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T107 0-118 Sentence denotes SHARPIN Is Essential for Cytokine Production, NF-κB Signaling, and Induction of Th1 Differentiation by Dendritic Cells
T1 0-153 Sentence denotes SHARPIN Is Essential for Cytokine Production, NF-κB Signaling, and Induction of Th1 Differentiation by Dendritic Cells Role of SHARPIN in Dendritic Cells
T2 156-164 Sentence denotes Abstract
T108 165-265 Sentence denotes Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases:
T3 165-265 Sentence denotes Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases:
T109 266-448 Sentence denotes Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines.
T4 266-448 Sentence denotes Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines.
T110 449-541 Sentence denotes The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive.
T5 449-541 Sentence denotes The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive.
T111 542-712 Sentence denotes Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity.
T6 542-712 Sentence denotes Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity.
T112 713-839 Sentence denotes Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen.
T7 713-839 Sentence denotes Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen.
T113 840-1074 Sentence denotes In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86.
T8 840-1074 Sentence denotes In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86.
T114 1075-1226 Sentence denotes However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide.
T9 1075-1226 Sentence denotes However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide.
T115 1227-1399 Sentence denotes Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact.
T10 1227-1399 Sentence denotes Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact.
T116 1400-1576 Sentence denotes A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4+ T cells.
T11 1400-1576 Sentence denotes A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4+ T cells.
T117 1577-1756 Sentence denotes In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.
T12 1577-1756 Sentence denotes In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.
T13 1758-1770 Sentence denotes Introduction
T978 1771-1946 Sentence denotes SHARPIN was originally identified in post-synaptic densities of excitatory synapses in the brain of rats [1], but this protein is widely expressed in a variety of tissues [2].
T14 1771-1946 Sentence denotes SHARPIN was originally identified in post-synaptic densities of excitatory synapses in the brain of rats [1], but this protein is widely expressed in a variety of tissues [2].
T979 1947-2253 Sentence denotes Two allelic, autosomal recessive mutations in the Sharpin gene occurred spontaneously in two inbred strains of mice, C57B/KaLawRij-Sharpincpdm/Sharpincpdm and CBy.Ocb3/Dem-Sharpincpdm-Dem/Sharpincpdm-Dem, resulting in premature termination of mRNA synthesis and absence of a functional protein product [2].
T15 1947-2253 Sentence denotes Two allelic, autosomal recessive mutations in the Sharpin gene occurred spontaneously in two inbred strains of mice, C57B/KaLawRij-Sharpincpdm/Sharpincpdm and CBy.Ocb3/Dem-Sharpincpdm-Dem/Sharpincpdm-Dem, resulting in premature termination of mRNA synthesis and absence of a functional protein product [2].
T980 2254-2449 Sentence denotes Despite different genetic backgrounds, both mutations cause similar inflammatory disease with severe chronic progressive dermatitis and defective development of secondary lymphoid organs [2]–[4].
T16 2254-2449 Sentence denotes Despite different genetic backgrounds, both mutations cause similar inflammatory disease with severe chronic progressive dermatitis and defective development of secondary lymphoid organs [2]–[4].
T981 2450-2520 Sentence denotes The dermatitis becomes clinically apparent at about four weeks of age.
T17 2450-2520 Sentence denotes The dermatitis becomes clinically apparent at about four weeks of age.
T982 2521-2796 Sentence denotes There are accumulations of eosinophils, neutrophils and macrophages in the skin of Sharpincpdm/Sharpincpdm mutant mice (hereafter referred to as cpdm mice) associated with increased expression of Th2 cytokines in the skin and in the supernatants of activated splenocytes [5].
T18 2521-2796 Sentence denotes There are accumulations of eosinophils, neutrophils and macrophages in the skin of Sharpincpdm/Sharpincpdm mutant mice (hereafter referred to as cpdm mice) associated with increased expression of Th2 cytokines in the skin and in the supernatants of activated splenocytes [5].
T983 2797-2984 Sentence denotes The mice have an impaired delayed type hypersensitivity response and decreased secretion of IFNγ [5], indicating a defect in Th1 immune responses and a bias towards a Th2 immune response.
T19 2797-2984 Sentence denotes The mice have an impaired delayed type hypersensitivity response and decreased secretion of IFNγ [5], indicating a defect in Th1 immune responses and a bias towards a Th2 immune response.
T984 2985-3087 Sentence denotes Systemic treatment of cpdm mice with recombinant IL12 caused complete remission of the dermatitis [5].
T20 2985-3087 Sentence denotes Systemic treatment of cpdm mice with recombinant IL12 caused complete remission of the dermatitis [5].
T985 3088-3294 Sentence denotes Neutralization of IL5 by antibody treatment or crosses with IL5-deficient mice reduced the number of circulating and cutaneous eosinophils, but failed to reduce the onset and severity of the dermatitis [6].
T21 3088-3294 Sentence denotes Neutralization of IL5 by antibody treatment or crosses with IL5-deficient mice reduced the number of circulating and cutaneous eosinophils, but failed to reduce the onset and severity of the dermatitis [6].
T986 3295-3492 Sentence denotes Recently, three independent groups identified SHARPIN as an essential component of the linear ubiquitin chain assembly complex (LUBAC) that regulates TNFα-induced canonical NF-κB signaling [7]–[9].
T22 3295-3492 Sentence denotes Recently, three independent groups identified SHARPIN as an essential component of the linear ubiquitin chain assembly complex (LUBAC) that regulates TNFα-induced canonical NF-κB signaling [7]–[9].
T987 3493-3667 Sentence denotes SHARPIN-deficient mouse embryonic fibroblast (MEF) were sensitized to TNFα-induced apoptosis and cell death was implicated as a factor in the dermatitis of cpdm mice [7]–[9].
T23 3493-3667 Sentence denotes SHARPIN-deficient mouse embryonic fibroblast (MEF) were sensitized to TNFα-induced apoptosis and cell death was implicated as a factor in the dermatitis of cpdm mice [7]–[9].
T988 3668-3820 Sentence denotes Dendritic cells (DC) have a sentinel role in sensing pathogen or danger signals and initiate and direct activation of the adaptive immune response [10].
T24 3668-3820 Sentence denotes Dendritic cells (DC) have a sentinel role in sensing pathogen or danger signals and initiate and direct activation of the adaptive immune response [10].
T989 3821-3970 Sentence denotes Activated and mature DC can carry processed antigenic peptides, migrate to lymphoid organs, and induce T-cell-mediated immune responses or tolerance.
T25 3821-3970 Sentence denotes Activated and mature DC can carry processed antigenic peptides, migrate to lymphoid organs, and induce T-cell-mediated immune responses or tolerance.
T990 3971-4102 Sentence denotes DC direct the differentiation of CD4+ T cells, and hence the type of immune response, through the selective secretion of cytokines.
T26 3971-4102 Sentence denotes DC direct the differentiation of CD4+ T cells, and hence the type of immune response, through the selective secretion of cytokines.
T991 4103-4238 Sentence denotes We hypothesized that defective cytokine secretion by DC contributed to the Th2-biased inflammatory phenotype in SHARPIN-deficient mice.
T27 4103-4238 Sentence denotes We hypothesized that defective cytokine secretion by DC contributed to the Th2-biased inflammatory phenotype in SHARPIN-deficient mice.
T992 4239-4420 Sentence denotes The studies reported here found that lack of SHARPIN protein in BMDC caused defective expression of pro-inflammatory mediators and impaired NF-κB activation upon ligand stimulation.
T28 4239-4420 Sentence denotes The studies reported here found that lack of SHARPIN protein in BMDC caused defective expression of pro-inflammatory mediators and impaired NF-κB activation upon ligand stimulation.
T993 4421-4526 Sentence denotes The ability of cpdm BMDC to stimulate Th1 cytokine production in allogeneic CD4+ T cells was compromised.
T29 4421-4526 Sentence denotes The ability of cpdm BMDC to stimulate Th1 cytokine production in allogeneic CD4+ T cells was compromised.
T994 4527-4724 Sentence denotes Taken together, these results reveal that SHARPIN is a novel regulatory molecule in DC biology and suggest that the dysregulated function of SHARPIN-deficient DC plays a role in the cpdm phenotype.
T30 4527-4724 Sentence denotes Taken together, these results reveal that SHARPIN is a novel regulatory molecule in DC biology and suggest that the dysregulated function of SHARPIN-deficient DC plays a role in the cpdm phenotype.
T31 4726-4733 Sentence denotes Results
T2217 4735-4762 Sentence denotes Characterization of Sharpin
T32 4735-4762 Sentence denotes Characterization of Sharpin
T2218 4764-4854 Sentence denotes Orthologs of SHARPIN protein are found in various species, including human, mouse and rat.
T33 4764-4854 Sentence denotes Orthologs of SHARPIN protein are found in various species, including human, mouse and rat.
T2219 4855-5066 Sentence denotes Motif prediction programming, using COILS [11] and MotifScan [12], suggests that SHARPIN contains a coiled-coil (CC) domain, a ubiquitin-like (UBL) domain, and a zinc-finger Ran-binding protein 2 (ZFRBP) domain.
T34 4855-5066 Sentence denotes Motif prediction programming, using COILS [11] and MotifScan [12], suggests that SHARPIN contains a coiled-coil (CC) domain, a ubiquitin-like (UBL) domain, and a zinc-finger Ran-binding protein 2 (ZFRBP) domain.
T2220 5067-5271 Sentence denotes These functional motifs constitute similar domain profiles that are present in the SHARPIN protein of all three origins (Fig. 1A), suggesting that SHARPIN exerts highly conserved functions across species.
T35 5067-5271 Sentence denotes These functional motifs constitute similar domain profiles that are present in the SHARPIN protein of all three origins (Fig. 1A), suggesting that SHARPIN exerts highly conserved functions across species.
T2221 5272-5508 Sentence denotes Spontaneous mutations in the mouse Sharpin gene results in a complex inflammatory phenotype characterized by severe dermatitis (Fig. 1B), systemic inflammation and an enlarged spleen (Fig. 1C) caused by extramedullary hematopoiesis [3].
T36 5272-5508 Sentence denotes Spontaneous mutations in the mouse Sharpin gene results in a complex inflammatory phenotype characterized by severe dermatitis (Fig. 1B), systemic inflammation and an enlarged spleen (Fig. 1C) caused by extramedullary hematopoiesis [3].
T2222 5509-5677 Sentence denotes The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS.
T37 5509-5677 Sentence denotes The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS.
T2223 5678-5800 Sentence denotes Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation.
T38 5678-5800 Sentence denotes Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation.
T2224 5801-5895 Sentence denotes There was a significant reduction of Sharpin mRNA (6–7-fold) in BMDC generated from cpdm mice.
T39 5801-5895 Sentence denotes There was a significant reduction of Sharpin mRNA (6–7-fold) in BMDC generated from cpdm mice.
T2225 5896-6051 Sentence denotes Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).
T40 5896-6051 Sentence denotes Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).
T41 6052-6137 Sentence denotes 10.1371/journal.pone.0031809.g001 Figure 1 In vivo and in vitro features of SHARPIN.
T12763 6096-6137 Sentence denotes In vivo and in vitro features of SHARPIN.
T12764 6138-6451 Sentence denotes (A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice.
T42 6138-6451 Sentence denotes (A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice.
T12765 6452-6711 Sentence denotes The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours.
T43 6452-6711 Sentence denotes The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours.
T12766 6712-6834 Sentence denotes The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC.
T44 6712-6834 Sentence denotes The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC.
T12767 6835-7015 Sentence denotes Bars represent the mean ± s.d. of 3 mice. * P<0.05; ** P<0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN.
T45 6835-7015 Sentence denotes Bars represent the mean ± s.d. of 3 mice. * P<0.05; ** P<0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN.
T12768 7016-7114 Sentence denotes After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody.
T46 7016-7114 Sentence denotes After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody.
T12769 7115-7145 Sentence denotes Nuclei were stained with DAPI.
T47 7115-7145 Sentence denotes Nuclei were stained with DAPI.
T12770 7146-7227 Sentence denotes In both transfected cell lines, FLAG-SHARPIN was found to be cytoplasm-localized.
T48 7146-7227 Sentence denotes In both transfected cell lines, FLAG-SHARPIN was found to be cytoplasm-localized.
T2872 7229-7282 Sentence denotes Phenotyping splenic DC and BMDC from WT and cpdm mice
T49 7229-7282 Sentence denotes Phenotyping splenic DC and BMDC from WT and cpdm mice
T2873 7283-7380 Sentence denotes DC are heterogeneous and can be categorized into multiple subtypes based on surface markers [13].
T50 7283-7380 Sentence denotes DC are heterogeneous and can be categorized into multiple subtypes based on surface markers [13].
T2874 7381-7616 Sentence denotes To determine if the Sharpin mutation affects DC development in lymphoid tissues, mouse spleens were examined for the distribution of conventional DC (cDC; CD11c+CD8α+ and CD11c+CD8α−) [13] and plasmacytoid DC (pDC; CD11c−PDCA-1+) [14].
T51 7381-7616 Sentence denotes To determine if the Sharpin mutation affects DC development in lymphoid tissues, mouse spleens were examined for the distribution of conventional DC (cDC; CD11c+CD8α+ and CD11c+CD8α−) [13] and plasmacytoid DC (pDC; CD11c−PDCA-1+) [14].
T2875 7617-7729 Sentence denotes The percentages for splenic cDC and pDC were both reduced in cpdm mice when compared with WT controls (Fig. 2A).
T52 7617-7729 Sentence denotes The percentages for splenic cDC and pDC were both reduced in cpdm mice when compared with WT controls (Fig. 2A).
T2876 7730-7856 Sentence denotes However, when gated on CD11c+ cDC, the percentages of CD8α+ and CD8α− cells were not affected by SHARPIN deficiency (Fig. 2A).
T53 7730-7856 Sentence denotes However, when gated on CD11c+ cDC, the percentages of CD8α+ and CD8α− cells were not affected by SHARPIN deficiency (Fig. 2A).
T2877 7857-8132 Sentence denotes Since the spleen of cpdm mice is markedly enlarged and contains three times as many cells (Fig. 1C), the different percentages of splenic cDC and pDC between WT and mutant mice reflect the increased number of total spleen cells rather than a reduction in cDC and pDC numbers.
T54 7857-8132 Sentence denotes Since the spleen of cpdm mice is markedly enlarged and contains three times as many cells (Fig. 1C), the different percentages of splenic cDC and pDC between WT and mutant mice reflect the increased number of total spleen cells rather than a reduction in cDC and pDC numbers.
T2878 8133-8253 Sentence denotes These data indicate that the Sharpin mutation does not affect the distribution of the examined DC subsets in the spleen.
T55 8133-8253 Sentence denotes These data indicate that the Sharpin mutation does not affect the distribution of the examined DC subsets in the spleen.
T56 8254-8361 Sentence denotes 10.1371/journal.pone.0031809.g002 Figure 2 Effect of Sharpin mutation on DC subpopulations and maturation.
T13265 8298-8361 Sentence denotes Effect of Sharpin mutation on DC subpopulations and maturation.
T13266 8362-8457 Sentence denotes (A) Spleens from WT and cpdm mice were isolated and subject to collagenase and DNase digestion.
T57 8362-8457 Sentence denotes (A) Spleens from WT and cpdm mice were isolated and subject to collagenase and DNase digestion.
T13267 8458-8569 Sentence denotes The obtained splenic homogenates were centrifuged over a Percoll gradient (35% and 55% density) for 15 minutes.
T58 8458-8569 Sentence denotes The obtained splenic homogenates were centrifuged over a Percoll gradient (35% and 55% density) for 15 minutes.
T13268 8570-8917 Sentence denotes The bands at the 35%-medium and the 35–55% interface were pooled, washed and stained with a combination of various antibodies to stain different DC subsets, conventional CD11c+CD8α+, CD11c+CD8α− and plasmacytoid DC (CD11c−PDCA-1+).The top panels were gated on FSChiSSClo cells to show separate populations of CD11c+PDCA-1− and CD11c−PDCA-1+ cells.
T59 8570-8917 Sentence denotes The bands at the 35%-medium and the 35–55% interface were pooled, washed and stained with a combination of various antibodies to stain different DC subsets, conventional CD11c+CD8α+, CD11c+CD8α− and plasmacytoid DC (CD11c−PDCA-1+).The top panels were gated on FSChiSSClo cells to show separate populations of CD11c+PDCA-1− and CD11c−PDCA-1+ cells.
T13269 8918-9058 Sentence denotes Further gating on the CD11c+PDCA-1− subpopulation gave the bottom panel that showed two distinct pools of CD11c+CD8α+ and CD11c+CD8α− cells.
T60 8918-9058 Sentence denotes Further gating on the CD11c+PDCA-1− subpopulation gave the bottom panel that showed two distinct pools of CD11c+CD8α+ and CD11c+CD8α− cells.
T13270 9059-9287 Sentence denotes Percentages were calculated based on the parental population and were additionally shown as bar graphs (n = 2) (B). (C) WT and cpdm BMDC (5×105) cells were stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T61 9059-9287 Sentence denotes Percentages were calculated based on the parental population and were additionally shown as bar graphs (n = 2) (B). (C) WT and cpdm BMDC (5×105) cells were stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T13271 9288-9405 Sentence denotes The cells were labeled with PE-labeled anti-CD40, anti-CD80, and anti-CD86, and subjected to flow cytometry analysis.
T62 9288-9405 Sentence denotes The cells were labeled with PE-labeled anti-CD40, anti-CD80, and anti-CD86, and subjected to flow cytometry analysis.
T13272 9406-9469 Sentence denotes The populations shown in histograms were gated on CD11c+ cells.
T63 9406-9469 Sentence denotes The populations shown in histograms were gated on CD11c+ cells.
T13273 9470-9514 Sentence denotes Unstained cells served as negative controls.
T64 9470-9514 Sentence denotes Unstained cells served as negative controls.
T13274 9515-9573 Sentence denotes Results are representative of two independent experiments.
T65 9515-9573 Sentence denotes Results are representative of two independent experiments.
T2879 9576-9697 Sentence denotes BMDC from in vitro cultures functionally resemble non-lymphoid tissue DC and monocyte-derived inflammatory DC [15], [16].
T66 9576-9697 Sentence denotes BMDC from in vitro cultures functionally resemble non-lymphoid tissue DC and monocyte-derived inflammatory DC [15], [16].
T2880 9698-9752 Sentence denotes The yields of BMDC from WT and cpdm mice were similar.
T67 9698-9752 Sentence denotes The yields of BMDC from WT and cpdm mice were similar.
T2881 9753-9851 Sentence denotes BMDC were CD11c+ and MHC II+ with low expression of co-stimulatory molecules CD40, CD80, and CD86.
T68 9753-9851 Sentence denotes BMDC were CD11c+ and MHC II+ with low expression of co-stimulatory molecules CD40, CD80, and CD86.
T2882 9852-10009 Sentence denotes The TLR3 ligand poly I:C and the TLR4 ligand LPS each activate overlapping but different signaling pathways and were used to induce DC maturation [17], [18].
T69 9852-10009 Sentence denotes The TLR3 ligand poly I:C and the TLR4 ligand LPS each activate overlapping but different signaling pathways and were used to induce DC maturation [17], [18].
T2883 10010-10232 Sentence denotes Incubation with the TLR agonists for 24 hours resulted in increased expression of CD40, CD80, and CD86 on BMDC; however, there was no difference in the expression levels of these markers between WT and cpdm BMDC (Fig. 2B).
T70 10010-10232 Sentence denotes Incubation with the TLR agonists for 24 hours resulted in increased expression of CD40, CD80, and CD86 on BMDC; however, there was no difference in the expression levels of these markers between WT and cpdm BMDC (Fig. 2B).
T2884 10233-10327 Sentence denotes Thus, SHARPIN deficiency did not influence the expression of co-stimulatory molecules by BMDC.
T71 10233-10327 Sentence denotes Thus, SHARPIN deficiency did not influence the expression of co-stimulatory molecules by BMDC.
T3823 10329-10393 Sentence denotes Production of proinflammatory mediators by cpdm BMDC is impaired
T72 10329-10393 Sentence denotes Production of proinflammatory mediators by cpdm BMDC is impaired
T3824 10394-10622 Sentence denotes Incubation with LPS or poly I:C resulted in secretion of IL6, IL12P70, and GMCSF from both WT and cpdm BMDC; however, BMDC from cpdm mice produced significantly less of all three cytokines compared with WT BMDC (Fig. 3A–C, E–F).
T73 10394-10622 Sentence denotes Incubation with LPS or poly I:C resulted in secretion of IL6, IL12P70, and GMCSF from both WT and cpdm BMDC; however, BMDC from cpdm mice produced significantly less of all three cytokines compared with WT BMDC (Fig. 3A–C, E–F).
T3825 10623-10838 Sentence denotes The amount of nitric oxide generated by mutant BMDC was also significantly reduced compared with WT cells (Fig. 3D), indicating severely disrupted production of proinflammatory mediators from Sharpin-deficient BMDC.
T74 10623-10838 Sentence denotes The amount of nitric oxide generated by mutant BMDC was also significantly reduced compared with WT cells (Fig. 3D), indicating severely disrupted production of proinflammatory mediators from Sharpin-deficient BMDC.
T3826 10839-11129 Sentence denotes In vivo complementation with a Sharpin gene-containing BAC reversed the phenotype of the mutant mice [2], and BMDC generated from these rescued mice secreted significantly more IL12P70 than BMDC from cpdm mice (Fig. 3G), supporting a necessary role of SHARPIN for the production of IL12P70.
T75 10839-11129 Sentence denotes In vivo complementation with a Sharpin gene-containing BAC reversed the phenotype of the mutant mice [2], and BMDC generated from these rescued mice secreted significantly more IL12P70 than BMDC from cpdm mice (Fig. 3G), supporting a necessary role of SHARPIN for the production of IL12P70.
T3827 11130-11345 Sentence denotes In addition, the transcript levels of the inflammatory cytokines Il6, Il12p40, Gmcsf, and Ifnb were examined, and these were all significantly reduced in stimulated cpdm BMDC when compared with WT controls (Fig. 4).
T76 11130-11345 Sentence denotes In addition, the transcript levels of the inflammatory cytokines Il6, Il12p40, Gmcsf, and Ifnb were examined, and these were all significantly reduced in stimulated cpdm BMDC when compared with WT controls (Fig. 4).
T77 11346-11467 Sentence denotes 10.1371/journal.pone.0031809.g003 Figure 3 Defective production of pro-inflammatory mediators from stimulated cpdm BMDC.
T13806 11390-11467 Sentence denotes Defective production of pro-inflammatory mediators from stimulated cpdm BMDC.
T13807 11468-11628 Sentence denotes (A–D) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T78 11468-11628 Sentence denotes (A–D) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T13808 11629-11868 Sentence denotes Supernatants were collected for ELISA of IL12P70, IL6, and GMCSF, and for quantification of nitric oxide (NO). (E–F) Gradient numbers (1×104, 2×104, and 4×104 cells in 0.1 mL complete medium) of BMDC were used for100 ng/mL LPS stimulation.
T79 11629-11868 Sentence denotes Supernatants were collected for ELISA of IL12P70, IL6, and GMCSF, and for quantification of nitric oxide (NO). (E–F) Gradient numbers (1×104, 2×104, and 4×104 cells in 0.1 mL complete medium) of BMDC were used for100 ng/mL LPS stimulation.
T13809 11869-12061 Sentence denotes After 24 hours, the amounts of IL12P70 and IL6 from the supernatant were measured. (G) The cpdm mice rescued by Sharpin-containing BAC had complete remission of the inflammatory phenotype [2].
T80 11869-12061 Sentence denotes After 24 hours, the amounts of IL12P70 and IL6 from the supernatant were measured. (G) The cpdm mice rescued by Sharpin-containing BAC had complete remission of the inflammatory phenotype [2].
T13810 12062-12179 Sentence denotes BMDC developed from cpdm and rescued cpdm mice were plated (1×106 cells in 0.3 mL) and stimulated with 100 ng/mL LPS.
T81 12062-12179 Sentence denotes BMDC developed from cpdm and rescued cpdm mice were plated (1×106 cells in 0.3 mL) and stimulated with 100 ng/mL LPS.
T13811 12180-12259 Sentence denotes After 24 hours, supernatants were collected for analysis of IL12P70 production.
T82 12180-12259 Sentence denotes After 24 hours, supernatants were collected for analysis of IL12P70 production.
T13812 12260-12339 Sentence denotes Data are representative of three independent experiments. * P<0.01; ** P<0.005.
T83 12260-12339 Sentence denotes Data are representative of three independent experiments. * P<0.01; ** P<0.005.
T84 12340-12447 Sentence denotes 10.1371/journal.pone.0031809.g004 Figure 4 Decreased mRNA levels of inflammatory cytokines from cpdm BMDC.
T14256 12384-12447 Sentence denotes Decreased mRNA levels of inflammatory cytokines from cpdm BMDC.
T14257 12448-12601 Sentence denotes Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS (A,C,E,G) or 25 µg/ml poly I:C (B,D,F,H).
T85 12448-12601 Sentence denotes Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS (A,C,E,G) or 25 µg/ml poly I:C (B,D,F,H).
T14258 12602-12758 Sentence denotes At 0, 1 and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the expression of Il12p40 (A,B), Il6 (C,D), Gmcsf (E,F), and Ifnb (G,H) mRNA.
T86 12602-12758 Sentence denotes At 0, 1 and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the expression of Il12p40 (A,B), Il6 (C,D), Gmcsf (E,F), and Ifnb (G,H) mRNA.
T14259 12759-12784 Sentence denotes Bars represent mean ± SD.
T87 12759-12784 Sentence denotes Bars represent mean ± SD.
T14260 12785-12840 Sentence denotes Data are representative of two independent experiments.
T88 12785-12840 Sentence denotes Data are representative of two independent experiments.
T4475 12842-12939 Sentence denotes Impaired cytokine production by cpdm BMDC is correlated with selective defects in NF-κB signaling
T89 12842-12939 Sentence denotes Impaired cytokine production by cpdm BMDC is correlated with selective defects in NF-κB signaling
T4476 12940-13284 Sentence denotes There are a number of possible explanations for the defective cytokine secretion in stimulated cpdm BMDC, including 1) reduced surface expression of the LPS receptor complex, 2) increased production of anti-inflammatory mediators, 3) increased expression of negative regulators of TLR pathways, and 4) impaired TLR-induced signaling activation.
T90 12940-13284 Sentence denotes There are a number of possible explanations for the defective cytokine secretion in stimulated cpdm BMDC, including 1) reduced surface expression of the LPS receptor complex, 2) increased production of anti-inflammatory mediators, 3) increased expression of negative regulators of TLR pathways, and 4) impaired TLR-induced signaling activation.
T4477 13285-13452 Sentence denotes We determined the surface expression of the LPS receptor complex that comprises TLR4, the accessory proteins CD14 and myeloid differentiation factor 2 (MD2/LY96) [19].
T91 13285-13452 Sentence denotes We determined the surface expression of the LPS receptor complex that comprises TLR4, the accessory proteins CD14 and myeloid differentiation factor 2 (MD2/LY96) [19].
T4478 13453-13580 Sentence denotes Flow cytometric analysis shows that the expression levels of CD14 and TLR4/MD2 between WT and cpdm BMDC were similar (Fig. 5A).
T92 13453-13580 Sentence denotes Flow cytometric analysis shows that the expression levels of CD14 and TLR4/MD2 between WT and cpdm BMDC were similar (Fig. 5A).
T4479 13581-13714 Sentence denotes We then quantified the secretion of the suppressive cytokines IL10 that can inhibit IL12 secretion in an autocrine manner [20], [21].
T93 13581-13714 Sentence denotes We then quantified the secretion of the suppressive cytokines IL10 that can inhibit IL12 secretion in an autocrine manner [20], [21].
T4480 13715-13931 Sentence denotes The supernatants from LPS-stimulated cpdm BMDC contained significantly lower levels of IL10 than stimulated WT BMDC (Fig. 5B), suggesting that IL10 was not responsible for decreased secretion of IL12P70 by cpdm BMDC.
T94 13715-13931 Sentence denotes The supernatants from LPS-stimulated cpdm BMDC contained significantly lower levels of IL10 than stimulated WT BMDC (Fig. 5B), suggesting that IL10 was not responsible for decreased secretion of IL12P70 by cpdm BMDC.
T4481 13932-14048 Sentence denotes Increased expression of a negative regulator of TLR signaling such as A20 [22] may also suppress cytokine secretion.
T95 13932-14048 Sentence denotes Increased expression of a negative regulator of TLR signaling such as A20 [22] may also suppress cytokine secretion.
T4482 14049-14239 Sentence denotes However, the transcript level of A20 was lower in LPS-activated cpdm BMDC than WT controls (Fig. 5C), thereby ruling out overexpression of A20 as a factor in the reduced cytokine production.
T96 14049-14239 Sentence denotes However, the transcript level of A20 was lower in LPS-activated cpdm BMDC than WT controls (Fig. 5C), thereby ruling out overexpression of A20 as a factor in the reduced cytokine production.
T97 14240-14376 Sentence denotes 10.1371/journal.pone.0031809.g005 Figure 5 Normal TLR4 and MD2 expression and decreased IL10 secretion and A20 expression by cpdm BMDC.
T14580 14284-14376 Sentence denotes Normal TLR4 and MD2 expression and decreased IL10 secretion and A20 expression by cpdm BMDC.
T14581 14377-14519 Sentence denotes (A) Unstimulated WT and cpdm BMDC (5×105) were labeled with PE-labeled anti-TLR4/MD2 or anti-CD14 and then subject to flow cytometry analysis.
T98 14377-14519 Sentence denotes (A) Unstimulated WT and cpdm BMDC (5×105) were labeled with PE-labeled anti-TLR4/MD2 or anti-CD14 and then subject to flow cytometry analysis.
T14582 14520-14722 Sentence denotes Unstained cells serve as negative controls. (B) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T99 14520-14722 Sentence denotes Unstained cells serve as negative controls. (B) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T100 14723-14907 Sentence denotes Supernatants were collected for ELISA of IL10. (C) Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS or 25 µg/ml poly I:C.
T14583 14723-15010 Sentence denotes Supernatants were collected for ELISA of IL10. (C) Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS or 25 µg/ml poly I:C. At 0, 1, and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the production of A20.
T101 14908-15010 Sentence denotes At 0, 1, and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the production of A20.
T4483 15014-15184 Sentence denotes The transcription of TLR3/4-induced proinflammatory intermediates is tightly regulated by cellular signaling pathways, in particular NF-κB, TBK1/IRF3, and MAPK [23]–[27].
T102 15014-15184 Sentence denotes The transcription of TLR3/4-induced proinflammatory intermediates is tightly regulated by cellular signaling pathways, in particular NF-κB, TBK1/IRF3, and MAPK [23]–[27].
T4484 15185-15342 Sentence denotes We next determined if disrupted NF-κB, TBK1/IRF3, and/or MAPK signaling may underlie the impaired cytokine production from stimulated Sharpin-deficient BMDC.
T103 15185-15342 Sentence denotes We next determined if disrupted NF-κB, TBK1/IRF3, and/or MAPK signaling may underlie the impaired cytokine production from stimulated Sharpin-deficient BMDC.
T4485 15343-15593 Sentence denotes Stimulus-induced phosphorylation of the IκB kinase (IKK1/2) is an essential step in NF-κB signaling, allowing phosphorylation and proteasome-mediated degradation of the NF-κB inhibitor IκBα to release the NF-κB transcription factors into the nucleus.
T104 15343-15593 Sentence denotes Stimulus-induced phosphorylation of the IκB kinase (IKK1/2) is an essential step in NF-κB signaling, allowing phosphorylation and proteasome-mediated degradation of the NF-κB inhibitor IκBα to release the NF-κB transcription factors into the nucleus.
T4486 15594-15775 Sentence denotes The amount of phosphorylated IKK1/2 (p-IKK1/2) and IκBα (p-IκBα) following incubation with LPS or poly I:C was severely decreased in cpdm BMDC as compared with WT controls (Fig. 6).
T105 15594-15775 Sentence denotes The amount of phosphorylated IKK1/2 (p-IKK1/2) and IκBα (p-IκBα) following incubation with LPS or poly I:C was severely decreased in cpdm BMDC as compared with WT controls (Fig. 6).
T4487 15776-15886 Sentence denotes The cpdm BMDC exhibited similar levels of TBK1, ERK1/2, and p38 phosphorylation to those of WT cells (Fig. 6).
T106 15776-15886 Sentence denotes The cpdm BMDC exhibited similar levels of TBK1, ERK1/2, and p38 phosphorylation to those of WT cells (Fig. 6).
T4488 15887-16040 Sentence denotes These results indicate that the absence of functional SHARPIN decreased NF-κB activation but did not affect TBK1/IRF3, ERK1/2, and p38 signaling in BMDC.
T107 15887-16040 Sentence denotes These results indicate that the absence of functional SHARPIN decreased NF-κB activation but did not affect TBK1/IRF3, ERK1/2, and p38 signaling in BMDC.
T108 16041-16128 Sentence denotes 10.1371/journal.pone.0031809.g006 Figure 6 Inhibition of NF-κB signaling in cpdm BMDC.
T14993 16085-16128 Sentence denotes Inhibition of NF-κB signaling in cpdm BMDC.
T14994 16129-16250 Sentence denotes WT and cpdm BMDC (2×106 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 µg/mL poly I:C (B).
T109 16129-16250 Sentence denotes WT and cpdm BMDC (2×106 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 µg/mL poly I:C (B).
T14995 16251-16439 Sentence denotes At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-κB, TBK1/IRF3, ERK1/2, and p38 signaling pathways.
T110 16251-16439 Sentence denotes At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-κB, TBK1/IRF3, ERK1/2, and p38 signaling pathways.
T14996 16440-16623 Sentence denotes Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IκBα in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines.
T111 16440-16623 Sentence denotes Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IκBα in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines.
T14997 16624-16691 Sentence denotes Results are representative of at least two independent experiments.
T112 16624-16691 Sentence denotes Results are representative of at least two independent experiments.
T5820 16693-16742 Sentence denotes Th2-biased immunogenicity of stimulated cpdm BMDC
T113 16693-16742 Sentence denotes Th2-biased immunogenicity of stimulated cpdm BMDC
T5821 16743-16956 Sentence denotes The defective IL12 production (Fig. 3A) and Th2-dominant cytokine profile in cpdm mice [5] suggest that the absence of SHARPIN affects the ability of cpdm BMDC to induce T cell differentiation into effector cells.
T114 16743-16956 Sentence denotes The defective IL12 production (Fig. 3A) and Th2-dominant cytokine profile in cpdm mice [5] suggest that the absence of SHARPIN affects the ability of cpdm BMDC to induce T cell differentiation into effector cells.
T5822 16957-17257 Sentence denotes Co-culture of allogeneic naïve CD4+ T cells with WT BMDC stimulated with LPS or poly I:C elicited robust IFNγ production, whereas the concentration of IFNγ in cpdm BMDC-T cell cultures was significantly lower after LPS stimulation (Fig. 7A), indicating impaired Th1-polarizing abilities of cpdm BMDC.
T115 16957-17257 Sentence denotes Co-culture of allogeneic naïve CD4+ T cells with WT BMDC stimulated with LPS or poly I:C elicited robust IFNγ production, whereas the concentration of IFNγ in cpdm BMDC-T cell cultures was significantly lower after LPS stimulation (Fig. 7A), indicating impaired Th1-polarizing abilities of cpdm BMDC.
T5823 17258-17394 Sentence denotes In addition to TLR3/4 agonists, the TLR2 ligand Pam3CYS was used since it has been shown to induce both Th1 and Th2 responses [28]–[30].
T116 17258-17394 Sentence denotes In addition to TLR3/4 agonists, the TLR2 ligand Pam3CYS was used since it has been shown to induce both Th1 and Th2 responses [28]–[30].
T5824 17395-17507 Sentence denotes Pam3CYS-matured WT BMDC induced robust IFNγ production at a significantly higher level than cpdm BMDC (Fig. 7A).
T117 17395-17507 Sentence denotes Pam3CYS-matured WT BMDC induced robust IFNγ production at a significantly higher level than cpdm BMDC (Fig. 7A).
T5825 17508-17688 Sentence denotes The reduced Th1 differentiation following Pam3CYS stimulation is consistent with the recent report of decreased IL12 production in cpdm macrophages following TLR2 stimulation [31].
T118 17508-17688 Sentence denotes The reduced Th1 differentiation following Pam3CYS stimulation is consistent with the recent report of decreased IL12 production in cpdm macrophages following TLR2 stimulation [31].
T5826 17689-17856 Sentence denotes In contrast, more Th2-specific IL4 cytokine was produced in the cpdm BMDC co-cultures than the WT control (Fig. 7B), suggesting Th2-skewed immunogenicity of cpdm BMDC.
T119 17689-17856 Sentence denotes In contrast, more Th2-specific IL4 cytokine was produced in the cpdm BMDC co-cultures than the WT control (Fig. 7B), suggesting Th2-skewed immunogenicity of cpdm BMDC.
T5827 17857-18025 Sentence denotes The production of Th17-specific cytokine IL17A following LPS stimulation of dendritic cells was similar between stimulated WT and cpdm BMDC cocultures (data not shown).
T120 17857-18025 Sentence denotes The production of Th17-specific cytokine IL17A following LPS stimulation of dendritic cells was similar between stimulated WT and cpdm BMDC cocultures (data not shown).
T5828 18026-18266 Sentence denotes Despite the distinct Th1- and Th2-stimulating abilities of WT and cpdm BMDC, they were equally effective in IL2 production from BMDC-T cell co-cultures except for poly I:C stimulation where WT BMDC induced more IL2 than cpdm BMDC (Fig. 7C).
T121 18026-18266 Sentence denotes Despite the distinct Th1- and Th2-stimulating abilities of WT and cpdm BMDC, they were equally effective in IL2 production from BMDC-T cell co-cultures except for poly I:C stimulation where WT BMDC induced more IL2 than cpdm BMDC (Fig. 7C).
T5829 18267-18447 Sentence denotes The Th2-biased stimulating capability of cpdm BMDC when co-cultured with allogeneic naïve CD4+ T cells is consistent with the Th2-dominant cytokine phenotype observed in cpdm mice.
T122 18267-18447 Sentence denotes The Th2-biased stimulating capability of cpdm BMDC when co-cultured with allogeneic naïve CD4+ T cells is consistent with the Th2-dominant cytokine phenotype observed in cpdm mice.
T123 18448-18576 Sentence denotes 10.1371/journal.pone.0031809.g007 Figure 7 Stimulated cpdm BMDC induced Th2-biased cytokine production from naïve CD4+ T cells.
T15334 18492-18576 Sentence denotes Stimulated cpdm BMDC induced Th2-biased cytokine production from naïve CD4+ T cells.
T15335 18577-18735 Sentence denotes WT and cpdm BMDC (5×104 cells in 0.1 mL complete medium; MHC haplotype: H-2b) were incubated with medium, 100 ng/mL LPS, 25 µg/mL poly I:C or 5 µg/mL Pam3CYS.
T124 18577-18735 Sentence denotes WT and cpdm BMDC (5×104 cells in 0.1 mL complete medium; MHC haplotype: H-2b) were incubated with medium, 100 ng/mL LPS, 25 µg/mL poly I:C or 5 µg/mL Pam3CYS.
T15336 18736-18908 Sentence denotes After 24 hours, cells were washed with PBS and incubated with freshly isolated allogeneic naïve CD4+ T cells (2.5×105 cells in 0.1 mL complete medium; MHC haplotype: H-2d).
T125 18736-18908 Sentence denotes After 24 hours, cells were washed with PBS and incubated with freshly isolated allogeneic naïve CD4+ T cells (2.5×105 cells in 0.1 mL complete medium; MHC haplotype: H-2d).
T15337 18909-19014 Sentence denotes After 5 days, supernatants were collected and the secretion of IFNγ, IL4, and IL2 were measured by ELISA.
T126 18909-19014 Sentence denotes After 5 days, supernatants were collected and the secretion of IFNγ, IL4, and IL2 were measured by ELISA.
T15338 19015-19172 Sentence denotes Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC.
T127 19015-19172 Sentence denotes Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC.
T15339 19173-19282 Sentence denotes Samples from both negative controls had no detectable production of the aforementioned cytokines (not shown).
T128 19173-19282 Sentence denotes Samples from both negative controls had no detectable production of the aforementioned cytokines (not shown).
T15340 19283-19342 Sentence denotes Results are analyzed based on 2–4 mice per group. * P<0.05.
T129 19283-19342 Sentence denotes Results are analyzed based on 2–4 mice per group. * P<0.05.
T130 19344-19354 Sentence denotes Discussion
T6766 19355-19536 Sentence denotes The present report showed that null mutations of the mouse Sharpin gene did not affect the steady-state distribution of splenic DC subsets nor the development and phenotype of BMDC.
T131 19355-19536 Sentence denotes The present report showed that null mutations of the mouse Sharpin gene did not affect the steady-state distribution of splenic DC subsets nor the development and phenotype of BMDC.
T6767 19537-19659 Sentence denotes However, loss of SHARPIN significantly diminished the capacity of BMDC to secrete inflammatory cytokines and nitric oxide.
T132 19537-19659 Sentence denotes However, loss of SHARPIN significantly diminished the capacity of BMDC to secrete inflammatory cytokines and nitric oxide.
T6768 19660-19830 Sentence denotes The attenuated cytokine production was not due to the presence of anti-inflammatory inhibitors, and can be largely explained by selective inactivation of NF-κB signaling.
T133 19660-19830 Sentence denotes The attenuated cytokine production was not due to the presence of anti-inflammatory inhibitors, and can be largely explained by selective inactivation of NF-κB signaling.
T6769 19831-19970 Sentence denotes Stimulated cpdm BMDC exhibited Th2-biased T cell-polarizing capabilities, consistent with the Th2 cytokine-dominant phenotype in cpdm mice.
T134 19831-19970 Sentence denotes Stimulated cpdm BMDC exhibited Th2-biased T cell-polarizing capabilities, consistent with the Th2 cytokine-dominant phenotype in cpdm mice.
T6770 19971-20152 Sentence denotes Together, these results indicate an indispensible role of SHARPIN in regulating DC immunological functions, disruption of which may contribute to the development of immune diseases.
T135 19971-20152 Sentence denotes Together, these results indicate an indispensible role of SHARPIN in regulating DC immunological functions, disruption of which may contribute to the development of immune diseases.
T6771 20153-20290 Sentence denotes Since WT and cpdm mice are both specific pathogen-free, the nature of the trigger of the severe inflammation in cpdm mice is not obvious.
T136 20153-20290 Sentence denotes Since WT and cpdm mice are both specific pathogen-free, the nature of the trigger of the severe inflammation in cpdm mice is not obvious.
T6772 20291-20484 Sentence denotes One such initiating factor could be endogenous apoptotic and/or necrotic cells that can release danger-associated molecular patterns (DAMPs) to launch and amplify an inflammatory response [32].
T137 20291-20484 Sentence denotes One such initiating factor could be endogenous apoptotic and/or necrotic cells that can release danger-associated molecular patterns (DAMPs) to launch and amplify an inflammatory response [32].
T6773 20485-20644 Sentence denotes Such ‘sterile’ inflammation could be initiated and take place in all organs and tissues affected in cpdm mice, thus causing multi-organ inflammatory disorders.
T138 20485-20644 Sentence denotes Such ‘sterile’ inflammation could be initiated and take place in all organs and tissues affected in cpdm mice, thus causing multi-organ inflammatory disorders.
T6774 20645-20700 Sentence denotes This hypothesis is supported by several recent studies.
T139 20645-20700 Sentence denotes This hypothesis is supported by several recent studies.
T6775 20701-20997 Sentence denotes Fibroblasts of cpdm mice are highly sensitive to TNFα-induced cell death and the cpdm phenotype can be partially rescued by deletion of TNF [7], [8], suggesting that deficiency of SHARPIN compromises the anti-apoptotic mechanisms in cpdm mice resulting in cell death-induced inflammatory disease.
T140 20701-20997 Sentence denotes Fibroblasts of cpdm mice are highly sensitive to TNFα-induced cell death and the cpdm phenotype can be partially rescued by deletion of TNF [7], [8], suggesting that deficiency of SHARPIN compromises the anti-apoptotic mechanisms in cpdm mice resulting in cell death-induced inflammatory disease.
T6776 20998-21156 Sentence denotes Apoptosis of keratinocytes is a prominent feature of the skin lesions in cpdm mice [33] and this is mediated by caspase-dependent mitochondrial pathways [34].
T141 20998-21156 Sentence denotes Apoptosis of keratinocytes is a prominent feature of the skin lesions in cpdm mice [33] and this is mediated by caspase-dependent mitochondrial pathways [34].
T6777 21157-21397 Sentence denotes These induced and/or intrinsic apoptotic cells can release various types of DAMPs that exert their pro-inflammatory properties by activating DC through pattern recognition receptors [32], [35], such as HMGB1 recognized by TLR2/4 [36], [37].
T142 21157-21397 Sentence denotes These induced and/or intrinsic apoptotic cells can release various types of DAMPs that exert their pro-inflammatory properties by activating DC through pattern recognition receptors [32], [35], such as HMGB1 recognized by TLR2/4 [36], [37].
T6778 21398-21580 Sentence denotes Consistent with the impaired Th1 immune response in cpdm mice [5], stimulated BMDC weakly polarized Th1 differentiation, but strongly supported the development of Th2 effector cells.
T143 21398-21580 Sentence denotes Consistent with the impaired Th1 immune response in cpdm mice [5], stimulated BMDC weakly polarized Th1 differentiation, but strongly supported the development of Th2 effector cells.
T6779 21581-21786 Sentence denotes Combined with the dramatic effect of IL12 treatment on the phenotype of these mice [5], this suggests that the Th2-biased systemic inflammation in cpdm mice is caused by reduced IL12P70 production from DC.
T144 21581-21786 Sentence denotes Combined with the dramatic effect of IL12 treatment on the phenotype of these mice [5], this suggests that the Th2-biased systemic inflammation in cpdm mice is caused by reduced IL12P70 production from DC.
T6780 21787-21996 Sentence denotes The importance of IL12P70 in regulating Th1 and Th2 immune responses in mice was clearly demonstrated in IL12P35- and IL12P40-deficient mice [38], [39] and through clinical studies in human patients [40]–[42].
T145 21787-21996 Sentence denotes The importance of IL12P70 in regulating Th1 and Th2 immune responses in mice was clearly demonstrated in IL12P35- and IL12P40-deficient mice [38], [39] and through clinical studies in human patients [40]–[42].
T6781 21997-22160 Sentence denotes NF-κB, TBK/IRF3, and MAPK signaling are important pathways activated by LPS or poly I:C and disruption of either pathway may lead to decreased cytokine expression.
T146 21997-22160 Sentence denotes NF-κB, TBK/IRF3, and MAPK signaling are important pathways activated by LPS or poly I:C and disruption of either pathway may lead to decreased cytokine expression.
T6782 22161-22429 Sentence denotes NF-κB activation was selectively inhibited in LPS and poly I:C stimulated cpdm BMDC compared to wild type, while activation of TBK1/IRF3, ERK1/2, or p38 was not, indicating that disrupted NF-κB signaling in cpdm DC is responsible for the defective cytokine expression.
T147 22161-22429 Sentence denotes NF-κB activation was selectively inhibited in LPS and poly I:C stimulated cpdm BMDC compared to wild type, while activation of TBK1/IRF3, ERK1/2, or p38 was not, indicating that disrupted NF-κB signaling in cpdm DC is responsible for the defective cytokine expression.
T6783 22430-22566 Sentence denotes These results seem to contradict a recent study that found increased levels of NF-κB activation and IL1 transcription in cpdm mice [43].
T148 22430-22566 Sentence denotes These results seem to contradict a recent study that found increased levels of NF-κB activation and IL1 transcription in cpdm mice [43].
T6784 22567-22738 Sentence denotes This difference can probably be attributed to the different cells and tissues used in these studies, and may point to cell- and tissue- type specific functions of SHARPIN.
T149 22567-22738 Sentence denotes This difference can probably be attributed to the different cells and tissues used in these studies, and may point to cell- and tissue- type specific functions of SHARPIN.
T6785 22739-22829 Sentence denotes This is consistent with recent reports of tissue-specific effects of NF-κB signaling [44].
T150 22739-22829 Sentence denotes This is consistent with recent reports of tissue-specific effects of NF-κB signaling [44].
T6786 22830-23063 Sentence denotes Ubiquitous activation of NF-κB by removing inhibitors such as A20 and ITCH through genetic manipulation results in widespread inflammation, consistent with the role of NF-κB in the production of pro-inflammatory mediators [45], [46].
T151 22830-23063 Sentence denotes Ubiquitous activation of NF-κB by removing inhibitors such as A20 and ITCH through genetic manipulation results in widespread inflammation, consistent with the role of NF-κB in the production of pro-inflammatory mediators [45], [46].
T6787 23064-23248 Sentence denotes However, selective inhibition of NF-κB activation in parenchymal cells of the skin, liver, and intestine results in chronic inflammation driven by NF-κB competent leukocytes [47]–[50].
T152 23064-23248 Sentence denotes However, selective inhibition of NF-κB activation in parenchymal cells of the skin, liver, and intestine results in chronic inflammation driven by NF-κB competent leukocytes [47]–[50].
T6788 23249-23441 Sentence denotes This indicates that a balance between the pro-inflammatory role of NF-κB in leukocytes and the anti-inflammatory role in parenchymal cells is critical in the maintenance of tissue homeostasis.
T153 23249-23441 Sentence denotes This indicates that a balance between the pro-inflammatory role of NF-κB in leukocytes and the anti-inflammatory role in parenchymal cells is critical in the maintenance of tissue homeostasis.
T6789 23442-23596 Sentence denotes Experiments with mice with cell- and tissue-specific deletion of Sharpin, currently under way, will help to elucidate the role of SHARPIN in inflammation.
T154 23442-23596 Sentence denotes Experiments with mice with cell- and tissue-specific deletion of Sharpin, currently under way, will help to elucidate the role of SHARPIN in inflammation.
T6790 23597-23778 Sentence denotes Despite defective NF-κB activation in the absence of Sharpin expression, there was no significant change in splenic DC populations or expression of co-stimulatory molecules on BMDC.
T155 23597-23778 Sentence denotes Despite defective NF-κB activation in the absence of Sharpin expression, there was no significant change in splenic DC populations or expression of co-stimulatory molecules on BMDC.
T6791 23779-23982 Sentence denotes Different NF-κB subunits involved in the canonical and non-canonical branches of the NF-κB signaling pathway have distinct functions to control specific aspects of DC development and function [51], [52].
T156 23779-23982 Sentence denotes Different NF-κB subunits involved in the canonical and non-canonical branches of the NF-κB signaling pathway have distinct functions to control specific aspects of DC development and function [51], [52].
T6792 23983-24227 Sentence denotes The non-canonical pathway (p100 processing to produce p52) appears to be intact in SHARPIN-deficient cells [7], [9] suggesting that the NF-κB heterodimer p52/RELB is sufficient to maintain the normal regulation of DC homeostasis and maturation.
T157 23983-24227 Sentence denotes The non-canonical pathway (p100 processing to produce p52) appears to be intact in SHARPIN-deficient cells [7], [9] suggesting that the NF-κB heterodimer p52/RELB is sufficient to maintain the normal regulation of DC homeostasis and maturation.
T6793 24228-24343 Sentence denotes The molecular basis by which the Sharpin mutation causes reduced NF-κB activation in BMDC remains to be determined.
T158 24228-24343 Sentence denotes The molecular basis by which the Sharpin mutation causes reduced NF-κB activation in BMDC remains to be determined.
T6794 24344-24449 Sentence denotes LPS and poly I:C used here are well-defined ligands that specifically engage TLR4 and TLR3, respectively.
T159 24344-24449 Sentence denotes LPS and poly I:C used here are well-defined ligands that specifically engage TLR4 and TLR3, respectively.
T6795 24450-24641 Sentence denotes The expression profile of surface TLR4 complexes is similar between WT and cpdm BMDC suggesting that defective NF-κB activation is not a result of differential TLR expression on target cells.
T160 24450-24641 Sentence denotes The expression profile of surface TLR4 complexes is similar between WT and cpdm BMDC suggesting that defective NF-κB activation is not a result of differential TLR expression on target cells.
T6796 24642-24796 Sentence denotes LPS engages TLR4 to activate MYD88-dependent and TRIF-dependent pathways, whereas TLR3 stimulated by poly I:C only triggers TRIF-dependent signaling [53].
T161 24642-24796 Sentence denotes LPS engages TLR4 to activate MYD88-dependent and TRIF-dependent pathways, whereas TLR3 stimulated by poly I:C only triggers TRIF-dependent signaling [53].
T6797 24797-24990 Sentence denotes The defective NF-κB activation by both stimuli suggests that the Sharpin mutation interferes with the protein adaptors or kinases shared by both signaling pathways, such as RIP1 and TRAF6 [18].
T162 24797-24990 Sentence denotes The defective NF-κB activation by both stimuli suggests that the Sharpin mutation interferes with the protein adaptors or kinases shared by both signaling pathways, such as RIP1 and TRAF6 [18].
T6798 24991-25229 Sentence denotes Recent studies demonstrated that SHARPIN interacts with HOIP to form LUBAC that exerts its linear-ubiquitin-chain-ligase activity on NF-κB signaling players RIP1 and NEMO [7], an essential step for intact TNFα-stimulated NF-κB activation.
T163 24991-25229 Sentence denotes Recent studies demonstrated that SHARPIN interacts with HOIP to form LUBAC that exerts its linear-ubiquitin-chain-ligase activity on NF-κB signaling players RIP1 and NEMO [7], an essential step for intact TNFα-stimulated NF-κB activation.
T6799 25230-25347 Sentence denotes The Sharpin null mutation disrupts the ubiquitylation process and abrogates the TNFα-induced NF-κB signaling pathway.
T164 25230-25347 Sentence denotes The Sharpin null mutation disrupts the ubiquitylation process and abrogates the TNFα-induced NF-κB signaling pathway.
T6800 25348-25532 Sentence denotes Since TNFR and TLR partially share their downstream signaling cascades, a similar ubiquitin-mediated regulation may hold true for SHARPIN in LPS- and poly I:C-induced NF-κB activation.
T165 25348-25532 Sentence denotes Since TNFR and TLR partially share their downstream signaling cascades, a similar ubiquitin-mediated regulation may hold true for SHARPIN in LPS- and poly I:C-induced NF-κB activation.
T6801 25533-25687 Sentence denotes In summary, the present study identified an indispensible role of SHARPIN in the production of pro-inflammatory mediators and TLR-induced NF-κB signaling.
T166 25533-25687 Sentence denotes In summary, the present study identified an indispensible role of SHARPIN in the production of pro-inflammatory mediators and TLR-induced NF-κB signaling.
T6802 25688-25818 Sentence denotes The impaired Th1-stimulating ability of Sharpin-deficient DC may account for the Th2-dominant inflammatory phenotype of cpdm mice.
T167 25688-25818 Sentence denotes The impaired Th1-stimulating ability of Sharpin-deficient DC may account for the Th2-dominant inflammatory phenotype of cpdm mice.
T6803 25819-25902 Sentence denotes The balance between Th1 and Th2 differentiation is critical for immune homeostasis.
T168 25819-25902 Sentence denotes The balance between Th1 and Th2 differentiation is critical for immune homeostasis.
T6804 25903-26127 Sentence denotes A better understanding of how such balance is maintained will help design cytokine treatment for human diseases with Th2-biased cytokine secretion similar to the mouse cpdm, such as allergies and hypereosinophilic syndromes.
T169 25903-26127 Sentence denotes A better understanding of how such balance is maintained will help design cytokine treatment for human diseases with Th2-biased cytokine secretion similar to the mouse cpdm, such as allergies and hypereosinophilic syndromes.
T170 26129-26150 Sentence denotes Materials and Methods
T9577 26152-26168 Sentence denotes Ethics Statement
T171 26152-26168 Sentence denotes Ethics Statement
T9578 26169-26297 Sentence denotes All mouse work was carried out in strict accordance with protocols approved by the Institutional Animal Care and Use Committees.
T172 26169-26297 Sentence denotes All mouse work was carried out in strict accordance with protocols approved by the Institutional Animal Care and Use Committees.
T9629 26299-26303 Sentence denotes Mice
T173 26299-26303 Sentence denotes Mice
T9630 26304-26497 Sentence denotes Specific-pathogen free colonies of C57BL/KaLawRij-Sharpincpdm/RijSunJ (JR#7599) and WT mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a barrier facility.
T174 26304-26497 Sentence denotes Specific-pathogen free colonies of C57BL/KaLawRij-Sharpincpdm/RijSunJ (JR#7599) and WT mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained in a barrier facility.
T175 26498-26558 Sentence denotes Normal littermate controls were either +/+ or +/Sharpincpdm.
T9631 26498-26645 Sentence denotes Normal littermate controls were either +/+ or +/Sharpincpdm. These control animals were phenotypically indistinguishable and are referred to as WT.
T176 26559-26645 Sentence denotes These control animals were phenotypically indistinguishable and are referred to as WT.
T9632 26646-26708 Sentence denotes Sex-matched WT and mutant mice were used at 6–10 weeks of age.
T177 26646-26708 Sentence denotes Sex-matched WT and mutant mice were used at 6–10 weeks of age.
T9633 26709-26890 Sentence denotes For some experiments, cpdm mice were crossed with transgenic mice with a bacterial artificial chromosome (BAC) containing the Sharpin gene (FVB/NJ-Tg(RP24-173I23)1Sun/Sun, JR#8279).
T178 26709-26890 Sentence denotes For some experiments, cpdm mice were crossed with transgenic mice with a bacterial artificial chromosome (BAC) containing the Sharpin gene (FVB/NJ-Tg(RP24-173I23)1Sun/Sun, JR#8279).
T9634 26891-27029 Sentence denotes These mice were backcrossed onto the C57BL/KaLawRij-Sharpincpdm/RijSunJ background and N4 mice were used in the experiments reported here.
T179 26891-27029 Sentence denotes These mice were backcrossed onto the C57BL/KaLawRij-Sharpincpdm/RijSunJ background and N4 mice were used in the experiments reported here.
T9635 27030-27161 Sentence denotes All mouse work was carried out in strict accordance with the approved protocols by the Institutional Animal Care and Use Committee.
T180 27030-27161 Sentence denotes All mouse work was carried out in strict accordance with the approved protocols by the Institutional Animal Care and Use Committee.
T9960 27163-27190 Sentence denotes Constructs and transfection
T181 27163-27190 Sentence denotes Constructs and transfection
T9961 27191-27298 Sentence denotes The complementary DNA (cDNA) of the mouse Sharpin gene was cloned and amplified from RAW264.7 RNA extracts.
T182 27191-27298 Sentence denotes The complementary DNA (cDNA) of the mouse Sharpin gene was cloned and amplified from RAW264.7 RNA extracts.
T183 27299-27428 Sentence denotes The primer sequences were forward, 5′-CC ATG GCG ATG TCG CCG CCC GCC GGC GGT; reverse, 5′- AAG CTT CTA GGT GGA AGC TGC AGC AAG A.
T9962 27299-27492 Sentence denotes The primer sequences were forward, 5′-CC ATG GCG ATG TCG CCG CCC GCC GGC GGT; reverse, 5′- AAG CTT CTA GGT GGA AGC TGC AGC AAG A. The Sharpin cDNA was cloned into expression vector pFLAG-CMV-2.
T184 27429-27492 Sentence denotes The Sharpin cDNA was cloned into expression vector pFLAG-CMV-2.
T9963 27493-27582 Sentence denotes Murine fibroblasts and macrophages were were used to express recombinant SHARPIN protein.
T185 27493-27582 Sentence denotes Murine fibroblasts and macrophages were were used to express recombinant SHARPIN protein.
T9964 27583-27663 Sentence denotes Cells (2×104) were seeded in 96-well treated plates the day before transfection.
T186 27583-27663 Sentence denotes Cells (2×104) were seeded in 96-well treated plates the day before transfection.
T9965 27664-27770 Sentence denotes After overnight incubation, 200 ng pFLAG-SHARPIN plasmids were transfected with 0.5 ul Lipofectamine 2000.
T187 27664-27770 Sentence denotes After overnight incubation, 200 ng pFLAG-SHARPIN plasmids were transfected with 0.5 ul Lipofectamine 2000.
T9966 27771-27834 Sentence denotes 24 hours later, cells were incubated with fresh culture medium.
T188 27771-27834 Sentence denotes 24 hours later, cells were incubated with fresh culture medium.
T9967 27835-27945 Sentence denotes After another 24 hours, cells were lysed to confirm the FLAG-SHARPIN expression by immunoblots with anti-FLAG.
T189 27835-27945 Sentence denotes After another 24 hours, cells were lysed to confirm the FLAG-SHARPIN expression by immunoblots with anti-FLAG.
T9968 27946-28081 Sentence denotes Cells with no transfection and transfected with empty vector pFLAG-CMV-2 were used as negative control in all transfection experiments.
T190 27946-28081 Sentence denotes Cells with no transfection and transfected with empty vector pFLAG-CMV-2 were used as negative control in all transfection experiments.
T10335 28083-28101 Sentence denotes Generation of BMDC
T191 28083-28101 Sentence denotes Generation of BMDC
T10336 28102-28156 Sentence denotes BMDC were developed as previously reported [54], [55].
T192 28102-28156 Sentence denotes BMDC were developed as previously reported [54], [55].
T10337 28157-28206 Sentence denotes Cells were collected after 10–12 days of culture.
T193 28157-28206 Sentence denotes Cells were collected after 10–12 days of culture.
T10338 28207-28263 Sentence denotes The cell yield was 2–3×107 cells/mouse with 80–95% BMDC.
T194 28207-28263 Sentence denotes The cell yield was 2–3×107 cells/mouse with 80–95% BMDC.
T10423 28265-28305 Sentence denotes Phenotype of BMDC and splenic DC subsets
T195 28265-28305 Sentence denotes Phenotype of BMDC and splenic DC subsets
T10424 28306-28409 Sentence denotes The phenotype of BMDC was determined before and after 24 hour culture in the presence of 100 ng/mL LPS.
T196 28306-28409 Sentence denotes The phenotype of BMDC was determined before and after 24 hour culture in the presence of 100 ng/mL LPS.
T10425 28410-28816 Sentence denotes The cells were incubated in PBS with 0.1% NaN3, 1% BSA and 10% normal rabbit serum for 20 minutes on ice, washed and incubated for 30 minutes on ice with Alex Fluor-labeled anti-CD11c (MCD11C20, CALTAG) in combination with PE- anti-CD40 (3/23, BD Biosciences), PE-anti-CD80 (16-10A1, eBioscience), PE-anti-CD86 (P03.1, eBioscience), PE-anti-CD14 (Sa2.8, eBioscience) and biotinylated anti-TLR4 (BioLegend).
T197 28410-28816 Sentence denotes The cells were incubated in PBS with 0.1% NaN3, 1% BSA and 10% normal rabbit serum for 20 minutes on ice, washed and incubated for 30 minutes on ice with Alex Fluor-labeled anti-CD11c (MCD11C20, CALTAG) in combination with PE- anti-CD40 (3/23, BD Biosciences), PE-anti-CD80 (16-10A1, eBioscience), PE-anti-CD86 (P03.1, eBioscience), PE-anti-CD14 (Sa2.8, eBioscience) and biotinylated anti-TLR4 (BioLegend).
T10426 28817-28939 Sentence denotes The cells incubated with biotinylated anti-TLR4/MD-2 were washed twice and incubated for 30 minutes on ice with avidin-PE.
T198 28817-28939 Sentence denotes The cells incubated with biotinylated anti-TLR4/MD-2 were washed twice and incubated for 30 minutes on ice with avidin-PE.
T10427 28940-29044 Sentence denotes To isolate splenic DC, spleens were collagenase digested and subject to Percoll gradient centrifugation.
T199 28940-29044 Sentence denotes To isolate splenic DC, spleens were collagenase digested and subject to Percoll gradient centrifugation.
T10428 29045-29216 Sentence denotes The bands at the 35–55% interface were collected to stain with PE-anti-CD11c (HL3, BioLegend), APC-anti-PDCA-1 (927, BioLegend) and FITC-anti-CD8α (53–6.7, BD BioScience).
T200 29045-29216 Sentence denotes The bands at the 35–55% interface were collected to stain with PE-anti-CD11c (HL3, BioLegend), APC-anti-PDCA-1 (927, BioLegend) and FITC-anti-CD8α (53–6.7, BD BioScience).
T10429 29217-29309 Sentence denotes The cells were washed twice, fixed in 2% paraformaldehyde, and stored at 4°C until analysis.
T201 29217-29309 Sentence denotes The cells were washed twice, fixed in 2% paraformaldehyde, and stored at 4°C until analysis.
T10430 29310-29372 Sentence denotes Flow cytometry was performed in an Excel (Coulter) instrument.
T202 29310-29372 Sentence denotes Flow cytometry was performed in an Excel (Coulter) instrument.
T10431 29373-29509 Sentence denotes Dead cells were omitted from the analysis by gating on forward and 90° light scatter, and 10,000 cells were analyzed by FlowJo software.
T203 29373-29509 Sentence denotes Dead cells were omitted from the analysis by gating on forward and 90° light scatter, and 10,000 cells were analyzed by FlowJo software.
T10960 29511-29543 Sentence denotes BMDC-T cell in vitro interaction
T204 29511-29543 Sentence denotes BMDC-T cell in vitro interaction
T10961 29544-29640 Sentence denotes WT and cpdm BMDC (5×104) were stimulated with medium, 1 µg/mL LPS, 25 µg/mL and 5 µg/mL Pam3CYS.
T205 29544-29640 Sentence denotes WT and cpdm BMDC (5×104) were stimulated with medium, 1 µg/mL LPS, 25 µg/mL and 5 µg/mL Pam3CYS.
T10962 29641-29698 Sentence denotes 24 hours later, cells were collected and washed with PBS.
T206 29641-29698 Sentence denotes 24 hours later, cells were collected and washed with PBS.
T10963 29699-29877 Sentence denotes Allogeneic naïve CD4+ T cells were isolated from spleens of BALB/c mice by negative selection kit (Invitrogen) and were then added at 2.5×105 and co-cultured with activated BMDC.
T207 29699-29877 Sentence denotes Allogeneic naïve CD4+ T cells were isolated from spleens of BALB/c mice by negative selection kit (Invitrogen) and were then added at 2.5×105 and co-cultured with activated BMDC.
T10964 29878-29983 Sentence denotes After 5 days, supernatant was collected and the secretion of IFNγ, IL4, IL2, and IL17A measured by ELISA.
T208 29878-29983 Sentence denotes After 5 days, supernatant was collected and the secretion of IFNγ, IL4, IL2, and IL17A measured by ELISA.
T10965 29984-30141 Sentence denotes Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC.
T209 29984-30141 Sentence denotes Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC.
T10966 30142-30212 Sentence denotes Both negative controls show no production of aforementioned cytokines.
T210 30142-30212 Sentence denotes Both negative controls show no production of aforementioned cytokines.
T11351 30214-30236 Sentence denotes RNA expression by BMDC
T211 30214-30236 Sentence denotes RNA expression by BMDC
T212 30237-30375 Sentence denotes BMDC were cultured at 106 cells/mL in 10 mL of RPMI-1640 complete medium in the presence or absence of 100 ng/mL LPS or 25 µg/mL poly I:C.
T11352 30237-30484 Sentence denotes BMDC were cultured at 106 cells/mL in 10 mL of RPMI-1640 complete medium in the presence or absence of 100 ng/mL LPS or 25 µg/mL poly I:C. After 1 and 2 hours, RNA was isolated with TRI-reagent (Sigma) according to the manufacturer's instructions.
T213 30376-30484 Sentence denotes After 1 and 2 hours, RNA was isolated with TRI-reagent (Sigma) according to the manufacturer's instructions.
T11353 30485-30567 Sentence denotes The expression of Il6, Il12p40, Gmcsf, and Sharpin mRNA was determined by qRT-PCR.
T214 30485-30567 Sentence denotes The expression of Il6, Il12p40, Gmcsf, and Sharpin mRNA was determined by qRT-PCR.
T215 30568-30626 Sentence denotes Primers and probes were purchased from Applied Biosystems.
T11354 30568-30970 Sentence denotes Primers and probes were purchased from Applied Biosystems. Reverse transcription was performed at 42°C for 60 minutes with the final denaturation step at 90°C for 5 minutes in 30 µl containing 0.5 µg of total RNA. dNTPs, oligo(dT)15 primer, recombinant RNasin Ribonuclease Inhibitor, and M-MLV Reverse Transcriptase (all from Promega, Madison, WI) were used according to the manufacturer's instruction.
T216 30627-30970 Sentence denotes Reverse transcription was performed at 42°C for 60 minutes with the final denaturation step at 90°C for 5 minutes in 30 µl containing 0.5 µg of total RNA. dNTPs, oligo(dT)15 primer, recombinant RNasin Ribonuclease Inhibitor, and M-MLV Reverse Transcriptase (all from Promega, Madison, WI) were used according to the manufacturer's instruction.
T11355 30971-31304 Sentence denotes Reverse transcription was done in a PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). qRT-PCR was performed in ABI Prism 7700 Sequence Detection System with TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA) for mouse Actb, IL6, IL12p40, Ifnβ, Gmcsf, and Sharpin according to the manufacturer's protocol.
T217 30971-31304 Sentence denotes Reverse transcription was done in a PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). qRT-PCR was performed in ABI Prism 7700 Sequence Detection System with TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA) for mouse Actb, IL6, IL12p40, Ifnβ, Gmcsf, and Sharpin according to the manufacturer's protocol.
T11356 31305-31378 Sentence denotes The endogenous standard for normalization of the target gene was β-actin.
T218 31305-31378 Sentence denotes The endogenous standard for normalization of the target gene was β-actin.
T11357 31379-31448 Sentence denotes Relative gene expression was calculated using the 2−ΔΔCt method [56].
T219 31379-31448 Sentence denotes Relative gene expression was calculated using the 2−ΔΔCt method [56].
T11921 31450-31485 Sentence denotes In vitro cytokine secretion by BMDC
T220 31450-31485 Sentence denotes In vitro cytokine secretion by BMDC
T11922 31486-31625 Sentence denotes The BMDC were cultured in triplicate wells of 24-well or 96-well plates at 106 or 105 cells/mL, respectively, in RPMI-1640 complete medium.
T221 31486-31625 Sentence denotes The BMDC were cultured in triplicate wells of 24-well or 96-well plates at 106 or 105 cells/mL, respectively, in RPMI-1640 complete medium.
T222 31626-31703 Sentence denotes The cells were washed and stimulated with 100 ng/mL LPS or 25 µg/mL poly I:C.
T11923 31626-31767 Sentence denotes The cells were washed and stimulated with 100 ng/mL LPS or 25 µg/mL poly I:C. After 24 hours, supernatants were harvested for ELISA analysis.
T223 31704-31767 Sentence denotes After 24 hours, supernatants were harvested for ELISA analysis.
T11924 31768-31852 Sentence denotes The presence of nitrite in the supernatants was determined using the Griess reagent.
T224 31768-31852 Sentence denotes The presence of nitrite in the supernatants was determined using the Griess reagent.
T12083 31854-31872 Sentence denotes Immunofluorescence
T225 31854-31872 Sentence denotes Immunofluorescence
T12084 31873-31941 Sentence denotes Fibroblast and macrophage cells were transfected with pFLAG-SHARPIN.
T226 31873-31941 Sentence denotes Fibroblast and macrophage cells were transfected with pFLAG-SHARPIN.
T12085 31942-32085 Sentence denotes 48 hours later, cells were washed briefly with PBS, then fixed in cold methanol at −20°C for 10 minutes and cold acetone at -20°C for 1 minute.
T227 31942-32085 Sentence denotes 48 hours later, cells were washed briefly with PBS, then fixed in cold methanol at −20°C for 10 minutes and cold acetone at -20°C for 1 minute.
T12086 32086-32162 Sentence denotes Incubate cells with PBS+1% BSA for 15 minutes to block non-specific binding.
T228 32086-32162 Sentence denotes Incubate cells with PBS+1% BSA for 15 minutes to block non-specific binding.
T12087 32163-32308 Sentence denotes Fixed cells were then incubated with anti-FLAG (1∶100) as the primary antibody at room temperature for 1 hour, followed by three 5-minute washes.
T229 32163-32308 Sentence denotes Fixed cells were then incubated with anti-FLAG (1∶100) as the primary antibody at room temperature for 1 hour, followed by three 5-minute washes.
T12088 32309-32473 Sentence denotes Cells were further incubated with goat anti-rabbit IgG-FITC (1∶100) as the secondary antibody at room temperature for 30 minutes, followed by three 5-minute washes.
T230 32309-32473 Sentence denotes Cells were further incubated with goat anti-rabbit IgG-FITC (1∶100) as the secondary antibody at room temperature for 30 minutes, followed by three 5-minute washes.
T12089 32474-32537 Sentence denotes Cells were then examined using inverse fluorescence microscope.
T231 32474-32537 Sentence denotes Cells were then examined using inverse fluorescence microscope.
T12382 32539-32550 Sentence denotes Immunoblots
T232 32539-32550 Sentence denotes Immunoblots
T12383 32551-32694 Sentence denotes After adding stimulating ligands, 100 ng/mL LPS or 25 µg/mL poly I:C, BMDC were collected and lysed at 0-, 15-, 30-, and 60-minute time points.
T233 32551-32694 Sentence denotes After adding stimulating ligands, 100 ng/mL LPS or 25 µg/mL poly I:C, BMDC were collected and lysed at 0-, 15-, 30-, and 60-minute time points.
T12384 32695-32901 Sentence denotes Immunoblots were performed with antibodies (Cell Signaling Technology) against p-IKK1/2(#2697), p-IκBα (#9246), IκBα (#4814), p-TBK1(#5483), p-p38 (#9216), p38 (#9212), p-ERK1/2 (#4376), and ERK1/2 (#4695).
T234 32695-32901 Sentence denotes Immunoblots were performed with antibodies (Cell Signaling Technology) against p-IKK1/2(#2697), p-IκBα (#9246), IκBα (#4814), p-TBK1(#5483), p-p38 (#9216), p38 (#9212), p-ERK1/2 (#4376), and ERK1/2 (#4695).
T12385 32902-32978 Sentence denotes Beta-actin (sc-47778, Santa Cruz Biotechnology) was used as loading control.
T235 32902-32978 Sentence denotes Beta-actin (sc-47778, Santa Cruz Biotechnology) was used as loading control.
T12679 32980-33000 Sentence denotes Statistical analysis
T236 32980-33000 Sentence denotes Statistical analysis
T12680 33001-33033 Sentence denotes Data are expressed as mean ± SD.
T237 33001-33033 Sentence denotes Data are expressed as mean ± SD.
T12681 33034-33150 Sentence denotes The statistical significance of differences of means between experimental groups was determined by Students' t-test.
T238 33034-33150 Sentence denotes The statistical significance of differences of means between experimental groups was determined by Students' t-test.