Background Soluble NSF attachment protein receptor (SNARE) proteins are a highly conserved family of intracellular transport proteins. They are essential for vesicular trafficking of cytokines and membrane proteins between organelles and the cell surface. We have identified an essential role for the SNARE Syntaxin11 (STX11) in the differentiation of Th17 cells, which have been implicated in the formation of Ectopic Lymphoid Structures (ELS). We have previously shown that STX11 is over expressed in the follicular structures of inflamed colonic tissue. Given the importance of these structures in synovitis, we wanted to investigate this mechanism in the context of Rheumatoid Arthritis (RA).
Objectives To confirm the role of STX11 in Th17 cell differentiation and investigate STX11 in ELS formation in rheumatoid arthritis.
Methods CD4+ cells were sorted from the lamina propria of mice with DSS-induced acute colitis by FACS. Naïve CD4+ Thelper cells isolated from WT and STX11-/- mice were stimulated in Th17 polarising conditions for 3 days and assessed for IL-17 production by intracellular flow cytometry. Synovial tissue biopsies from established RA and OA patients were obtained from the EMR tissue biobank. Tissue was scored for ELS using immunohistochemical (IHC) staining for CD3, CD20, CD68 and CD21. Synovial tissue biopsies from 40 DMARD naïve patients in the Pathobiology of Early Arthritis Cohort (PEAC) study were sent for transcriptional analysis via micro-array based gene expression profiling.
Results Lymphoid structures were increased in number and size in the colons of DSS induced colitis mice. These structures stained positive for the SNARE STX11. CD4+ T-cells isolated from the colon of these mice had increased STX11 and IL-17 expression. Differentiation of naïve CD4+ T-cells from STX11-/- mice into a Th17 cell subset in vitro was significantly inhibited when measured by IL-17 production and mRNA expression. In RA, STX11 mRNA expression was increased in synovial tissue when compared to established OA patients. Furthermore, ELS positive synovial tissue from RA patients also stained for STX11 in B-and T-cell aggregates. Syntaxin Binding Protein 2 (STXBP2) is a regulatory chaperone protein required for STX11 stability and function in T-cells. In the PEAC study of early arthritis, there appears to be no change in STX11 expression between ELS positive and negative patient tissue, however STXBP2 expression was significantly increased in the ELS positive cohort of patients indicating an important role for these proteins in ELS structure regulation in auto immunity.
Conclusions STX11 is essential for Th17 differentiation and is clearly expressed in the germinal centre structures of both mouse and human inflamed tissue. STX11 is up regulated in RA synovitis and STXBP2 regulation of STX11 expression also appears to be important in ELS formation in synovitis. Further work is needed to identify if the function of STX11 and STXBP2 expression in these structures is due to its role in Th17 differentiation or differentiation of other Thelper cell subsets in ELS. Understanding these mechanisms will lead to a better understanding of ELS formation in rheumatoid arthritis.
Disclosure of Interest None declared
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