Background/Purpose: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation and joint destruction caused by pro-inflammatory cytokines. Progressive joint damage is also an outcome of the enhanced osteoclast function in synovial microenvironment mediated by IL-6. However, the molecular mechanisms through which IL-6 propels synovial fibroblasts to contribute to bone loss in RA are not fully characterized.

Methods: Human synovial tissue derived RA synovial fibroblasts (RASFs) were treated with IL-6 and IL-6 receptor (IL-6/IL-6R, 100 ng/ml each) alone or in combination with M-CSF (2 ng/ml) and RANKL (5 ng/ml) for 9 days to study differentiation mechanisms to osteoclast-like precursor cells. Osteoclast-like phenotype of RASFs was confirmed by a Tartrate-resistant acid phosphatase (TRAP) staining method. Osteoclast-like characteristics of RASFs were determined using quantitative RT-PCR or Western immunoblotting for markers such as RANKL, OPG, NFATc1, OSCAR and MITF. Signaling mechanisms that contribute to osteoclastogenesis in RASFs were confirmed by transient small-interfering RNA (siRNA). Effect of IL6/IL-6R was further examined on bone marrow-derived macrophages (BMDM) from 7-week old C57BL/6 mice and compared to M-CSF/RANKL treatment groups.

Results: Treatment of human RASFs with IL-6/IL-6R for 9 days resulted in the phenotypic changes to osteoclast-like features confirmed using TRAP staining (p< 0.05; n=3). IL6/IL-6R treatment of RASFs also increased the expression of osteoclast-specific expression of OPG, Cathepsin K, and Cathepsin B, which was comparable to the expression in M-CSF/RANKL-stimulated samples (p< 0.05; n=4). The increased TRAP staining and osteoclastogenic factors by IL-6/IL-6R were independent of M-CSF receptor expression in human RASFs. In addition to increasing Cathepsin K, Cathepsin B, and traditional osteoclast-specific markers (MITF, RANKL, and OSCAR), IL-6/IL-6R increased the expression transcription factor ETS2 by 3-5-fold when compared to M-CSF/RANKL-differentiated human RASFs. In vitro studies using mouse BMDMs showed a significantly higher expression of Cathepsin B, OPG, MITF and ETS2 in response to IL-6/IL-6R activation when compared to M-CSF/RANKL treatment group (p< 0.05; n=3). Silencing ETS2 expression during human RASF differentiation abrogated IL-6/IL-6R-induced Cathepsin K and Cathepsin B production, and osteoclastogenic features of these synovial cells.

Conclusion: IL-6 uniquely contributes to osteoclast-like phenotype of RASFs by directing the molecular reprogramming in these cells mediated through ETS2. Targeting ETS2 to suppress this phenotypic switch of RASFs to osteoclast-like cells may serve as regulatory mechanism of limiting bone resorption in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-6-promotes-osteoclast-like-phenotype-in-human-rheumatoid-arthritis-synovial-fibroblasts/