Career situation of first and presenting authorStudent for a master or a PhD.IntroductionTumour necrosis factor (TNF) is important in immune-mediated inflammatory diseases such as spondyloarthritis (SpA). Transmembrane (tm)TNF-transgenic (tg) mice1 that overexpress tmTNF develop SpA symptoms, including inflammation, ectopic lymphoid structures (ELS) in bone marrow (BM), bone destruction and bone formation. SpA patients have extensive angiogenesis in inflammatory and bone forming regions.ObjectivesTo investigate whether there is a link between pathological angiogenesis and ELS in tmTNF tg mice in the BM.MethodsAnkles, femora, tibiae, vertebrae and spleens from 6 and 12 weeks and 8 months old tmTNF tg mice and wild-type (WT) littermates (n=5 per age per group) were dissected and analyzed with confocal microscopy and analyzed 12 week old tmTNF tg and WT mice (n=4) with flow cytometry. To study the importance of TNF-R signaling, tmTNF tg mice lacking TNF-RI (tmTNF tgxTNF-RI-/-) or TNF-RII (tmTNF tgxTNF-RII-/-) (n=4 per group) were used.ResultsImmunofluorescent evaluation demonstrated that BM of tmTNF tg mice contained significantly more and extensive ELS. These ELS are limited to BM of the vertebrae and ankles, and are in close proximity of MECA79+high endothelial venules (HEVs). ELS predominantly consisted of B220+ B cells, of which most are IgD+ naive B cells. Preliminary flow cytometric analysis revealed a trend towards an increase in IgD-CD95+ germinal center B cells and CXCR5+PD-1+FoxP3-CTLA4- T follicular helper cells and CXCR5+PD-1+FoxP3+CTLA4+ in the vertebrae of tmTNF tg mice compared to WT littermates. Meanwhile, B cell lineages in the BM of tmTNF tg hind limbs were not altered. Furthermore, preliminary data indicates that BM and spleen from tmTNF tg mice contain more IgA+ plasma cells compared to WT littermates. tmTNF tgxTNF-RI-/- mice did not display lymphoid aggregates or HEVs in the BM, while tmTNF tgxTNF-RII-/- mice did, although to a lesser extent than tmTNF tg mice.ConclusionstmTNF overexpression in mice results in extensive ELS associated with HEVs in the BM, which is likely to be mediated through TNF-RI signaling. HEV formation may lead to persistence of inflammation in the BM which contributes to pathology.ReferenceAlexopoulou L, et al. Eur J Immunol 1997;27(10):2588–92Disclosure of InterestNone declared
Ann Rheum Dis 2013;72(Suppl 1):A1-A88 A23 EWRR abstractsCellular distance mapping revealed that T IH cells were spatially related to CD20 + B cells across LuN (42.9% of cells within 0.27 microns, n = 10 biopsies), control tonsil tissue (65.3%, n = 2 biopsies), and mixed RT cases (70.3%, n = 7 biopsies) as compared to other T cells (less than 20% for all groups, respectively). These associations remained unchanged after correction for total cellular density and T:B cell ratios. Low-density TCM cases showed a comparatively low rate of T IH :B cell association (15.0%, n = 8 biopsies) and these results were statistically significant (p < 0.0001 versus mixed RT and tonsil cases, p = 0.002 versus LuN). Comparing the above results against a theoretical model of random T IH :B-cell distribution revealed that the likelihood of our observations in LuN being due to chance was approximately 8.12 × 10 -41 . Conclusions Our data reveal that T IH cells are present in similar rates across cases of renal allograft rejection as well as LuN. Their presence is associated with a higher degree of TII as well as worse renal function at time of biopsy in LuN. T IH cells are more likely to form proximal conjugates with naïve and activated B cells in tissues of diseases associated with aberrant autoantibody production (SLE, mixed RT) but not in processes where autoantibodies are absent (TCM). Background and Objectives Vitamin D has suppressive effects on autoimmune diseases, such as rheumatoid arthritis (RA). Within these diseases, T-helper-17 (Th17) cells have been implicated to play a crucial role in the development and progression of chronic inflammation. Recently, we have found that the active vitamin D compound, 1.25(OH) 2 D 3 , has direct suppressive effects on both human and mouse Th17 cytokine expression and activity. Using gene-expression profiling, we aim to identify molecular targets of 1.25(OH) 2 D 3 signalling underlying this suppressive action of 1.25(OH) 2 D 3 in Th17 cells. Methods Primary Th17 cells were sorted from peripheral blood of treatment naïve patients with early RA and cultured with or without 1.25(OH) 2 D 3 . From these cultures gene-expression profiles were generated. Expression of genes of interest was confirmed by Q-PCR and/or specific ELISA. Results In the presence of 1.25(OH) 2 D 3 , protein expression of Th17 associated cytokines IL-17A and IL-22 was inhibited, while in contrast the anti-inflammatory cytokine IL-10 was induced. These findings were supported by the gene-expression profiles from these cultures. Furthermore, 1.25(OH) 2 D 3 inhibited transcription of the cytokine receptors IL-23R and IL-7R, which are involved in Th17 survival and proliferation. Chemokines CCL20 and CXCL10 were down-regulated and chemokine receptors CCR2, CXCR6, CXCR3 and CCR10 were up-regulated. Importantly, RORγ t, which is critically involved in Th17 differentiation and function and the cell-size regulator and oncogene c-Myc were down-regulated by 1.25(OH) 2 D 3 . Conclusions: From these findings, we concluded that 1.25(OH)...
BackgroundSites of chronic inflammation, such as rheumatoid arthritis synovial tissue, are characterized by neovascularization and often contain tertiary lymphoid structures with characteristic features of lymphoid organs such as endothelial venules (HEV), and sometimes even true germinal centers. Ligation of the lymphotoxin (LT)-β receptor (LTβR) results in activation of both canonical and NF-κB-Inducing Kinase (NIK)-dependent non-canonical NF-κB signaling in endothelial cells (ECs) and plays a crucial role in lymphoid neogenesis. Non-canonical NF-κB signaling in ECs promotes inflammation-induced angiogenesis and triggers the development of the cuboidal HEV appearance. However, the relative contribution of the individual pathways to the acquisition of leukocyte traffic-regulating properties by ECs is less well understood.ObjectivesTo identify the molecular pathways by which LTβR drives inflammatory activation of ECs to promote interactions with leukocytes.MethodsPrimary human ECs were treated with LTβ or LIGHT to activate LTβR. Induction of downstream signaling pathways was assessed by western blot analysis and NF-κB transcription factor ELISA. The expression of adhesion molecules, inflammatory cytokines and chemokines, such as CXCL1, CXCL5, CXCL8 and GM-CSF in ECs was measured by RT-qPCR and cytokine antibody arrays. EC interactions with leukocytes were determined by an adhesion assay, and EC barrier integrity was assessed by a permeability assay. To repress canonical NF-κB signaling pathway, a small molecule inhibitor of IKKβ was used, and inactivation of non-canonical NF-κB signaling was achieved with siRNAs targeting NFκB2. The role of NIK in LTβR signaling was investigated using small molecule inhibitors of NIK, siRNAs targeting NIK and adenoviral vectors encoding wild type and kinase-deficient NIK.ResultsLTβR triggering in ECs resulted in activation of both canonical and non-canonical NF-κB signaling pathways and induced the expression of inflammatory cytokines and chemokines (CXCL1, CXCL5, CXCL8, MCP-1, GM-CSF, CCL5). Consistent with inflammatory activation of ECs, LTβR ligation also induced adhesion of immune cells to activated endothelium and increased permeability across EC monolayers. IKKβ inhibition completely repressed LTβR-induced inflammatory activation of ECs, indicating that this process was mediated through canonical NF-κB signaling. Interestingly, inactivation of NIK with small molecule inhibitors and siRNAs significantly decreased LTβR-induced expression of inflammatory cytokines and adhesion of immune cells to endothelium, whereas silencing of NFκB2 had no effect. This suggests that the non-canonical pathway is dispensable for NIK-dependent activation of endothelial cells through the canonical NF-kB pathway. Further analyses, including silencing of NIK and NIK overexpression, demonstrated a role for NIK in activation of the canonical NF-kB pathway by amplifying IKK complex activity.ConclusionsThese findings suggest that in addition to its pivotal role in the non-canonical pathway, NIK can ser...
BackgroundInterleukin (IL)-17A is a pro-inflammatory cytokine and is involved in the pathogenesis of psoriatic arthritis (PsA) (1,2). Various cells can produce IL-17A. However, it is not clear which cell types in PsA patients are responsible for the production of IL-17A. In addition, the expression of IL-17RA and IL-17RC on different cell types is not well defined.ObjectivesTo identify IL-17A, IL-17RA and IL-17RC positive cells in blood of first diagnosed PsA patients with arthritis and in synovial fluid of established PsA patients with active disease.MethodsFresh blood was taken from first diagnosed DMARD and steroid naïve PsA patients (n=10), having arthritis in 1 or more joints (PsA blood). The diagnosis was made by a rheumatologist according to the CASPAR-criteria. In addition, fresh synovial fluid was obtained from established PsA patients (PsA SF) with active disease (n=10) and treated with either methotrexate (n=3) or adalumimab (n=3) or NSAIDs (n=4). Multicolor flow cytometric analysis was performed on PsA blood and PsA SF. For the detection of IL-17A, IL-17RA or IL-17RC the following antibodies were used: IL-17A-PE (eBioscience), IL-17RA or isotype control IgG1k (both Biolegend), IL-17RC or isotype control IgG2b (both R&D systems). The following markers were used to discriminate between different cell populations: T cell subsets (CD3, CD4, CD8, CD45RO, CCR6, TCRγδ), B cells (CD19), NK cells (CD15-CD16+), neutrophils (CD15+CD16+), monocytes (CD33+CD14+CD16+/-), mast cells (CD117+FcER1a+) and eosinophils (CD15+FcER1a+).ResultsDifferent lymphoid and myeloid cell types were IL-17A positive in PsA blood of first diagnosed PsA patients such as CD3+, TCRγδ+, CD4+, CD8+ lymphoid cells, CD14+ monocytes and eosinophils. In PsA SF of established PsA patients TCRγδ+ T cells, neutrophils, NK cells and eosinophils were IL-17A positive.In both groups, no difference in expression of IL-17RA and IL-17RC was found on CD4+, CD8+, CD4+CD45RO+CCR6+/-, TCRγδ+ and CD19+ lymphoid cells compared to their isotype control. In contrast, the expression of IL-17RA and IL-17RC was increased compared to their isotype control on neutrophils and monocytes in PsA blood and on neutrophils, monocytes, mast cells and eosinophils in PsA SF.ConclusionsThese preliminary data show that not only lymphoid cells but also specific myeloid cell types may be sources of IL-17A in PsA. Furthermore, not lymphoid cells but IL-17RA/IL-17RC positive myeloid cells such as monocytes, neutrophils, mast cells and eosinophils may be potential target cells for IL-17A.Together, these data suggest a more broad, but specific IL-17A-IL-17RA/RC signaling network between different cell types important in the IL-17A-driven pathogenesis of PsA.References Lubberts E. Nat Rev Rheumatol 2015, 11: 415–29.McInnes IB, et al. Lancet 2015, 386: 1137–46. Disclosure of InterestNone declared
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