E076 To investigate the mechanisms of action of novel compounds ex vivo in cells obtained from the inflammatory synovium of patients with rheumatoid arthritis

Abstract

Abstract Background/Aims To investigate the ex vivo effect of JAK1/2 inhibitor baricitinib on the expression of pro-inflammatory mediators in normal and RA diseased fibroblasts stimulated with oncostatin M (OSM) as well as in cells derived from RA inflamed synovial membrane cells (RA SMC). Methods RA SMC and fibroblast like synoviocytes (RA FLS) were isolated from synovial membranes of patients undergoing surgery using enzymatic digestion and cell propagation. Normal fibroblast-like synoviocytes (FLS) and normal human skin fibroblasts (HSF) were purchased from commercial sources. All fibroblasts were cultured in media supplemented foetal bovine serum and antibiotics in incubators using established laboratory procedures, and serum-starved prior to stimulation with OSM with or without baricitinib. RA SMC were cultured in the presence of baricitinib without OSM as they spontaneously release pro-inflammatory cytokines. Levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, CCL5 and IP-10) were determined by qPCR, ELISA and MSD. Western blot was used to measure levels of phospho-JAK specific components. Results qPCR analysis found that baricitinib inhibited OSM induced IP-10 and MCP-1 mRNA expression but not RANTES mRNA expression in normal human skin fibroblasts and RA FLS. In normal FLS however, baricitinib was found to inhibit RANTES and MCP-1 mRNA expression, but not IP-10 mRNA expression. Expression levels of IL-8 were too low for accurate conclusions to be drawn in all cell types apart from the RA SMCs, and in this case baricitinib did not inhibit IL-8 expression. ELISA results showed that baricitinib inhibited OSM induced MCP-1 expression in all cell types investigated. OSM induced IL-6 expression was inhibited in RA FLS but no other cell type. Conclusion Baricitinib inhibits OSM stimulated pro-inflammatory cytokine production through JAK1, 2. It has a varying inhibitory effect on the production of different cytokines: it is shown to particularly inhibit MCP-1. Baricitinib was found to have no effect on the production of spontaneously released pro-inflammatory cytokines in RA mixed cell populations. Disclosure M. Turner: None.