell activator CpG 2006 was added to the B cells. 60 IgGpositive B cell clones were cultured for the production of monoclonal antibodies. Screening of clones by immunohistochemistry on muscle tissue showed immunoreactivity to striated muscle proteins in 30%. Nevertheless, no anti-AChR antibody producing B-cell was found in our immortalized clones. To further study the specificity of the non positive striated muscle antibodies we performed protein array techniques.
In conclusion, the increased immortalization efficiency of EBV transformation by the addition of a polyclonal B cell activator allowed us to immortalize memory B cells from MG patients and subsequently to produce monoclonal antibodies. Interestingly we showed that the thymus does not contain an enriched population of mature B-cells with AChR specificity. Finally, with this improved method further insights into the pathology of this autoimmune disease might be obtained by helping to identify autoantibodies in both seropositive and in double seronegative MG patients. Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We investigated the role of a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. The anti-inflammatory property of IgG4 antibodies was tested in vivo in a rhesus monkey model of experimental autoimmune myasthenia gravis.
Passive transfer of a pathogenic, patient-derived, IgG1 antibody against acetylcholine receptor (AChR) induced autoimmune myasthenia gravis in rhesus monkeys. Immunoprohylaxis with an IgG4 anti-AChR antibody prevented IgG1-induced myasthenia gravis as well as the characteristic structural damage and accompanying electro-physiological changes to neuromuscular junctions. IgG4 Fab arm exchange was shown to dampen receptor down-modulation, which may prevent disease by reducing receptor cross-linking and internalization.
IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies. Our study provides clues for novel approaches for the development of antigen-specific immunotherapeutic treatments for patients suffering from MG and possibly other antibody-mediated autoimmune disorders.
Multiple sclerosis (MS) is an immune-mediated, demyelinating disease of the central nervous system (CNS). While MS and its animal model, experimental autoimmune encephalomyelitis (EAE) are thought to be mediated and regulated by CD4+ Tcells, several reports from us and others show that CD8+ T cells play an important role in MS/EAE immunology. Glatiramer acetate (GA, Copaxone) is an immunomodulatory MS therapy that is believed to mediate its effects through Th1 to Th2 immune deviation. We have shown that GA therapy induces robust CD8+ T cell responses, which are suppressor and cytotoxic in nature and may mediate immune effects through HLA-E-mediated killing of activated CD4+ T cells. We further show that CD8+ T cells are, in fact, essential in mediating the effects of GA on EAE. We also address the role of CNS-specific CD8+ T cells in the disease process. While these cells are widely believed to be pathogenic in nature, we show that both in human MS and in EAE, CNS-targeted CD8+ T cells have an immune suppressor role. We also demonstrate that untreated MS patients are functionally deficient in not only CD4+, but also CD8+, regulatory T cells (T reg s). Interestingly, all human CD4+ and CD8+ T cells attain a transient FOXP3+ state following multiple modes of stimulation. Importantly, in this state they behave unequivocally like bonafide T reg s as assessed by in vitro suppressor assays. Thus, we believe that MS is a disease of perturbed immune regulation characterized by the inability to sustain induced T reg s. We are therefore evaluating the interplay between antigen presenting cells and T cells to dissect the ability of immune populations to modulate T cell development into suppressor vs. effector cells.
These studies were supported by grant awards from the NIH and National MS Society. NJK is a Harry Weaver Neuroscience Scholar of the NMSS.
CONCURRENT SYMPOSIUM 1: Neuroprotective strategies in neuroimmunologic disease Chairs: S. Khoury, S. Appel
Interactions between the immune system and neural stern cells
Brigham and Women's Hospital, and Harvard Medical School, Boston, MA, USA Multiple sclerosis (MS) is an autoimmune disease that causes demyelination and axonal damage in the central nervous system. The majority of the patients experience relapsing-remitting symptoms followed by a secondary progressive phase leading to permanent disability once chronic disease has set in. The current treatment of MS relies on immunologic manipulation. However, it is becoming clear that repair should be considered as a goal for treatment. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS, and provides a powerful tool for investigating the pathogenesis of MS. Although current evidence shows that remyelination and regeneration occur spontaneously to some extent, it is not nearly sufficient enough in MS or EAE. Remyelinating oligodendrocytes arise from differentiation of neural progenitors and lack of progenitor function has been proposed to play a role in remyelination failure in MS. Understanding how inflammation can impact the function of neural stem cells (NSCs) acutely and chronically is critical for the development of therapeutic strategies targeting endogenous and exogenous NSCs.
T cell astrocyte crosstalk in health and disease J. Kipnis Recent evidence suggests that T cells can influence neuronal plasticity by supporting learning and memory. For example, T celldepleted mice exhibit impaired cognitive performance, which could be reversed following injection of exogenous T cells from genetically matched donors. The mechanisms underlying the beneficial effects of T cells in the brain are not well understood. We now suggest that cognitive impairment is a result of a malfunctioning meningeal immune surveillance in T cell deficient mice. Myeloid cells, occupying subarachnoid and ventricular spaces, exhibit a skewed pro-inflammatory phenotype in the absence of T cells and produce high levels of proinflammatory cytokines. Pro-inflammatory cytokines produced by meningeal myeloid cells are proposed to diffuse into brain parenchyma and to affect astroglial activation. Immune deficient mice possess higher levels of activated astrocytes along with a reduced expression of the synaptogenic protein, thrombospondin1 (TSP1), as compared to wild type mice. Moreover, immune deficient mice have fewer hippocampal synapses when compared to wild type counterparts. We show that while anti-inflammatory cytokine, interleukin (IL)-4, increases TSP1 expression in astrocytes, pro-inflammatory cytokines, such as IL-1beta and TNF-alpha inhibit its expression, which is, supposedly, responsible for reduced synapses and for cognitive impairment observed in immune deficient mice. Our results not only shed a light on a potential mechanism underlying cognitive dysfunctions associated with immune decline but might also lead to a development of new future therapies for cognitive and mental conditions that target meningeal. Neuroinflammation, marked by gliosis and infiltrating T-cells, is a prominent pathological feature in human ALS as well as in models of neurodegenerative diseases. Transgenic mice ubiquitously overexpressing mutant Cu 2+ /Zn 2+ superoxide dismutase (mSOD1), a chronic neurodegenerative model of inherited amyotrophic lateral sclerosis (ALS), exhibit such neuroinflammatory changes. We have documented that the innate immune system plays a pivotal role in determining the rate of disease progression in mSOD1 mice by demonstrating that microglia either lacking or with reduced mSOD1 expression enhance motoneuron protection and slow disease progression in such models. These studies were carried out in mice that are unable to develop myeloid cells, but in which both peripheral and central immune systems are modified.
To provide evidence for a potential role of the adaptive immune system in these models, we bred mSOD1 mice with RAG2 −/− mice that are unable to develop mature and functional T-and B-cells. mSOD1/ RAG2 −/− mice died earlier than mSOD1/RAG2 +/− mice, and no T-cells were present within the spinal cord. The onset of disease was unchanged. Following irradiation and transplantation with mSOD1 or WT-derived bone marrow, survival was extended in mSOD1/RAG2 −/− mice, to ages identical to mSOD1/RAG2 +/− mice. Immunohistochemical analyses of spinal cord sections demonstrated that CD4+ T-cells had been restored by BMT, but there was no evidence for the presence of B cells either in mSOD1/RAG2 −/− mice or in mSOD1/RAG2 +/− mice.
When CD4 −/− mice were crossed with mSOD1 mice, the CD4 −/− /mSOD1 mice died earlier than CD4 +/− /mSOD1 mice, confirming the importance of CD4+ T-cells in mediating neuroprotection. Mice lacking functional T-cells, or CD4+ T-cells also had increased mRNA levels for pro-inflammatory cytokines and NOX2, and decreased levels of trophic factors and glial glutamate transporters. Bone marrow transplants reconstituted mice with T-cells, prolonged survival, suppressed cytotoxicity, decreased mRNA levels for pro-inflammatory cytokines and NOX2, and increased mRNA expression of trophic factors and glial glutamate transporters. These results demonstrate for the first time in a model of chronic neurodegeneration that CD4+ Tcells may provide neuroprotection by modulating the trophic/ cytotoxic balance of glia. These glial/T-cell interactions establish a novel target for therapeutic intervention in ALS and possibly other neurodegenerative diseases.
Regenerating the brain: The role of neural stem cells
Neuroimmunology Unit -Institute of Experimental neurology (INSpe), DIBIT -San Raffaele Hospital, Milan, Italy 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 distinctalthough strictly interrelatedcategories: 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 pathways (cortical maps)occurring mainly via axonal branching and synaptogenesis takes 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 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, neuroprotection). It is still a matter of investigation whether (or not) equally robust brain repair/protection can occur following the recruitment within the CNS of trans-differentiating stem cells of a different embryonic origin (e.g. developmental plasticity vs. cell fusion). Multiple sclerosis (MS)the most common demyelinating disease of the CNS affecting young adultsis an autoimmune disease encompassing both inflammatory (myelin and axon destruction) and degenerative (axonal and neuronal loss) features. Spontaneous brain repair (remyelination) occurs early during MS evolution but fails over time. Recent evidence clearly indicate that there are immunological, developmental and cellular events aiming at restoring tissue integrity in MS patients that operate early during disease evolution but lose their efficacy over time. This supports the ensuing idea that MS progression can be viewed as a dysfunction of the intrinsic brain repair mechanisms rather than caused by an uncontrolled, and still undiscovered, pathogenic alien(s). Antibodies against nicotinic acetylcholine receptors (AChR) are implicated in myasthenia gravis as well as in autoimmune autonomic ganglionopathy (AAG). AAG presents as severe generalized sympathetic and parasympathetic autonomic failure. This disorder is also known as acute pandysautonomia or idiopathic subacute autonomic neuropathy. The most disabling manifestations are orthostatic hypotension (OH) and gastrointestinal dysmotility. An autoimmune pathogenesis was suggested by the subacute onset, frequent association with antecedent viral illness, occasional association with cancer or other autoimmune disorders, and spontaneous recovery in some patients. Many cases have a rapid onset, but others have a chronic progressive course.
Several clinical and experimental observations indicate that AAG is an antibody-mediated disorder. (1) About 50% of AAG patients have high levels of serum antibodies specific for the neuronal AChR in autonomic ganglia. This ganglionic (α3-type) AChR mediates fast synaptic transmission in all autonomic ganglia. Ganglionic AChR antibodies found in AAG patients are distinct from muscle AChR antibodies found in patients with MG. (2) Ganglionic AChR antibody levels correlate with severity of autonomic deficits. Patients with high antibody levels have a combination of OH, dry eyes and dry mouth, Adies pupil, gastroparesis, and neurogenic bladder. Patients with low antibody levels may have mild or restricted autonomic deficits or postural tachycardia syndrome (POTS). (3) Removal of antibodies using plasmapheresis may result in dramatic clinical improvement.
(4) Induction of ganglionic AChR antibodies in rabbits by immunization leads to experimental AAG which reproduces many of the cardinal features of the human disease. (5) Passive transfer of ganglionic AChR antibodies to mice causes transient autonomic failure. In electrophysiological studies, ganglionic AChR antibodies produce a reduction in ganglionic AChR current in cultured neuroblastoma cells, inhibit synaptic transmission in isolated autonomic ganglia due to a reduction in the amplitude of the cholinergic excitatory postsynaptic potentials. AAG is an antibody-mediated channelopathy that produces a severe, but potentially treatable, form of autonomic failure.
Department of Neurology, Kinki University School of Medicine, Osaka, Japan
Anti-ganglioside antibodies are frequently present in some autoimmune neuropathies. In particular, they are present in about 60% of the acute-phase sera of Guillain-Barré syndrome (GBS). Some of them may be directly involved in the pathogenetic mechanisms by biding to the regions where the respective target ganglioside is localized. We have recently found the presence of the IgG antibody that specifically recognizes a new conformational epitope formed by two gangliosides (ganglioside complex, GSC) in the acute-phase sera from some GBS patients. The IgG antibodies against GD1a/GD1b and/or GD1b/GT1b complexes are associated with severe GBS requiring artificial ventilation. In contrast, the binding activity of the antibody highly specific to GD1b is weaker towards GSC comprising GD1b than to GD1b alone because GD1b undergoes conformational change in that GSC. GD1b is localized in large sensory neurons in dorsal root ganglia. Monospecific anti-GD1b IgG has been shown as a causative factor of sensory ataxic neuropathy. However, GBS patients with monospecific anti-GD1b IgG do not always have ataxia. We investigated whether the reactivities of anti-GD1b IgG to such GSCs are different between GBS with ataxia and that without ataxia. As a result, the reactivities to GD1b mixed with GD1a, GT1b, GQ1b and GalNAc-GD1a were significantly more reduced in ataxic patients than in non-ataxic patients (p b 0.001). It suggests that the IgG antibodies associated with ataxia are highly specific to GD1b. Thus, highly specific anti-GD1b IgG may bind to GD1b tightly to cause ataxia in GBS. Most of the patients with Miller Fisher syndrome (MFS) have anti-GQ1b IgG antibody. Our recent investigation showed that some MFS patients have antibodies with higher titer against GSCs including GQ1b; such as GQ1b/GM1 and GQ1b/GD1a. Based on the results of anti-GSC antibody assay, there are three types of IgG antibodies in MFS; those specific to GQ1b itself, those specific to GQ1b/GM1 complex, and those specific to GQ1b/GD1a. Gangliosides along with other components as cholesterol are known to form lipid rafts, in which the carbohydrate portions of two different gangliosides may form a new conformational epitope. Within the rafts, gangliosides are considered to interact with important receptors or signal transducers. More study is needed to elucidate the mechanisms how the antibodies against gangliosides or ganglioside complexes cause nerve dysfunction. Increasing evidence suggests that CD8 + T cells play an important role in the pathogenesis of multiple sclerosis (MS). CD8 + T cells are found in greater number than CD4 + T cells in acute and chronic MS lesions, and preferential oligoclonal expansion of CD8 + T cells has been observed in the blood, CSF and brains of MS patients. Furthermore, therapeutic depletion of CD4 + T cells did not improve disease in MS patients, however, depleting all lymphocytes reduced new lesions and relapses. We have previously demonstrated in a murine model that CD8 + T cells specific for myelin basic protein (MBP) induce central nervous system (CNS) autoimmunity upon adoptive transfer. To investigate the pathogenesis of MBP-specific CD8 + T cell-mediated autoimmune disease, we generated T cell receptor (TCR) transgenic models using two different MBP-specific CD8 + T cell clones. One model exhibits strong central tolerance while the other model exhibits a unique form of tolerance that allows MBP-specific CD8 + T cells to populate the periphery without inducing disease. Using the first model, we show that negative selection is mediated in the thymus by cross-presentation of MBP by non-bone marrow derived cells, and by synthesis and direct presentation of MBP by bone marrow-derived cells. Using the second model, we demonstrate that the unique state of tolerance, which is maintained during exposure to many immunogenic stimuli, can be broken by viral infection. Our data indicate that the ability of viral infection to trigger disease depends upon expression of endogenous TCR chains on the MBP-specific TCRtransgenic T cells. Expression of a particular endogenous TCR alpha chain confers specificity to a viral epitope, and T cell activation via the virusspecific TCR allows the T cells to respond to endogenous MBP using the transgenic TCR. Interestingly, viral infection of CD4 + MBP-specific TCR transgenic mice does not trigger autoimmune disease. These data provide a potential mechanism by which infectious agents may activate myelin-specific CD8 + T cells to trigger CNS autoimmunity.
The dual-face role of alpha B-crystallin in multiple sclerosis
Hans van Noort, Delta Crystallon BV, Leiden, The Netherlands
The role of T cells in multiple sclerosis (MS) has remained somewhat confusing. On the one hand, infiltrated and locally activated T cells against one or more central nervous system (CNS) antigens are likely to be crucial in lesion formation. On the other hand, no abnormality in the T-cell repertoire of MS patients appears to exist which would explain an autoimmune attack on the CNS. These seemingly paradoxical elements can be reconciled by assuming that MS lesions do not result from an autoimmune attack by an abnormal immune system, but instead, by autoimmune provocation driven by a neurodegenerative process within the CNS itself. Data on the role of alpha B-crystallin (CRYAB) support the latter model.
In response to stress of various kinds, glial cells including oligodendrocytes produce strongly increased levels of CRYAB, whose prime intracellular role is anti-apoptotic. Even before lymphocyte infiltration is apparent in the CNS of MS patients, CRYAB accumulation occurs in glial cells, reflecting some form of neurodegenerative stress in the absence of peripheral immune elements. CRYAB is also secreted and as a chaperokine it triggers microglia to produce TNF-alpha and RANTES. By doing so, CRYAB helps recruit immune surveillance. The point is to protect the CNS. Neuroinflammation in CRYAB-deficient mice causes more damage than in normal mice, and soluble CRYAB itself prevents or inhibits neuroinflammation.
Unfortunately, however, humans are selectively immune reactive to CRYAB, much more so than to other CNS antigens. In MS patients and healthy subjects alike, marked numbers of pro-inflammatory memory T cells against CRYAB are present in the circulation, as well as anti-CRYAB antibodies. This immune-reactive state is therefore common in humans and thought to be a bystander effect of prior viral infection. Combining these data suggests that immune surveillance provoked by a stressed CNS will inadvertently recruit also CRYAB-reactive elements. Since local antigen-presenting cells in the CNS of MS patients offer high levels of CRYAB they could well trigger an inflammatory reaction which overwhelms the originally protective role of CRYAB, and creates an MS lesion.
While therefore common among humans and not the cause of MS per se, we consider CRYAB-reactive T cells to be the key elements which locally fuel an inflammatory lesion, when provoked to do so by a stressed CNS. As a possible solution, CRYAB itself may be used to eliminate the T cells against it. Controversial debate continues as to whether T cells are able to promote central nervous system repair. It has been suggested that T cell injections and vaccination strategies with encephalitogenic antigens contribute to protection from secondary damage after noninflammatory brain and spinal cord injury, however, these studies have been challenged by recent findings demonstrating that the approach includes severe detrimental effects. These contradictory data suggest that a specific molecular crosstalk between the immune system and the nervous system critically determines the ultimate outcome of T cell actions in the brain. Here, we have investigated the use of T cells and their marker cytokine IL-4 for the treatment of spinal cord injury.
Here we report that IL-4, released by T cells independent of their antigen specificity, stimulates substantial fiber regrowth in the lesioned brain and spinal cord in vivo. IL 4 receptor −/− animals, mice with selective mutations in the IL-4 signal transduction cascade, inhibitory antibodies, and delivery of IL-4 both in vitro and in vivo were used to identify this cytokine as the molecule responsible for T cell mediated axon outgrowth. We could show that IL-4-dependent AKT/MAPK pathways are crucial for T cell-mediated axon regeneration, while the classical Stat-6 pathway does not play a role. This effect was not the result of increased local release of neurotrophins but of T cells conveying regeneration via IL 4 dependent modulation of neurotrophin signaling. Locomotion analysis revealed significantly improved neurological outcome after lesion in mice treated with IL-4+ T cells.
Our results feature IL-4 as a key player in T cell mediated axon reorganization and open the door to the design of novel CNS repair strategies. Demyelination of central nervous system (CNS) axons can cause devastating neurological impairment, but is often followed by myelin repair, which restores function and protects irreplaceable nerve fibres from degenerating. For example, inflammatory demyelinating lesions of multiple sclerosis (MS) are repaired in many, but not all patients. Although strategies to promote myelin repair are eagerly sought, factors that govern remyelination in vivo have been elusive. Chemokine receptor CXCR2 facilitates inflammatory demyelination, by promoting myeloid leukocyte entry into the CNS. Blocking CXCR2 on these cells might suppress inflammatory tissue damage. During postnatal CNS organogenesis, however, CXCR2 positions oligodendrocyte progenitor cells (OPCs) in the spinal cord, to receive timely growth-factor stimulation. CXCR2 is present in clinical and experimental demyelinating lesions, raising the question whether reengaging CXCR2's developmental properties might enhance remyelination. If that were the case, inhibiting CXCR2 signalling could antagonize tissue repair.
We show that the action of CXCR2 towards non-hematopoietic cells, unexpectedly, delays myelin repair. Bone marrow chimeric mice (Cxcr2ht to Cxcr2 wt and Cxcr2ht to Cxcr2ko) were subjected to two distinct models of myelin injury. In both cases, myelin repair was more efficient in Cxcr2ht to Cxcr2ko animals. OPCs in demyelinated lesions of Cxcr2ko recipient proliferated earlier and more vigorously than in tissues from Cxcr2wt recipient animals. In-vitro demyelinated CNS slice cultures also showed faster myelin repair when CXCR2 was blocked with neutralizing antibodies or genetically deleted. We propose that CXCR2-dependent developmental cues function aberrantly during CNS myelin repair. Instead, our results suggest that CXCR2-inactivation permits optimal spatiotemporal positioning of OPCs in demyelinating lesions, to receive local proliferative and differentiating signals.
Given its actions towards inflammatory myeloid leukocytes and non-hematopoietic cells, our present findings identify CXCR2 as a unique drug target for clinical demyelinating disorders. Four different patterns of demyelination have been described in active demyelinating lesions of multiple sclerosis (MS) patients that were biopsied shortly after disease onset. These patterns were suggested to represent heterogeneity of the underlying pathogenesis. The aim of the present study was to determine if lesion heterogeneity also exists in an unselected collection of autopsy material from patients with established MS.
All post-mortem MS brain tissue available in the VU medical center was assessed for the presence of active demyelinating lesions using magnetic resonance imaging (MRI)-guided sampling and immunohistochemistry. Tissue blocks containing active demyelinating lesionsdefined by the presence of activated macrophages containing intracellular myelin proteinswere evaluated for the presence of complement and antibody deposition, oligodendrocyte apoptosis, differential loss of myelin proteins and hypoxia-like damage using histology, immunohistochemistry and confocal microscopy. Sixty-three tissue blocks from 27 MS patients contained active demyelinating lesions. Blocks with active demyelinating lesions were compared to blocks with active (non-demyelinating) and inactive lesions.
Deposition of antibodies and activated complement components was consistently associated with active demyelination. Antibodies and complement colocalized with myelin proteins in phagocytic vesicles in macrophages. This pattern was observed in all tissue blocks containing active demyelinating lesions, lesion heterogeneity between patients was not found. Preferential loss of myelin proteins, extensive hypoxialike damage and oligodendrocyte apoptosis were absent or very rare in our tissue sample.
We conclude that the immunopathological appearance of active demyelinating lesions in established MS is uniform. Initial heterogeneity of demyelinating lesions in the earliest phase of MS lesion formation may disappear over time as different pathways converge in one general mechanism of demyelination. Consistent presence of complement, antibodies and Fcgamma receptors in phagocytic macrophages suggests that antibody-and complement mediated myelin phagocytosis is the dominant mechanism of demyelination in established MS. Metalloproteinase-disintegrins (ADAMs) represent a family of membrane-anchored metalloproteinases with both proteolytic and disintegrin characteristics, playing an important role in the development and neurobiology of the nervous system. ADAMs have been implicated in ectodomain shedding of surface proteins, growth factors, and remodeling of components of extracellular matrix (ECM). In the present study, we investigated the role of ADAMs in axonal outgrowth and myelination of the peripheral nervous system (PNS).
Rat dorsal root ganglia (DRG) and Schwann cells (SCs) were studied in vitro. Expression patterns of ADAM7, -8, -10, -12, -17, -19, -28, and -33, which were predicted to be active metalloproteinases, were investigated in SCs by qPCR. Furthermore, mRNA analysis was performed in the co-culture of DRG and SCs, from the day 1 after seeding the cells up to day 21 after induction of myelination using qPCR. Protein expression was assessed by immunostaining. The involvement of ADAM10 in the process of axonal outgrowth and myelination was studied in greater details by applying the hydroxamate-based inhibitor GI254023X during the pre-myelination stage as well as myelination process. The mean length of the axons was measured and compared to the control group. Using western blotting, P0 protein expression level was used as a marker for myelin synthesis.
Among all ADAMs investigated, ADAM10 revealed the highest level of mRNA expression in SCs. ADAM10 mRNA level was up-regulated consistently (p b 0.001) in DRG-SCs co-cultures after seeding the cells. Neurons as well as SCs widely expressed ADAM10 at the protein level. In neurons, the expression of ADAM10 was exclusively limited to the axons during the pre-myelination stage in which axonal outgrowth takes place. Inhibition with GI254023X resulted in a significant dosedependent decrease in the mean length of the axons (p b 0.0001), leading to nearly no outgrowth at the highest dose. The cultures treated with GI254023X from the beginning of the seeding, significantly expressed much less P0 in comparison to those inhibited from the beginning of myelination induction and the control ones.
Our data suggest that ADAMs are differentially expressed in DRGs and SCs. Among all ADAMs investigated, ADAM10 seems to play a critical role in axonal outgrowth in the peripheral nerve.
While inflammation can damage the CNS, there is an increasing appreciation that there are beneficial aspects of neuroinflammation that can promote neuroprotection or repair. In particular, leukocytes are sources of neurotrophic factors and they could be used to deliver a multitude of neurotrophic factors into the injured CNS. The challenge of harnessing beneficial aspects of neuroinflammation is how to avoid its potential detrimental consequences.
The multiple sclerosis (MS) medication, glatiramer acetate (GA), has been shown to be an immuno-modulator through mechanisms including the generation of CD4+ T helper 2 (Th2) cells. Th2 cells produce anti-inflammatory cytokines such as interleukin-5 (IL-5), are not toxic to neurons, and they have been shown to produce BDNF in an animal model of MS, experimental autoimmune encephalomyelitis (Aharoni et al., PNAS 102:19045-19050, 2005) . Thus, there is the prospect of harnessing beneficial inflammation for repair by using cells that are generated/modulated by GA.
Remyelination may occur in spinal cord injury. The process of remyelination requires the generation of oligodendrocyte precursor cells (OPCs) that mature into myelin forming oligodendrocytes. Several growth factors are involved in the generation of OPCs, and important ones include insulin-like growth factor-1 (IGF-1), plateletderived growth factor (PDGF) and BDNF. Here, we have addressed whether GA-reactive Th2 cells or GA-treated macrophages produce these growth factors. We have also examined whether GA treatment of mice with a demyelinating injury to the spinal cord could promote the generation of OPCs and remyelination in vivo.
Our results show that GA-reactive Th2 cells and GA-treated macrophages have elevated levels of growth factors, particularly IGF-1, and that the conditioned medium from GA-reactive T cells promotes the maturation of OPCs from progenitors cultured from E15 mouse brains. Moreover, demyelinated mice injected daily with GA for the first 7 days have increased number of OPCs around the injury site at day 7 compared to vehicle-treated animals, correspondent with elevated mRNA levels for growth factors and a polarization of inflammatory markers to one that is anti-inflammatory. Finally, we determined that GA increased indices of remyelination 28 days after injury.
These experiments highlight the feasibility of promoting spinal cord repair through harnessing beneficial inflammation, using medications such as GA. Pregnancy reduces multiple sclerosis (MS) disease activity, especially during the third trimester. This mitigation is often followed by a relapse within the first 3 months post-partum. The biological mechanisms underlying this amelioration and rebound disease largely remain unclear. The Dutch MS pregnancy study aims to identify biological parameters of MS disease course during pregnancy. The first parameter addressed focused on antigen presenting cells (APC) which drive autoreactive T cells and therefore play a major role in MS disease progression.
The second parameter addressed the analysis of Treg versus T helper 17 (Th17) cell subsets during the course of pregnancy.
Using Affymetrix microarray, gene expression of CD14+ cells from pregnant MS patients and from pregnant healthy controls was compared. Using stringent statistical analysis five inflammation related genes, CD64, STAT1, JAK2, CD38 and SLAMF7 were upregulated more than twofold. Furthermore 4 genes, IL-8, FCER1A, PTX3, CXCL2 were downregulated more than twofold. The microarray data was confirmed by qPCR analysis.
Multicolor flowcytometry analysis was performed in order to identify Treg and Th17 cells. Preliminary results indicate that frequencies of Treg and Th17 are altered in pregnant MS patients.
Overall the data shows differential gene expression in APC in MS patients during pregnancy. Furthermore preliminary data suggests that pregnancy influences the numbers of Th17 and Treg cells in MS patients during pregnancy. The Dutch MS pregnancy study contributes to the further understanding of the complex MS disease course. P-selectin glycoprotein ligand-1 (PSGL-1) and alpha (1,3) fucosyltransferases (FucT), enzymes that glycosylate PSGL-1 and control its binding capacity, are involved in the migration of leukocytes in sites of inflammation. Although some previous studies have shown that PSGL-1 ligands have also a protective role in models of autoimmune diseases, some controversies emerged from studies performed in experimental autoimmune encephalomyelitis (EAE). Our goal was to clarify the role of mucin PSGL-1 and glycosylation-dependent mechanisms in EAE.
Among other METHODS: Two photon microscopy was performed in draining lymph nodes of immunized mice. Proliferation and bioplex assays for cytokine production were performed. Adhesive interactions were quantified in inflamed brain microcirculation by using intravital microscopy.
MOG35-55 specific T cells produced from FucT-VII−/− and PSGL-1 −/− mice transferred a significantly more severe disease than WT cells. Co-cultures performed with CD4+CD25+ Tregs and effector T cells showed that deficiency of PSGL-1 and FucT-VII leads to a marked decrease of suppression capacity on the proliferation and production of pro-inflammatory cytokines by effector T cells. Two-photon microscopy experiments performed inside draining lymph nodes of EAE mice show that the locomotion of FucT-VII and PSGL-1 deficient MOG-specific T cells is significantly decreased while the arrest coefficient is increased when compared to WT effector T cells suggesting that the lack of PSGL-1 and FucT activity leads to prolonged contacts with DCs and increased activation of effector T cells. Moreover, FucT-VII deficient Tregs displayed altered behavior by increased average velocity and mean motility coefficient. In addition to these dysfunctions, Tregs from FucT-VII−/− and PSGL-1−/− mice presented decreased migration capacity to inflamed brain when compared to WT Tregs. Moreover, intravital microscopy experiments showed that FucT-VII and PSGL-1 deficient Tregs display a dramatic decrease of adhesive interactions in inflamed brain microcirculation. Finally, Tregs deficient in PSGL-1 and FucT-VII have a reduced capacity to suppress EAE when compared to WT cells.
Our data suggest that PSGL-1 and fucosyltransferase activity have multiple roles in the censorship of EAE by CD4+CD25+ Tregs by participating to cell-cell contacts required for efficient suppression in lymph nodes and by controlling Treg migration into the inflamed brain. 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 decreased during pregnancy with the greatest reduction in disease observed during the third trimester. We have studied pregnancy in the murine model of MS, experimental autoimmune encephalomyelitis (EAE). Disease severity is significantly reduced when pregnancy is induced during established EAE. We also observed a post-partum flare in disease scores, with EAE suppression only occurring during the gestation period. A serum factor has been implicated as the primary mediator of disease suppression. Exosomes are small lipid-bound microvesicles that are found in peripheral blood and are important in intercellular communication. Exosomes are able to modulate the immune response by relaying molecular signals from their cell of origin to target cells bearing specific adhesion molecules.
We have shown that pregnancy-associated exosomes are able to suppress antigen-specific T cell proliferation. We have also observed that the concentration of circulating serum exosomes is increased during pregnancy. Exosomes are hypothesized to dampen immune responses during pregnancy to protect the semiallogeneic fetus, causing a global immunosuppressive state. Thus, we examined exosomal proteins that are known to influence T cell activity such as Fas ligand, heat shock protein 70, TGF-β, IL-10, IL-17, and IFN-gamma.
Harnessing the mechanism by which exosomes suppress immunity, and consequently EAE, can have extraordinary implications for therapy development in MS. (Supported by NIH grant AI 064320, National MS Society grant RG3272 and NIH grant T32 AI055411).
3 -CD4+FOXP3+ T regulatory cells inhibit experimental autoimmune encephalomyelitis via distinct IL-10 dependent and IL-10 independent mechanisms Davidson Todd⁎ 1 , Shevach Ethan 1 1 National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD, United States CD4+Foxp3+ T regulatory cells (Tregs) are important for controlling a diverse array of immunological responses. Adoptive transfer of these cells into mice results in protection from experimental autoimmune encephalomyelitis (EAE) while depletion of this population results in exacerbation of disease. Tregs have been shown to produce IL-10, a cytokine with immunosuppressive potential that may be important in their ability to inhibit disease in vivo.
Adoptive transfer of IL-10−/− T-regs does not inhibit EAE, underlining the importance of this cytokine in Treg mediated suppression. Tracking of adoptively transferred congenically labeled Tregs demonstrated their presence not only in the lymph nodes and spleen, but also in the CNS at time-points both before and after the onset of clinical symptoms, suggesting a role in preventing immune responses in situ. Analysis of IL-10 production by intracellular staining indicated that Tregs do not produce IL-10 in either lymph nodes or spleen but rather express this cytokine almost exclusively in the CNS. Consistent with this result, we found antigen specific TGF-β induced Tregs are capable of limiting the proliferation and activation of effector cells in the draining lymph nodes of immunized mice, but that this effect does not require IL-10.
Thus, Tregs inhibit EAE via two distinct mechanisms. The first being an IL-10 independent mechanism whereby Tregs inhibit priming in lymph nodes. The second, IL-10 dependent mechanism requires production of this cytokine by Tregs within the CNS.
A rational approach to treat autoimmune diseases such as multiple sclerosis (MS) requires the restoration of self tolerance. We hypothesize that over-representation of self-constituents in the thymus is required to avoid autoimmunity and by extension, MS. As such we have exploited thymus-based tolerance mechanisms to purge autoreactive T-cells and to reinstall immune tolerance. Thus the central aim of this study was to assess whether intrathymic delivery of a recombinant lentiviral vector (LV) engineered to overexpress myelin oligodendrocyte glycoprotein (MOG), a key autoantigen in MS, ameliorates the signs of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, an animal model for MS.
Animals were intrathymically injected with concentrated lentiviral vectors engineered to express MOG (LV-MOG) or the hydrogen potassium ATPase -an irrelevant autoantigen (LV-HKATPase) or PBS. As a further control aged-matched naive animals were also used. Animals were subsequently immunized with the encaphalotogenic MOG35-55 peptide 12 weeks post-treatment and monitored for a further 6 weeks. Thereafter, animals were humanely killed and tissues and organs analyzed. Neurological assessment indicated that untreated and control animals injected with LV-HKATPase or with PBS, developed fulminant symptoms of EAE after immunization. In contrast, animals receiving the therapeutic LV-MOG vector manifest significantly reduced symptoms. Histological analysis of the brain and spinal cords from control animals showed inflammatory lesions that correlated with severe areas of demyelination and axonal loss. Notably LV-MOG-treated animals demonstrated significantly fewer cellular infiltrates that corresponded with a lower number of demyelinating areas with little, or no axonal loss. Functional in vitro activity of MOG35-55-stimulated T cell responses from LV-MOG-treated animals demonstrated a 34% to 50% decrease in proliferative responses in comparison to controls. Finally, quantification of cytokine immunoreactivity in activated spleen cell supernatants revealed that the T-cell pro-inflammatory differentiation cytokine, IL-6, was significantly reduced in LV-MOG treated animals in comparison to controls.
Collectively these data indicate that intrathymic delivery of a LV-MOG represents a novel form of tolerance induction for the treatment of MS. Our approach further adds to the armamentarium of nontoxic gene-based strategies for the treatment of this debilitating disease. Th17 cells represent an highly proinflammatory population that have been recently shown to play a crucial role in autoimmune diseases, such as Multiple Sclerosis. The break of the balance that generally exists between the effector lymphocytes and the T regulatory compartment, has been associated with autoimmunity. We have recently detected a strong immunosuppressive function within the CD4+CD25HighCD39+ subpopulation of Treg cells in human. Furthermore we showed that this subset of T cell was decreased in Multiple Sclerosis patients. The aim of this study is to characterize effector T cell subsets which release proinflammatory cytokines such as IL17 and IFNgamma as well as CD39+ Treg cells in healthy individuals age matched with patients affected by the relapsing-remitting form of Multiple Sclerosis.
We performed a multicolor cytometric analysis and assays in vitro in order to phenotipically and functionally characterized the cells populations mentioned above. Surprisingly we found, within the CD25high cells, IFN-gamma+ and IL-17+ T cell lymphocytes, suggesting that recently activated proinflammatory cells contaminate the pool of Treg cells in the CD25high compartment. Interestingly these Th1 and Th17 cells were confined in the CD39 negative T cell population thus confirming that CD39 expression represent a valuable marker for Treg cells within the CD25high compartment. We then monitored the frequency of both CD39-Th1 and Th17 cells and CD39+ Treg cells in healthy donors and in MS patients. We found that the decrease of the frequency of the CD39+ cells among the CD25high subset in these patients is associated to an increase in the CD39-CD25high effector cells releasing proinflammatory cytokines.
Our results highlighted a contamination within the CD25high regulatory compartment by recently activated effector T cell lymphocytes in human. Furthermore the analysis of the frequency of these populations suggest that in MS patients the equilibrium between effector cells and regulatory subsets is shifted towards the proinflammatory cytokine producing cells. The implications of our findings will be discussed.
Esposito Marianna⁎ 1 , Ruffini Francesca 1 , Bellone Matteo 1 , Martino Gianvito 1 , Furlan Roberto 1 1 San Raffaele Scientific Institute, Milano, Italy
Rapamycin is a novel macrolide immunosuppressive drug, widely used in preventing clinical allograft rejection, and has been previously reported to efficiently suppress autoimmune diseases by increasing CD4+Foxp3+ Treg cell number and functionality. While its effect has been extensively characterized in vitro, the in vivo effect on the level and function of Treg cell population is still awaiting elucidation.
We have treated SJL mice affected by relapsing EAE induced by immunization with PLP139-151, with rapamycin using different administration routes, including oral, and schedules, and studied its effect on clinical, pathological, and immunological parameters.
We found that rapamycin treatment, independently from the route and schedule of administration, significantly ameliorates the clinical course of EAE. The protective effect was confirmed also by the finding of reduced demyelination and axonal loss. The therapeutic effect of rapamycin was associated to a decrease of infiltrating CNS cells, especially macrophages, but not with an increase of Treg cells, or in the CNS or in peripheral lymphoid organs. Rapamycin administration, however, was associated with decreased proliferation and inhibited release of IFN-gamma and IL-17 by antigen-specific T cells in draining lymph nodes and spleens from EAE rapa-treated mice as compared to the control group.
These pre-clinical results indicate rapamycin as an efficient drug in controlling experimentally induced autoimmune responses, provide evidence of the immunological mechanism, and suggest this compound as a plausible candidate for the treatment of multiple sclerosis. Natalizumab is a humanized monoclonal antibody directed against alpha-4-integrin, which reduces the relapse rate and MRI evidence of disease activity in patients with multiple sclerosis (MS) by blocking the transmigration of T cells and B cells into the central nervous system (CNS). Little is know about effects of natalizumab on the activation stage and function of circulating leukocytes.
We used flow cytometry to study the phenotype of peripheral blood mononuclear cells (PBMCs) in 28 patients with relapsing-remitting MS (21 females and seven males) before and during treatment with natalizumab (Tysabri, Biogen Idec Inc; 300 mg IV once a month). Blood was obtained at baseline and immediately before natalizumab infusions at month 1, 3, 6 and 12. None of the patients had been treated with any immunomodulatory drugs for at least 1 month before inclusion in the study. PBMCs were stained directly ex vivo and after in vitro stimulation with anti-CD3 and anti-CD28 (for studies of CD4+ and CD8+ T cells) or IFN-g and LPS (for studies of CD19+ B cells and CD14+ monocytes). The pretreatment measurement was compared to the on-treatment measurements using a mixed effects model. We observed that the expression of IFN-g increased on all cell types under study (CD4+, CD8+, CD19+ and CD14+ cells) early after initiation of natalizumab treatment and remained elevated throughout the follow-up (p b 0.05-0.005). In addition, CD4+ T cells expressed higher levels of TNF-a (p b 0.05), while stimulated CD19+ B cells displayed increased expression of TNF-a, IL-6 and IL-23 (p b 0.05-0.005) during natalizumab treatment. The expression of CCR5 and CCR6, two chemokine receptors associated with previously activated effectormemory cells were also increased during natalizumab treatment (p b 0.005-0.0005). The frequency of CCR6+/CD4+ T cells doubled during the first month of treatment, while increased frequencies of CCR5+/CD8+ T cells were detected later during treatment.
In conclusion, we observed increased numbers of leukocytes expressing proinflammatory cytokines in peripheral blood during natalizumab treatment, presumably due to sequestration of activated cells in the peripheral circulation. An accumulation of potentially aggressive leukocytes in blood may lead to rapid influx of inflammatory cells to the CNS after discontinuation of natalizumab treatment and may be of clinical concern.
2 -Different chaperone usage by IL-12 AND IL-23 during their assembly reveals novel targets for intervention with cytokine secretion in neuroinflammation IL-12, IL-23 and IL-27 are members of a unique dimeric cytokine family. IL-12 is composed of disulphide-linked p40 and p35 while IL-23 is composed of disulphide-linked p40 and p19. In addition, the p40 subunit can homodimerize to form p80, a cytokine with as yet poorly understood chemotactic activity in humans. The promiscuity of the p40 subunit is indicative for complex regulatory mechanisms operating at the level of partner selection, assembly and maturation during folding in the endoplasmic reticulum (ER) prior to secretion. IL-12 is involved in Th1 differentiation while IL-23 induces development of IL-17-secreting CD4+ T cells. Both cytokines are linked to stages in the development and maintenance of CNS inflammation, such as seen in MS or EAE. All known therapeutic agents, including the mAb ustekinumab or drugs interfering with transcription of the p40 subunit via inhibition of NFkappab, block simultaneously IL-12, p80, p40 and IL-23 via the p40 subunit. As yet, no drugs are available that can selectively interfere with either IL-12, p80 or IL-23.
We investigated the assembly stages of these cytokines using in-lab established recombinant cell lines specifically producing IL-12, p80 or IL-23. We identified geldanamycin and its more soluble version 17-AAG as drugs capable of blocking secretion of IL-12 and p80, through a mechanism involving the ER chaperone GRP94 rather than the cytosolic homologue HSP90 (IC50 50 nM). Geldanamycin/17-AAG did not inhibit IL-23 secretion nor secretion of p40 monomers at tested concentrations up to 1 μM. Geldanamycin is a benzoquinone ansamycin that has been shown to inhibit HSP90 and GRP94. In immunoprecipitation experiments geldanamycin/17-AAG inhibited interaction of p80 with GRP94. siRNA knockdown of GRP94 completely abolished p80 secretion, but not that of IL-23. Other GRP94 inhibitors including herbimycin A and radicicol also inhibited p80.
Geldanamycin research has concentrated on its ability to target cancer cells susceptible to Hsp90 inhibition. Our work shows that the ER chaperone GRP94 is vital for folding of IL-12 and p80 but dispensible for folding of IL-23. Experiments are underway in our lab to test geldanamycin/17-AAG in EAE models of MS. A full comparative proteomic analysis of the ER "foldosomes" of IL-12, p80, IL-23 and IL-27 is being performed. Natalizumab, a humanized anti-a4 integrin monoclonal antibody, reduces relapses and disease progression in patients with multiple sclerosis (MS). Whereas its presumed mode of action is inhibition of T cell/monocyte entry into the brain, little is known about its specific effect on B cells, which are increasingly recognized to participate in MS pathogenesis.
We obtained serial blood samples from 17 patients before and during natalizumab therapy for MS for up to 16 months, and blood samples from 10 untreated MS patients and 13 healthy donors. We determined numbers of mature and immature lymphocyte subsets by flow cytometry for CD3, CD4, CD8, CD19, CD138, and CD10 in 111 samples. We analyzed marker transcripts for immature hematopoietic cells by quantitative PCR for CD34, Vpre-beta-1 (pre-B lymphocyte gene 1) and DNTT (terminal deoxynucleotidyltransferase) in 65 samples.
Natalizumab therapy increased CD19+ mature B cells more than other lymphocytes/monocytes in blood (2.8-fold versus1.3-1.8-fold increase in cells/µl; pb 0.01)). Even greater was the increase of immature CD19+ CD10+ pre-B cells (7.4-fold; p b 0.01). This pattern remained stable during treatment for up to 16 months. Transcripts of lymphocyte precursors (Vpre-beta-1 and DNTT) were elevated more than transcripts for CD34.
Circulating B cells and especially pre-B cell are most prominently elevated among the studied immune cell subsets, raising the possibility that the effects and side effects of natalizumab are partly mediated by actions on B cells. This longitudinal systematic gene expression study focuses on multiple sclerosis (MS) and the effects of medication by subcutaneous recombinant Interferon-beta-1a (IFNβ-1a). It is the aim to attain human peripheral blood derived gene transcripts that are differentially regulated throughout therapy and to infer accessory regulatory pathways of the applied drug.
Employing the Affymetrix HGU133Plus2.0 array (55.000 transcript species), we performed a full genome study monitoring 12 RRMS patients receiving IFNβ-1a (s.c.) over a period of 2 years. Peripheral blood samples were collected and directly processed (Ficoll) before first treatment (t0) and correspondingly before each upcoming injection after 2 days (t1), 4 weeks (t2), 1 (t3) and 2 (t4) years. Transcriptome analyses were realized including data curation by MAS 5.0 (Absent-and No change-calls, respectively), probeset quality by GeneAnnot (specificity and sensitivity) and a self-made algorithm (intensity dependent fold change criterion) as well as available R software packages (paired t-test, principal component analysis). Gene regulatory network inference and transcription factor binding site identification (TRANSFAC, JASPAR) analyses are underway.
Consistently upregulated throughout analyzed time points were four genes, namely interferon-induced protein 44 (IFI44) and SP110 nuclear body protein (alias IFI41), protein associated with topoisomerase II homolog 2 (PATL2; DNA transcription and replication related), and CD20 antigen-like 1 (MS4A4A) (3 probesets).
Groups of functionally related genes changed over time in the majority of analyzed individuals. Whereas earlier during therapy mainly typical IFNβ induced genes were filtered, such as IFIT-1, -2, -3, -5, MX-1, -2, OAS-1, -2, -3, -L, other sets of up-regulated genes (FCRL1, TCL1A) dominate at years 1 and 2. Only HERC6 and JUP were consistently upregulated until 1 year. Of interest are up-regulated killer cell genes (KLRF1, KLRD1, KIR2DL3, KIR2DS2, KIR3DL-1, -2, -3) at 2 days, and downregulated integrin genes (ITGA2B, ITGB-3, -5) at 1 month post-therapy.
Filtered genes represent promising candidates to be actively involved in IFNβ regulated effects. The dynamic expression changes of individual RNA profiles reflect the intertwining of disease and therapy course complexities.
5 -Diversity in the major ass B-cell epitope among different mammals: Affect on vaccine design, safety, and efficacy of immunotherapy for Alzheimer's disease Cribbs David⁎ 1 , Head Elizabeth 1 , Vasilevko Vitaly 1 1
The failure of the first immunotherapy trial in Alzheimer's disease (AD) patients, due to meningoencephalitis in a few patients that received the AN1792 vaccine has provided an opportunity to develop novel second-generation vaccines that reduce the potential for adverse events. The translational effectiveness of immunotherapy for AD is likely to be dependent on how well animal models predict the type of immune response that will occur in humans. Notably, the evidence from the AN1792 clinical trial indicates that the overwhelming antibody response was to the free N-terminus of the amyloid-beta (Aβ) peptide (DAE). However, previous studies in mice identified an internal sequence, EFRH, near the N-terminus as the major B-cell epitope in Aβ. Importantly, anti-Aβ antibodies generated in mice bind the amyloid precursor protein (APP), which is expressed on the surface of cells, whereas the antibodies generated in the AN1792 clinical trial do not recognize APP. Thus differences in the B cell epitope may have significant consequences for the efficacy, as well as safety of immunotherapy.
To explore the diversity of immune responses in mammals, we immunized mice, rats, guinea pigs, rabbits, goats, and dogs with the AN-1792 vaccine. We then performed fine epitope mapping to determine the major anti-Aβ B-cell epitopes, and Western blot analysis to determine whether the anti-Aβ antibodies cross-reacted with amyloid APP. Surprisingly, we found that the major anti-Aβ B-cell epitope in mice, rats, a goat and guinea pigs is located in an internal sequence of Aβ1-42, and serum from these species recognizes APP on a Western blot. Conversely, anti-Aβ antibodies from monkeys, canines and rabbits all closely resembled the reported immune response in humans by recognizing only the free N-terminus of Aβ1-42, which is dependent on the free aspartic acid at position one of the peptide. As expected, the serum from these species does not recognize APP by Western blot.
Thus, careful selection of animal models for testing new vaccine candidates may facilitate effective translation to human clinical trials. Moreover, developing immunogens that induce neo-epitope antibodies that recognize free N-or C-terminal, or naturally occurring truncated forms of Aβ, but not APP, may improve the safety as well as the efficacy of new active immunization approaches. Neural stem/precursor cells (NPCs) have the capacity to self-renew and generate functional differentiated cells (e.g., neurons, astrocytes and oligodendrocytes). Due to their plasticity, NPCs could potentially be implemented in cell-based therapy to treat neurodegenerative diseases, such as multiple sclerosis. Besides the beneficial role provided by cell replacement, NPCs exert neuroprotection from inflammatory damage acting through multiple mechanisms alternative to cell replacement. Transplantation of NPCs in mice with experimental autoimmune encephalomyelitis (EAE), has been demonstrated to ameliorate the disease. NPCs injected intravenously or subcutaneously migrate in the central nervous system (CNS) but also accumulate, survive and persist in non-CNS sites. Undifferentiated NPCs are found within lymph nodes even after 100 days from transplantation. At these peripheral districts, NPCs seem to have immunomodulatory effects. Indeed, NPCs inhibit the activation of dendritic cells (DC) and prevent the expansion of antigen-specific encephalitogenic T cells in vitro.
To elucidate the crosstalk between NPCs and cells of the immune system, we are performing the ex vivo characterization of EAE mouse lymphnode DCs. DC subsets from different body districts of NPC-injected or not treated mice were analyzed, especially for their activation state. Given the extensive data and insights obtained from the mouse system, our aim is to extend these studies to human NPCs from fetal human brain, also with the perspective of a clinical trial. Immunomodulatory properties have already been demonstrated for human mesenchymal stem cells, although the exact mechanisms remain elusive.
There are clearly other genes that are crucial in the development of HSE that have yet to be revealed. The identification of other genes involved in HSE will present important biological and medical implications, providing a dissection of immunity to HSV-1 in natural conditions and a rational understanding of HSE pathogenesis.
Hosking Martin⁎ 1 , Lane Thomas 1 1 University of California, Irvine, Irvine, United States Inoculation with the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice results in an acute encephalomyelitis associated with an immune-mediated demyelinating disease. JHMV infection induces transcription of the chemokine receptor CXCR2 and signaling ligands CXCL1 and CXCL2 during acute and chronic disease yet the functional significance of these molecules to either host defense or defense is not defined. Therefore, the goals of this study were to define the functional role of ELR+ chemokines in host defense and disease following viral infection of the CNS.
Treatment with anti-CXCR2 antibody during acute disease abrogated blood-brain barrier (BBB) breakdown associated with diminished MMP-9 expression and virus-specific T cell infiltration correlating with elevated viral titers in the brain and increased mortality. Silencing CXCR2 signaling during chronic disease was associated with increased mortality independent of either viral recrudescence, modulating leukocyte entry into the CNS, or affecting the severity of demyelination. However, anti-CXCR2 treatment resulted in increased numbers of TUNEL-positive oligodendrocytes and oligodendrocyte precursor cells. Cultures of oligodendrocytes were protected from apoptosis in response to exposure to MHV following inclusion of the CXCR2 ligands CXCL1 and CXCL2 in a dose-dependent manner.
These findings reveal previously unrecognized and differential roles for ELR+ chemokines in host defense by increasing BBB permeability during acute disease and protecting cells of the oligodendrocyte lineage from apoptosis during a chronic neuroinflammatory demyelinating disease. The migration of lymphocytes into the central nervous system (CNS) during viral encephalitis is hindered by the blood-brain barrier (BBB) such that most infiltrating cells remain localized to perivascular spaces. This sequestration of leukocytes away from the parenchyma is believed to protect the CNS from immunopathologic injury. Infections of the CNS with highly cytopathic neurotropic viruses, such as West Nile virus (WNV), however, require the parenchymal penetration of T lymphocytes for virus clearance and survival, suggesting that perivascular localization might hinder antiviral immune responses during WNV encephalitis.
Using human and murine brain specimens from individuals with WNV encephalitis, we evaluated the expression of CXCL12 and its receptor, CXCR4, at the BBB and tested the hypothesis that inhibition of CXCR4 would promote T lymphocyte entry into the CNS parenchyma and increase viral clearance. Antagonism of CXCR4 significantly improved survival from lethal infection through enhanced intraparenchymal migration of WNV-specific CD8+ T cells within the brain, leading to reduced viral loads and, surprisingly, decreased immunopathology at this site.
The benefits of enhanced CD8+ T cell infiltration suggest that pharmacologic targeting of CXCR4 may have therapeutic utility for the treatment of acute viral infections of the CNS.
6 -Both TLR2 and TLR4 are required for the effective immune response in Staphylococcus aureus-induced experimental murine brain abscess - Toll-like receptors (TLRs) play central roles in the innate reaction to bacterial products, and transmit specific immune responses against these pathogens. TLRs are expressed on numerous cell types including innate immune cells, and on astrocytes, neurons, and microglial cells of the CNS. Lipoproteins and lipopolysaccharides are specifically recognized by TLR2 and TLR4, respectively. We examined the in vivo role of TLR2 and TLR4 in Staphylococcus (S.) aureus-induced brain abscess.
After intracerebral inoculation with bacteria, we performed survival studies followed by histopathological qualitative and flow-cytometrical quantitative as well as molecular qPCR studies on relevant chemokines and cytokines.
Phenotypically, 87% of TLR2−/− mice, and 43% of TLR4−/− mice died, while all WT mice recovered. Clearance of bacteria from the CNS was significantly delayed in TLR2−/− mice as compared to TLR4−/− and WT animals. Recruitment of granulocytes and macrophages to the CNS, as well as microglial activation and expansion was upregulated in TLR2−/− mice. While inflammation persisted especially in the CNS of TLR2−/−, but also of TLR4−/− mice, WT mice terminated the infection more effectively.
Collectively, these data show that the immune response to experimental S. aureus-induced brain abscess depends crucially on the recognition of S. aureus by TLR2, but that TLR4 is also required for an optimal intracerebral immune response in this disorder. Although there is no curable treatment for Alzheimer disease (AD), based on the growing understanding of the pathological mechanisms, several novel therapeutic strategies are being developed, and it is widely accepted that Aβ immunotherapy is one of the potential methods. Active and passive Aβ immunotherapies can reduce AD-like pathology and improve cognitive performance in animal models of the disease and potentially in AD patients. Hock et al. (2004) suggested that tissue amyloid plaque immuno-reactive (TAPIR) antibodies correlated well with the slower cognitive decline in AD patients who had received active immunization with Aβ and aduvant. Thus, we wished to develop such monoclonal antibody for passive immunization therapy in AD.
We used a hybridoma technique to develop a TAPIR-like anti-human Aβ mouse monoclonal antibody, characterized it in vitro and evaluated its therapeutic effects on 18 months old APP transgenic Tg2576 mice by intraperitoneal administration of the antibody for 8 weeks. The obtained monoclonal antibody, 3.4A10 was an IgG2b isotype, and recognized Nterminal portion of Aβ1-42 without binding denatured or native AβPP fragments. It had higher affinity to Aβ1-42 than to Aβ1-40 (the dissociation constant was 5.64 × 10-11 M and 3.77 × 10-8 M, respectively). It stained preferably the peripheral part of senile plaques of AD brain sections and recognized the plaque core less than 4G8 (7.5% versus 14.93%, P b 0.05). It inhibited the Aβ1-42 fibril formation as well as degraded pre-aggregated Aβ1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. It decreased amyloid burden compared to the control group by a quantitative image analysis (P b 0.05) and significantly reduced Aβ42 levels rather than Aβ40 levels in brain lysates by a quantitative ELISA assay as well as the Aβ⁎56 oligomer (12mer) in TBS fraction of the brain lysates by western blot. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia after 8 weeks administration. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage examined by Berlin blue staining.
3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody to Aβ, and has a potential of therapeutic application for AD. Recent genetic studies show an independent role of HLA class-I genes in conferring susceptibility to chronic human inflammatory diseases such as type-1 diabetes and multiple sclerosis (MS). These data provide strong incentive to further investigate the contribution of MHC class-I-restricted CD8 T cells in the induction and development of these diseases. We are investigating the contribution of MHC class-I restricted CD8 T cells in mouse models of central nervous system inflammation in which CD8 T cells specifically target oligodendrocytes, the primary cellular target of MS lesion.
To assess the pathogenic impact of CD8 T cells on oligodendrocytes, we generated mice, referred as double knock in (DKI) mice, in which the well-studied model antigen influenza hemagglutinin (HA) is selectively expressed on oligodendrocytes.
Introduction of HA-specific CD8 T cells by TCR transgenesis did not result in any sign of central or peripheral tolerance, or any CNS pathology in the DKI animals. The exclusive expression of HA in oligodendrocytes, and the resulting lack of T-cell silencing, is very reminiscent of some natural myelin self-antigens such as MOG. Transfer of pre-activated HA-specific CD8 T cells from CL4 TCR transgenic mice in DKI recipients led to inflammatory lesions in the optic nerve, spinal cord, and brain. These lesions associating CD8 Tcell infiltration with focal loss of oligodendrocytes, demyelination, and microglia activation, were very reminiscent of active MS lesions. In order to trace these HA-specific CD8 T cells in vivo, we labeled them with green florescent protein (GFP). Staining for GFP and Granzyme B revealed that HA-specific Tc1 cells containing Granzyme B granules were detected within CNS lesions of DKI mice, some of the Granzyme B+ T cells were found in close apposition to carbonic anhydrase II or cyclic nucleotide 3′ phosphodiesterase+ oligodendrocytes. We also detected GFP+ Tc1 cells with polarized Granzyme B+ granules facing an oligodendrocyte, suggesting directed degranulation.
These findings can provide important new insights with regard to CNS tissue damage mediated by CD8 T cells specific for a sequestered antigen and in extension for the understanding of the role of CD8 T cells in human MS.
3 -Tim-3:Galectin-9 pathway protects against CNS autoimmunity by expanding myeloid-derived suppressor cells Tim-3 is a cell surface molecule expressed on IFN-g-producing Th1 cells; upon interaction with its ligand, galectin-9, it terminates Th1 immunity. Tim-3/Tim-3 ligand interactions also play an important role in immune tolerance as mice treated with Tim-3 Ig fusion protein and Tim-3−/− mice cannot be tolerized. Here we sought to study more precisely the mechanism by which Tim-3 induces tolerance in the immune system.
We therefore generated mouse strains that overexpress either Tim-3 or galectin-9. We find that in both strains T cell responses are suppressed and that this is associated with an increased frequency of CD11b+Ly-6G+ cells that exhibit morphology consistent with myeloidderived suppressor cells (MDSC). We further find that Tim-3 transgenic mice are protected from Experimental Autoimmune Encephalomyelitis (EAE), a murine model for multiple sclerosis (MS), and that protection from disease is associated with an increased frequency of MDSC among central nervous system (CNS)-infiltrating cells. Lastly, we find that loss of Tim-3 reverses the MDSC expansion observed in Galectin-9 transgenic mice and restores normal immune responses.
MDSC accumulate in large numbers in cancer patients and in tumor-bearing mice in which they are potent inhibitors of anti-tumor immunity. Accumulation of MDSC has also been observed after exposure to bacterial, parasitic, and viral antigens. Despite the biological importance of MDSC in regulating immune responses, the molecular pathways that expand and regulate MDSC are unknown. It is known that IFN-g plays an important role as MDSC fail to accumulate in IFN-g Receptor−/− mice and require IFN-g for activating suppressor functions. Here, we have made a novel observation that the Tim-3/galectin-9 pathway plays a role in the expansion and/or activation of MDSC. As Tim-3 is a cell surface receptor expressed on IFN-g secreting Th1 cells, our findings are consistent with the previous studies implicating IFN-g in MDSC-mediated immune suppression. Since MSC have been shown to divert immune responses in autoimmune, cancer, and infectious diseases, targeting of the galectin-9-Tim-3 pathway may represent an important therapeutic target in many immune-mediated diseases.
4 -CNS-specific, autoreactive CD8+ T cells have a regulatory role in autoimmune demyelination -York Nathan⁎ 1 , Mendoza Jason 1 , Benagh Andrew 1 , Firan Mihail 1 , Karandikar Nitin 1
A great deal of our current knowledge about the immune pathogenesis of multiple sclerosis (MS) derives from work in its murine model, experimental autoimmune encephalomyelitis (EAE). The vast majority of studies in MS and EAE have focused on the role of CD4+ T-cells in these diseases, with the underlying assumption that they are CD4+ Th1-mediated and Th2-regulated autoimmune diseases. However, several reports from us and others have implicated an important role for CD8+ T-cells in these diseases.
In this study, we specifically dissected the role of autoreactive, CNSspecific CD8+ T-cells in EAE. Similar to our observations in human MS, we saw robust induction of CNS-specific CD8+ T-cell responses in several established models of EAE. However, unlike some recent reports, these CD8+ T cells, when purified, were unable to adoptively transfer disease into naïve mice. Thus, in vitro activated MOG35-55-specific CD4+ T-cells, but not CD8+ T-cells, were capable of adoptively transferring EAE to naïve B6 mice. In contrast, transfer of MOG-stimulated CD8+ T-cells resulted in inhibition of EAE induction, suggesting a regulatory, rather than pathogenic, role for these cells. MOG-loaded target cells were specifically killed in MOG-immunized mice in a CD8+ T-cell-dependent manner. Both CD4+ T cells as well as CD4-depleted APC were appropriate cytolytic targets of MOG-specific CD8+ T cells, suggesting a cytotoxic/ suppressor mechanism for these cells.
In conclusion, our studies reveal a novel immune regulatory function for CNS-specific CD8+ T-cells that are reproducibly generated during the induction of EAE. The neuropathology of Parkinson's disease (PD) includes microglial inflammatory responses and T cell modulation of disease. We previously showed that nitration of alpha-synuclein creates novel antigenic epitopes capable of inducing both innate and adaptive immune responses that exacerbate PD pathobiology. We now demonstrate that the pathogenic activities of nitrated alpha-synuclein-mediated immunity are attenuated by CD4+CD25+ regulatory T cells (Tregs).
Mice immunized with nitrated alpha-synuclein generate robust T cell proliferative and pro-inflammatory secretory responses, and transfer of these cells to MPTP-intoxicated recipients exacerbates microglial-associated neurodegeneration. We now show that transfer of T cells from mice injected with tolerogenic doses of vasoactive intestinal peptide (VIP) to MPTP-intoxicated recipients attenuates microglial activation and promotes neuronal survival. These T cells also show enhanced regulatory function and anti-inflammatory secretory responses. Surprisingly, we found that pooled splenocytes from synuclein-immunized and VIP-injected donors afforded greater neuroprotection upon adoptive transfer to MPTP-intoxicated recipients than those from VIP-treated donors alone. This was due in part to greater suppressive capabilities of Tregs within the population.
Using both proteomic and cell biological assays, we found that Tregs were capable of modulating microglial neurotoxic activities through regulation of phagocytosis and induction of redox-active enzymes and bioenergetic proteins in activated microglia while suppressing reactive oxygen species and nuclear factor kappa B activation.
The data demonstrate that the effects of Tregs on nitrated alphasynuclein-mediated immune activities are multifaceted and of therapeutic benefit.
6 -Primary oligodendrocyte death does not cause autoimmunity - The presence of axon growth inhibitors in the adult CNS also appears to play a prominent role in limiting axonal regeneration after CNS injury. Growth inhibitors include both CNS myelin-derived and astrocyte glial-scar derived proteins. The Nogo protein is a significant component of this inhibition, since CNS myelin lacking Nogo possesses reduced axonal growth inhibition. Axonal regeneration and improved recovery is observed in certain strains of genetically engineered mice lacking the Nogo protein after spinal cord injury (SCI). Since the degree of regeneration varies with age and mutation, Nogo appears to collaborate with other factors appear to determine the degree of fiber regeneration after SCI. In mice lacking two other myelin proteins, myelin associated glycoprotein (MAG) and olgodendrocyte myelin glycoprotein (OMgp), as well as Nogo, there is greater recovery after axonal damage in the spinal cord.
An axonal receptor for Nogo (NgR1) has been identified. This one receptor also mediates inhibition of axonal outgrowth by two other myelin inhibitors, MAG and OMgp. A short NEP1-40 peptide functions as a NgR antagonist of Nogo function. Administration of this peptide promotes significant axonal regeneration in the rodent SCI. Moreover, this regeneration allows improved walking function and the treatment succeeds even when the compound is administered systemically, up to a week after the injury. To block all NgR function, the extracellular domain of the receptor was delivered to mice with SCI by transgenic means and to rats with SCI intrathecally. The degree of fiber sprouting was moderately greater than with the selective NEP1-40 blockade. Mice lacking NgR1 also show enhanced plasticity of the raphespinal and rubrospinal (but not corticospinal) fiber systems after spinal cord injury. In contrast to poor regeneration of cut corticospinal fibers, the growth of contralateral spared corticospinal fibers is robustly increased in mice lacking NgR1. Further work demonstrates that Nogo/NgR blockade after stroke promotes axonal sprouting and improves functional neurological recovery in rodents. While these myelin inhibitors limit axon regeneration after injury they play a physiological role in limiting brain experience-dependent plasticity.
Advances in understanding the molecular determinants of axonal growth within the adult CNS are likely to lead to therapeutic methods to promote neurological recovery after acquired injuries of the brain and spinal cord. Clinical trials of oncolytic viruses (OVs) for cancers have revealed that these agents are well tolerated. However, clear-cut evidence of efficacy has been elusive. A fundamental issue continues to limit the efficacy of cancer virotherapy: the response of the host to the ongoing viral infection. This response occurs at the level of intracellular tumor defenses to the viral infection, extracellular stromal reactions to the propagating virus, and, importantly, active host defenses consisting of innate immune as well as inflammatory angiogenic responses. The combination of these host responses severely limit and curtail tumor cell infection and viral replication, thus limiting anticancer efficacy. We thus believe that such host responses will have to be circumvented, even temporarily and/or partially, in order to fulfill the promising anticancer effects of these agents. Immune privilege and a paucity of dendritic cells (DC) in the brain contribute to immunological ignorance against glioblastoma multiforme (GBM), an aggressive intracranial primary brain tumor. Herein we uncovered that activation of GBM antigenspecific T-cell dependent immune responses were mediated through the release of endogenous high-mobility-group box 1 (HMGB1) from dying tumor cells in response to herpes simplex type 1 thymidine kinase (TK) gene therapy, chemotherapy and radiation therapy. Tumor-derived HMGB1 activated TLR2 signaling on GBM infiltrating, bone marrow derived DCs, which induced a systemic CD8+ T cell dependent anti-GBM immune response. DCs were recruited to the tumors following intratumoral expression of Fms-like Tyrosine Kinase 3 ligand (Flt3L) and TK. Using chimeric mice we demonstrated that tumor infiltrating DCs were derived from the bone marrow. TLR2 signaling was required for the infiltration of DC into the tumor and for the subsequent activation of DCs for efficient antigen presentation. ELISPOT and antigen specific T-cell proliferation assays indicated that clonal expansion of tumor antigen specific T-lymphocytes was induced by treatment of glioma bearing mice with Ad-Flt3L and Ad-TK (+GCV); crucially, this did not occur in TLR2−/− mice. We then identified the endogenous TLR2 ligand responsible for TLR2 activation on tumor infiltrating DCs. HMGB1 was released from tumor cells in vitro, in response to Ad-TK (+GCV) and in the serum of GBM bearing Ad-TK treated mice in vivo. TLR2 signaling induced by media from Ad-TK (+GCV) treated tumor cells; could be blocked with either glycyrrhizin, which binds to both of the box domains on HMGB1 and prevents subsequent HMGB1 signaling, or with antibodies to HMGB1. These data indicate that HMGB1 is an endogenous, GBM derived TLR2 ligand. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor bearing, Ad-Flt3L and Ad-TK treated mice, abolished GBM regression and long term survival, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit T-cell dependent tumor regression. In summary, our data provide the first experimental evidence for the role of TLR2 signalling, and its endogenous ligand, i.e., HMGB1, in regulating immune surveillance against brain tumor antigens. The discovery of a central role for HMGB1 and TLR2 signalling in overcoming immune ignorance to brain tumor. Epilepsy has an incidence of about 1-3%, affecting an estimated 2.5 million Americans and more than 50 million individuals worldwide. Seizures can occur singly or temporally. Seizure disorders in some adults and children are often associated with infections such as viral infections. Many of these infectiontriggered seizure disorders are often refractory to many currently established anti-epileptic drugs. About 30% of patients with epilepsy are refractory to current anti-seizure medications. The goal is to define and characterize a novel virus infection-induced seizure model in C57BL/6 mice and determine the virus-host interactions that lead to seizure and epilepsy. C57BL/6 mice were infected with the DA virus (DAV) strain of Theiler's murine encephalomyelitis virus or mock infected. Mice were followed for seizures, weight change, body temperature, motor function (righting reflex, rotorod) and neurologic changes including inflammation, perivascular cuffing and pyknotic/dying neurons. In addition, various knockout mice were infected to determine the role for IL-1, TNF-alpha, IL-6 and IFN-gamma. C57BL/6 mice are susceptible to seizures induced by DAV infection. Approximately 50% of C57BL/6 mice develop acute behavior seizures 3-10 days p.i. Following a variable latent period, about half of the seized mice had spontaneous seizures, epilepsy. Motor function and coordination are impaired in seized mice. Pyramidal neuron pyknosis/death and inflammatory changes in the hippocampus correlated with seizure activity. IL-1R1 and MyD88 deficient mice developed seizures in similar numbers to wild-type infected mice but fewer numbers of infected IL-6 and TNF-R1 deficient mice developed seizures than wild-type infected mice. Interestingly more IFN-gammaR deficient mice developed seizures than wild-type infected mice. Cytokine mRNA levels were monitored by array analyses and real-time PCR. Innate immune responses to infection by DAV results in neuronal cell death and seizures. These seizures resolve and then following a latent period mice develop epilepsy. The characterization of this model will enable the investigation of viral and immune contributions in the central nervous system to the development of seizure disorders in humans.
Immunological synapses: The cellular and molecular biology of antiviral immune responses in the brain
Board of Governors' Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, and Depts. of Medicine, and Molecular and Medical Pharmacology, Brain Research Institute, and Jonsson Comprehensive Cancer Center, David Geffen School of Medicine; UCLA, Los Angeles, CA, United States
Immunological synapses (ISs) mediate cell to cell communication in the immune system; in the brain, they mediate communication between anti-viral T cells and infected brain cells, and between antitumor T cells and brain tumors. Adaptive immune responses clear adenovirally infected cells from the brain, or brain tumor cells, with high efficiency. In order to unravel the cell and molecular biology of T cell mediated clearance of virally infected cells from the brain, we have developed a novel approach to elucidate the communication pathways between T cells and infected brain cells, or brain tumor glioma cells. We previously demonstrated that T cell-mediated clearance of virally infected cells in the brain is IFNγ dependent, and does eventually lead to the death of infected brain cells. We have also shown that ISs mediate intercellular communication between T-cells and infected target antigen presenting cells in the brain. We have demonstrated the polarization of IFNγ in CD8 T cells which had established close synaptic contacts with virally infected astrocytes. To determine whether IFNγ only acts exclusively on target cells contacted by T cells, or whether it is secreted non-specifically into the extracellular space within the brain, we developed a novel method to detect vectorial IFNγ release at the level of ISs. Here we describe the construction of novel adenoviral bi-cistronic vectors encoding a constitutively expressed gene (e.g. eGFP; used as a marker to identify infected brain cells), and a second gene under the control of the IFNγ inducible GAS element (e.g. Cre recombinase, or luciferase). These vectors were tested both in vitro and in vivo for IFNγ inducible reporter gene expression. When injected into Rosa26 mice the antiviral T cells induced the expression of β-galactosidase, indicating that they established contacts with infected cells and released IFNγ, which induced expression of Cre, and removal of the STOP codon within the Rosa26 locus that allowed expression of β-galactosidase; the release of IFNγ at immunological synapses is currently being examined. Our method allows us to utilize IFNγ regulated transgene expression as a means to elucidate the existence of targeted vectorial release of T cell cytokines, and to evaluate the function of immunological synapses in orchestrating the function of neuro-immunological circuits during antiviral and anti-tumoral immune responses in the brain in vivo. Support: NINDS/NIH. While viral infections can promote or diminish autoimmunity in type 1 diabetes (T1D), their relationship with this disease is not understood. We found that prevention of autoimmune diabetes by acute viral infection results from regulation of antiviral immunity. Following challenge during the prediabetic phase with lymphocytic choriomeningitis virus (LCMV), Programmed death-ligand 1 (PD-L1) is increased while PD-1-expressing autoreactive CD8 + T cells decline and CD4 + CD25 + regulatory T cells (Tregs) are enhanced in terms of number, TGF-β production and capacity to control T1D even when disease is virally induced. PD-L1 up-regulation delays overt diabetes and facilitates the action of virally enhanced Tregs in a synergistic phenomenon enabled by inflammation and Toll-like receptor 2. These results reconcile current discordance about the role of viruses in T1D and provide novel insight into disease etiology and potential treatment.
Innate immune evasion mechanisms of neurovirulent murine coronavirus Susan R. Weiss The neurovirulent murine coronavirus, mouse hepatitis virus (MHV), possesses mechanisms to ensure propagation and persistence despite the existence of host defense strategies. The type I IFN response is an important host defense in vivo as demonstrated by the increased susceptibility to MHV of IFN receptor knockout mice. We have shown that MHV induces IFN-β mRNA synthesis, albeit only at very late times post-infection, but does not induce IFN-β protein synthesis in mouse L2 fibroblast cultures. The only cell type, prior to our studies, reported to produce type I IFN in response to MHV is plasmacytoid dendritic cells. However, we have observed that, while MHV does not induce IFN-β in several types of primary cell cultures derived from the murine central nervous system, it does induce IFN-β in macrophages/microglia in the brain as well as in cultures of bone marrow derived macrophages (BMM). Furthermore MHV induction of IFN-β, at least in BMM, is dependent on MDA5. We and others have reported that MHV is highly resistant to the antiviral effects of IFNs in fibroblast cultures, but that coinfection with MHV, does not provide protection to the IFN-sensitive Sendai virus when IFN is administered prior to MHV-Sendai co-infection. However, pre-treatment of cells with MHV, can provide protection from the anti-viral effect of IFN-β on Sendai, but not on other IFN sensitive viruses including NDV, Sindbis, and VSV. Using a reporter linked to an interferon-stimulated response element (ISRE), we found that MHV infection prevents activation of the ISRE by IFN-α/β and by Sendai infection. Interestingly, MHV does not prevent translocation of STAT1 to the nucleus following IFN-β treatment, suggesting that MHV is able to actively block STAT1 at a later step such as binding to the ISRE. Similarly, MHV prevents type I interferon independent activation by Sendai of an ISRE driven reporter gene. However, it does not inhibit translocation of IRF-3 to the nucleus, suggesting that binding of IRF-3 to the ISRE may be inhibited by MHV infection. Using quantitative real-time PCR, we have shown that MHV-infection can downregulate a number of wellcharacterized ISGs important for recognition of viral dsRNA and antiviral defense. We are currently investigating the significance of these IFN-induced genes in the defense against MHV and the mechanism by which MHV downregulates their expression. Peroxisome proliferator-activated receptors (PPAR) belong to a family of nuclear hormone receptors involved in inflammation, lipid and glucose metabolism, cell proliferation and differentiation. Previous work from our laboratory has examined the efficacy of using a PPARg agonist after spinal cord injury (SCI) and noted modest behavioral and anatomical improvements. In this study, we examined the expression pattern after SCI of PPARd, the most prevalent PPAR isotype in the central nervous system where it is involved in brain lipid metabolism, differentiation of oligodendrocyte progenitor cells (OPC) and myelination. It also appears to play a protective role in certain disease models like experimental autoimmune encephalomyelitis (EAE), Parkinson's and cerebral ischemia. In this study we used a rat spinal contusion model to investigate whether PPARd expression is changed after spinal cord injury (SCI). Contusive SCI was performed at the T8 level on Sprague-Dawley rats and spinal cord tissue was collected at 1-28 day post-injury (dpi); uninjured spinal cords served as controls. Cross-sections spanning the injury site were used to examine the number, distribution and phenotype of cells expressing PPARd. It was observed that after injury, PPARd+ cell numbers gradually increased throughout the first week. By 7 dpi, expression levels were significantly elevated and reached almost twice that of uninjured controls. Cell numbers remained elevated through at least 28 dpi. PPARd was localized primarily in neurons, NG2+ cells (putative oligodendrocyte progenitors), oligodendrocytes and astrocytes. This rise in expression could reflect an endogenous neuroprotective mechanism that may influence cell survival and promote myelination. Future studies will examine whether providing a PPARd agonist after SCI is beneficial and will test the role of endogenous PPARd signaling in CNS repair by performing SCI and demyelination injuries in PPARd knock-out mice. Supported by Christopher and Dana Reeve Foundation, NS043494 and P30-NS045758.
Alzheimer Research Laboratory, Case Western Reserve University, Department of Neurosciences, Cleveland, OH, United States Alzheimer's disease (AD) is typified by deposition of Aβ peptides in the brain owing to an imbalance between production and clearance of Aβ peptides. Apolipoprotein E isoforms are associated with age-related risk for Alzheimer's disease and play critical roles in Aβ homeostasis in the brain although the basis of this remains a central unanswered question in AD pathogenesis. Liver X Receptors (LXRs) are ligandactivated transcription factors that regulate basal and inducible expression of the key cholesterol homeostatic genes ABCA1 and ApoE. Importantly, LXRs also act as potent inhibitors of inflammatory gene expression. We report that LXR signaling impacts the development of AD-related pathology. Oral treatment of one year old Tg2576 mice with the LXR agonist GW3965 resulted in a significant reduction in amyloid plaque burden and Abeta peptide levels in the brain that was accompanied by suppression of microglial-mediated inflammation. Genetic loss of either Lxrα or Lxrβ in APP/PS1 transgenic mice results in increased amyloid plaque load and a significantly enhanced inflammatory response. These findings demonstrate that LXRs play critical roles in Abeta clearance and deposition from the brain. Importantly, LXR agonist treatment of Tg2576 mice dramatically attenuated behavioral deficits in these mice. LXR activation enhanced the clearance of Aβ by facilitating the proteolytic degradation of soluble Abeta by neprilysin and IDE through an ABCA1 and ApoE-dependent mechanism. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. These data demonstrate a novel mechanism through which ApoE facilitates the clearance of Abeta peptides from the brain and suggest that LXR agonists may represent a novel therapy for AD through this mechanism. The enhanced expression of lipidated forms of ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Aβ peptide levels and plaque load. LXR agonist treatment also reversed contextual memory deficits observed in these mice. We report that ApoE plays a previously unappreciated role in facilitating the proteolytic clearance of soluble Abeta from the brain. Abnormal or excessive inflammation is thought to contribute to the progression of neurodegenerative conditions. Although this topic has been widely studied in both basic and clinical enterprises, the mechanisms of brain inflammation and indeed the relationship between neurodegeneration and inflammation are poorly understood. Microglia are the principal brain-resident immunocompetent cells, and are relatively potent sources of reactive oxygen and nitrogen intermediates. Thus, experiments were designed to better understand the intricate relationship between intracellular oxidative pathways in microglia and intercellular inflammatory signaling. To this end, the production and/or disposition of oxygen free radicals in microglia were manipulated to determine the resultant effects on oxidative and inflammatory indices. Data show that the production of intracellular oxidants in microglia and macrophages is generally crucial to the subsequent production and release of cytokines and the adoption of a neurotoxic phenotype. However, the development of significant oxidative stress in microglia can also be associated with decreases in the release of inflammatory mediators, and the ability of free radicals to control inflammatory signaling in microglia/macrophages may be based on oxidative alterations to the multicatalytic proteasome. Together, these findings suggest that free radical production in brainresident monocytic cells could be a key mechanism regulating brain inflammation in response to various stimuli, and that interventions aimed at free radical regulation in microglia may provide novel and effective therapeutic avenues to ameliorate neurotoxic brain inflammation. Supported by RO1 NS046267, P20 RR15592, and P01 DA19398. The biological significance of post-traumatic spinal cord inflammation is controversial. Resident microglia and infiltrating leukocytes (e.g., monocytes/T-cells) cause neuronal cell death, axonal injury and demyelination. However, these same cells can promote neuron survival, axon regeneration, remyelination and revascularization. Over the past few years, we have used numerous strategies to manipulate resident and recruited immune cells in an effort to understand their reparative and injurious function in the context of rodent models of traumatic spinal cord injury (SCI). Data will be presented that illustrate the conflicting effects of activated CNS macrophages with an emphasis on newer data that suggest a role for a subset of toll-like receptors (TLRs) in regulating various aspects of CNS repair (e.g., axonal regeneration, remyelination, gliosis) and/or down-regulating destructive neuroinflammation. Myelin-reactive and non-CNS reactive lymphocytes are also recruited to the injured spinal cord where they have been reported to have beneficial and detrimental effects. Data will be presented illustrating the pathogenic potential of B lymphocytes and self-reactive antibodies in models of SCI. The experimental limitations and clinical implications of these data will be discussed. Supported by NIH grants NS37846 and NS047175.
Therapeutic strategies against microglia-induced neurotoxicity
Department of Neuroimmunology, RIEM, Nagoya University, Nagoya, Japan Accumulation of activated microglia and reactive astrocytes were observed around degenerated neurons. Cytokines from these activated glial cells may be involved in the pathophysiology of neuronal degeneration and regeneration. In this study, we have examined the neurotoxicity by microglia-derived factors. The most neurotoxic factor from activated microglia was glutamate. All the recombinant cytokines examined was not toxic to neurons. However, TNF-a exerted neurotoxicity via inducing glutamate in microglia and IFN-g induced minor neurotoxicity that was mediated through IFN-g-R and AMPA receptor complex. Upon stimulation with TNF-a, microglia produced glutamate by glutaminase using extracellular glutamine, and released it through gap-junction, but not through glutamate transporters. Both glutaminase and gap-junction inhibitors suppressed glutamate release and neurotoxicity by microglia. We then examined whether glutaminase inhibitor (6-diazo-5-oxo-L-norleucine: DON) and gapjunction inhibitor (Carbenoxolone: CBX) suppress neuronal damage in vivo, using animal models. Both DON and CBX significantly suppressed delayed neuronal cell death in hippocampus after brain ischemia, and also increased survival of the animal model of amyotrophic lateral sclerosis (ALS). Blockade of the unique mechanisms in glutamate production and release by microglia may be useful therapeutic strategy against neuronal degeneration.
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