OP0165 Joint-specific differences in the activation of the jak-stat pathway in rheumatoid arthritis
Authors: T. Masterson, K. Klein, E. Karouzakis, O. Distler, C. Ospelt, M. Frank Bertoncelj
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Synovial fibroblasts (SF) promote chronic joint inflammation and joint destruction in rheumatoid arthritis (RA). We have shown recently that SF from different joints exhibit profound differences in their transcriptomes, epigenomes and functions, which creates a unique microenvironment in each joint. This might influence the susceptibility of distinct joints to develop RA or lead to joint-specific differences in the disease severity or therapeutic response. <h3>Objectives</h3> To analyse differences in the JAK-STAT pathway in SF from different joints. <h3>Methods</h3> SF were isolated from knee, shoulder and hand joints of RA and osteoarthritis patients undergoing joint replacement surgery and from knee synovial biopsies of non-arthritic subjects with arthralgia. Transcriptomes and epigenomes of SF were determined by RNA-seq, Illumina HiSeq 2000 n=21), ChIP-seq (Illumina HiSeq 2500, n=7) and Infinium HumanMethylation450 BeadChip (n=12). We used MetaCore (Thomson Reuters) for the pathway enrichment analysis of RNA-seq data. SF were stimulated with IL-6/soluble IL-6 receptor (IL-6/sIL-6R, 50 ng/ml each). The amount of STATs and phospho-STAT3 (p-STAT3) was measured by Western blot with normalisation to α-tubulin. <h3>Results</h3> The JAK-STAT pathway was enriched in knee SF versus hand and shoulder SF (FDR&lt;0.05). JAK1 (normalised reads – mean ±SD: 10673±2084) and STAT1 (15520±2678) were the top expressed Janus kinase and STAT mRNAs in SF, respectively, whereas the expression of JAK3 (40±26) and STAT4 (164±91) mRNAs was low. Looking into joint-specific differences, STAT1 mRNA was higher in knee SF and shoulder SF compared with hand SF (p&lt;0.05, FDR&lt;0.15). Accordingly, STAT1 protein was increased in knee SF (STAT1/α-tubulin ratio: 0.83±0.02, p=0.02, n=4) and shoulder SF (1.02±0.02, p=0.001, n=5) versus hand SF (0.57±0.02, n=3). JAK1, STAT2 and STAT5B mRNAs were higher in knee compared with hand SF (p&lt;0.05, FDR&lt;0.05) and STAT2 and STAT6 mRNAs were higher in knee versus shoulder SF (p&lt;0.05, FDR&lt;0.05). TYK2 mRNA was high in hand SF compared with shoulder and knee SF (p&lt;0.05, FDR&lt;0.05). SF from different joints exhibited comparable DNA methylation at the promoters of these genes. Activating histone marks H3K4me3 and/or H3K27ac were enriched at the promoters of JAK1, STAT1, STAT2 and STAT5B in knee versus hand SF. This indicated that the abundance of activating histone marks at gene promoters might shape joint-specific expression of a subset of Janus kinase and STAT genes. Stimulation of SF with IL-6/sIL-6R increased the phosphorylation of STAT3 in knee (p-STAT3/α-tubulin ratio 1.8±1.0, p=0.03, n=5) and shoulder SF (1.8±0.7, p=0.03, n=6) compared with hand SF (0.9±0.5, n=6). The basal amount of STAT3 protein and the ratio pSTAT3/STAT3 was higher in knee SF (STAT3/α-tubulin ratio: 0.8±0.1; p-STAT3/STAT3: 2.2±0.8, n=3) and shoulder SF (0.6±0.4; 2.1±1, n=4) versus hand SF (0.3±0.02; 1.0±0.4, n=2). <h3>Conclusions</h3> Here we show substantial quantitative and qualitative differences in the JAK-STAT signalling pathway in SF from different joints. Knee SF, in particular, exhibit increased expression of Janus kinase and STAT genes and enhanced JAK-STAT signalling upon stimulation with IL-6/sIL-6R. This suggests that RA in different joints might not be equally sensitive to Janus kinase inhibitors or blockade of IL-6. This has important implications in clinical practice and drug discovery in RA. <h3>Disclosure of Interest</h3> T. Masterson: None declared, K. Klein: None declared, E. Karouzakis Grant/research support from: BTCure, GSK, O. Distler Grant/research support from: Abbvie, Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, iQone, Lilly, medac, MedImmune, Mepha, MSD, Mitsubishi Tanabe Pharma, Novartis, Pfizer, Pharmacyclics, Sanofi, Sinoxa and UCB, Consultant for: Abbvie, Actelion, Bayer, BiogenIdec, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, iQone, Lilly, medac, MedImmune, Mepha, MSD, Mitsubishi Tanabe Pharma, Novartis, Pfizer, Pharmacyclics, Sanofi, Sinoxa and UCB, C. Ospelt Grant/research support from: euroTEAM, BTCure, CABMM, IRR, Promedica, M. Frank Bertoncelj Grant/research support from: AbbVie Rheumatology grant 2017 euroTEAM, BTCure, IRR, Promedica, Georg und Berta Schwyzer Winiker Grant