THU0006 PROINFLAMMATORY RESPONSES IN THE JAK-STAT PATHWAY IN SYNOVIAL FIBROBLASTS ARE STIMULUS-SPECIFIC AND ONLY PARTIALLY INHIBITED BY THERAPEUTIC DOSES OF TOFACITINIB

Background: The Jak-STAT signalling pathway has a key role in the pathogenesis of rheumatoid arthritis (RA). First Jak inhibitors, including tofacitinib, have been approved for the treatment of RA. Whereas Jak inhibitors exhibit pleiotropic effects on the immune system, their “in situ” activities on synovial fibroblasts (SF), the resident synovial cells, are less well understood. Objectives: To characterise the Jak-STAT pathway and its inhibition by tofacitinib in RA synovial tissues and SF from different joints across diverse pro-inflammatory stimuli. Methods: Synovial tissues and SF were isolated from knee (n=4), shoulder (n=4) and hand (n=4-6) joints of RA patients undergoing joint replacement surgery. To activate and inhibit the Jak-STAT pathway, SF were stimulated with TNF (0.1 ng/ml, 1ng/ml) ± IL-6 receptor (IL-6R, 50ng/ml) or IL-6 (50ng/ml) + IL-6R for 24h in the presence or absence of 80nM, 180nM and 1000nM tofacitinib. 80nM and 180nM tofacitinib mimic the plasma drug concentrations in subjects on therapeutic doses of tofacitinib. 1000nM is the in vitro used tofacitinib concentration. Gene expression in synovial tissues and SF (n=12 each) was measured using the low-density gene expression arrays containing 6 housekeeper genes and 90 probes for the core components, inhibitors and target genes of the Jak-STAT pathway. Clustering analysis of the array data [principal component analysis (PCA), heatmap] was performed using the ClustVis web tool. PCA identified one outlier SF sample, driven by distinct housekeeper gene expression that was omitted from further analysis. The production of IL-6 was determined by ELISA. Results: RA synovial tissues and quiescent SF exhibited highly similar expression of the core components of the Jak-STAT signalling pathway, suggesting that SF contribute significantly to the synovial expression of JAK and STAT genes. Clustering analysis of the array data showed that SF stimulated with 0.1ng/ml TNF+IL-6R, SF treated with IL-6+IL-6R and quiescent SF formed separate clusters (Figure 1), pointing towards stimulus-specific transcriptional outputs in the Jak-STAT pathway. Moreover, TNF+IL-6R increased the expression of multiple core components in the Jak-STAT pathway (IL6ST, JAK2, JAK3, STAT3, STAT4) with simultaneous downregulation of pathway inhibitors (PIAS1, PIAS2) (p Conclusion: Here we show that soluble IL-6R strongly augments the pro-inflammatory effects of TNF in SF. Furthermore, percent inhibition of the IL-6 production by tofacitinib remained constant across diverse proinflammatory conditions despite different amounts of IL-6 being produced. Thus, higher pre-treatment inflammatory responses to TNF ± IL-6R predict higher residual inflammatory activity in SF following Tofacitinib therapy, pointing towards a saturation of anti-inflammatory effects of Tofacitinib in SF in RA. Disclosure of Interests: Ana Županic Grant/research support from: EULAR Scientific Bursary, Blaž Burja: None declared, Kerstin Klein: None declared, Raphael Micheroli: None declared, Oliver Distler Grant/research support from: Prof. Distler received research funding from Actelion, Bayer, Boehringer Ingelheim and Mitsubishi Tanabe to investigate potential treatments of scleroderma and its complications, Consultant for: Prof. Distler has/had consultancy relationship within the last 3 years with Actelion, AnaMar, Bayer, Boehringer Ingelheim, ChemomAb, espeRare foundation, Genentech/Roche, GSK, Inventiva, Italfarmaco, iQvia, Lilly, medac, MedImmune, Mitsubishi Tanabe Pharma, Pharmacyclics, Novartis, Pfizer, Sanofi, Serodapharm and UCB in the area of potential treatments of scleroderma and its complications. In addition, he had/has consultancy relationship within the last 3 years with A. Menarini, Amgen, Abbvie, GSK, Mepha, MSD, Pfizer and UCB in the field of arthritides and related disorders, Caroline Ospelt: None declared, Mojca Frank-Bertoncelj : None declared