Background Rheumatoid arthritis (RA) is a chronic and progressive autoinflammatory disorder characterized by the infiltration of inflammatory cells, including B-cells, T-cells and macrophages, in the synovial membrane ultimately leading to cartilage destruction and bone erosion. Clinical disease activity in RA correlates strongly with macrophage numbers in synovial tissue and expression of macrophage-derived cytokines. Bruton’s tyrosine kinase (Btk) is important not only in B cell activation, but also mediates immune complex-dependent activation of monocytes. Pharmacological inhibition of Btk has been shown to be effective in suppressing pathology in murine models of arthritis
Objectives The objective of this study was to determine the potential role of Btk in the pathology of RA.
Methods Btk expression was detected by immunohistochemistry and digital image analysis in synovial tissue from 16 RA and 12 PsA patients, naïve to treatment with biologicals. Immunofluorescent double labelling confocal microscopy was performed to identify which cell types express Btk in RA synovial tissue. Validating qRT-PCR and immunoblotting experiments were performed on isolated relevant cell populations. Effects of a specific Btk inhibitor, RN486, on activation-dependent human macrophage IL-6 production (n=8) and RA synovial tissue explant cultures (n=6) were assessed by ELISA. RN486 influence on macrophage expression of genes related to angiogenesis, cellular adhesion, tissue remodelling and innate immune responses was determined using low density qPCR arrays.
Results Btk was expressed at equivalent levels in patients with RA and PsA. No relationship was observed between expression levels and patient clinical characteristics (CRP, ESR, DAS28, RF-positivity). In RA, but not PsA, Btk expression was significantly related to numbers of synovial macrophages (R= 0.63, p< 0.01) and T cells (R= 0.79, p< 0.001), but not fibroblast-like synoviocytes (FLS), B cells, plasma cells, or endothelial cells. qPCR and immunoblotting experiments confirmed that Btk was expressed in B cells, monocytes, and macrophages, but not T cells or RA FLS. RN486 (1μM) inhibited macrophage IL-6 production induced by Fc receptor stimulation (40% inhibition, p< 0.01) and anti-CD40 antibodies (50%, p< 0.05), but not TNFα or LPS stimulation. qPCR analysis of human macrophages demonstrated that RN486 inhibited by more than 2-fold 12 of 21 genes induced by IgG, 11 of 52 genes induced by CD40 stimulation, and 6 of 25 genes induced by RA SF in 3 independent experiments. RN486 also inhibited spontaneous IL-6 production by cultured RA synovial explants (65%, p< 0.01).
Conclusions Btk is expressed in RA synovial tissue and macrophages would be prominent synovial targets of strategies aimed at inhibiting Btk in RA. Btk activity is needed to drive macrophage activation in response to multiple stimuli relevant to RA, and drives IL-6 production in RA synovial tissue. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA.
Disclosure of Interest L. Hartkamp: None Declared, I. van Es: None Declared, J. Fine Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, M. Smith Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. Woods Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, S. Narula Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, J. DeMartino Shareholder of: Hoffmann-La Roche, Employee of: Hoffmann-La Roche, P. Tak Consultant for: GlaxoSmithKline, Employee of: GlaxoSmithKline, K. Reedquist Grant/research support from: Hoffmann-La Roche