Dual role of IL6 mediated by mesenchymal stromal cell signaling to joint macrophages in osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is a joint disease affecting > 5 million Canadians and patients have limited palliative and joint-replacement surgical options. Stromal cell therapy is emerging as a compelling treatment for OA. Our group led the first-in-Canada Ph1/2 trial with bone marrow mesenchymal stromal cells (BM-MSCs) in OA patients successfully; and significant improvements in patient outcomes were observed. This was accompanied with reduction in pro-inflammatory monocytes/macrophages (MΦs) in the synovial fluid (SF), suggestive of a clinical MSC anti-inflammatory effect. Understanding mechanism of action of MSCs in OA will enable effective donor MSC screening, and generation of more efficient engineered MSCs. While IL6 is a known pro-inflammatory mediator in the joint, and anti-IL6 therapeutics have been effective in treatment of Rheumatoid Arthritis, blocking single pro-inflammatory cytokines has been less effective in OA. This leads us to believe that IL6 might have complex roles. Indeed, MSCs-induced polarization of macrophages to inflammation resolving subtypes via IL6 signaling. This effect is not well studied in an OA joint where high levels of IL6 may have pro-inflammatory effects. We hypothesize that IL-6, which is known as pro-inflammatory mediator in OA is mediating anti-inflammatory function of MSCs through signaling via membrane-bound, but not soluble receptors. Methods: We co-cultured peripheral blood-derived MΦs with OA BM-MSCs in the presence of early and late SF from OA patients; or in the presence of IL6 antibody. Polarization of MΦs were assessed by analysis of surface markers and IL10 secretion. Levels of IL6 soluble IL6 receptor (sIL6R) were measured by ELISA. Results: Preliminary quantification of BM- MSC from OA patients revealed high IL6 secretion is associated with positive patient-reported outcomes. IL6 antibody abrogated MSCs-induced anti-inflammatory polarization of MΦs as shown by reduction of CD163 and CD206, suggesting a key role for IL6 in MSC anti-inflammatory effects on MΦs. Paradoxically, IL6 levels were increased in SF from late stage OA, whereas levels of sIL6R were comparable in early and late OA-SF. Further, the effect of MSCs on anti-inflammatory polarization of MΦs was attenuated in the presence of late OA-SF compared to early OA-SF. This could be due to enhanced IL6 trans-signaling (mediated by sIL6R) in late-stage OA SF expected to occur at the expense of reduced IL6 classical pathway (mediated by membrane bound IL6 receptor, mIL6R). To discriminate between classical vs. trans-signalling, we are using soluble gp130, a non-signaling decoy receptor which complexes IL6-sIL6R. Our preliminary result showed that sgp130 treatment enhanced the expression of CD163 in MΦs exposed to pooled OA SF. The classical IL6 pathway will be studied specifically by silencing mIL6R in MΦs using siRNA Conclusions: MSCs exert their anti-inflammatory action in OA, at least partly through polarization of MΦs to anti-inflammatory phenotype mediated by IL6. The ratio of IL6 signaling through trans-vs. classical signaling pathways is an important arbiter of MSC-modulation of joint inflammation.