Abstract
Rheumatoid arthritis (RA) is an autoimmune disease characterized by persistent synovial inflammation. The major drivers of synovial inflammation are cytokines and chemokines. Among these molecules, TNF activates fibroblast-like synoviocytes (FLSs), which leads to the production of inflammatory mediators. Here, we show that TNF regulates the expression of the transcription factor interferon regulatory factor 1 (IRF1) in human FLSs as well as in a TNF transgenic arthritis mouse model. Transcriptomic analyses of IRF1-deficient, TNF-stimulated FLSs define the interferon (IFN) pathway as a major target of IRF1. IRF1 expression is associated with the expression of IFNβ, which leads to the activation of the JAK-STAT pathway. Blocking the JAK-STAT pathway with the Janus kinase inhibitor (JAKinib) baricitinib or tofacitinib reduces the expression of IFN-regulated genes (IRGs) in TNF-activated FLSs. Therefore, we conclude that TNF induces a distinct inflammatory cascade, in which IRGs are key elements, in FLSs. The IFN-signature might be a promising biomarker for the efficient and personalized use of new treatment strategies for RA, such as JAKinibs.
Highlights
1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects the synovium of diarthrodial joints
TNF-induced interferon regulatory factor 1 (IRF1) expression in RA-fibroblast-like synoviocytes (FLSs) In line with previous observations[5], we found IRF1 to be highly expressed in the synovium in RA patients, but not in osteoarthritis (OA) patients (Fig. 1a) when we analyzed IRF1 expression by IHC
STAT1 phosphorylation and interferon-regulated genes (IRGs) expression in the TNF-treated FLSs were decreased when we targeted IFNAR by using either specific siRNA pools (IFNAR1, Fig. 4f and Supplementary Fig. 3) or a blocking antibody specific for IFNAR (Supplementary Fig. 4). These data suggest that the TNFmediated upregulation of IRF1 expression induces the expression of IFNβ, which in turn activates the transcription factor STAT1 to induce the expression of IRGs in RA-FLSs
Summary
Patients and synovial tissue samples Synovial tissue samples were obtained from RA (fulfilling the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for RA18) or osteoarthritis (OA) patients undergoing joint replacement or synovectomy. Synovial IRF1 expression was detected with a polyclonal rabbit anti-IRF1 antibody (Cell Signaling Technology). Paraffin-embedded sections were used for immunohistochemical staining for synovial IRF1 expression using the antibodies and protocol mentioned above. Isolation and culture of FLSs FLS single cell suspensions were obtained by digesting minced synovial tissue samples with collagenase type II (Merck). The protein lysates were fractioned on polyacrylamide gels, followed by electrotransfer to nitrocellulose membranes, which were blocked with either 5% BSA or 5% nonfat dry milk and incubated with primary antibodies (Cell Signaling Technology: anti-IκBα, anti-IRF1, anti-BAFF, anti-p-STAT1, and anti-STAT1; Sigma-Aldrich: anti-ACTIN). Nonimmune immunoglobulins of the same isotype and concentration as the primary antibody (anti-rabbit IgG, Novus Biologicals; rat IgM, Thermo Fisher Scientific) served as controls. Library concentrations were quantified with the Qubit Fluorometric Quantitation system (Thermo Fisher Scientific), and the size distribution was assessed using the Experion Automated Electrophoresis System (Bio-Rad). For graphing and statistical analysis, we used Graph Pad Prism 6 software
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