Role of MDA5 in regulating CXCL10 expression induced by TLR3 signaling in human rheumatoid fibroblast-like synoviocytes

Abstract

C-X-C motif chemokine 10 (CXCL10) is an inflammatory chemokine and a key molecule in the pathogenesis of rheumatoid arthritis (RA). Melanoma differentiation-associated gene 5 (MDA5) is an RNA helicase that plays a role in innate immune and inflammatory reactions. The details of the regulatory mechanisms of CXCL10 production and the precise role of MDA5 in RA synovitis have not been fully elucidated. The aim of this study was to examine the role of MDA5 in regulating CXCL10 expression in cultured human rheumatoid fibroblast-like synoviocytes (RFLS). RFLS was stimulated with Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA mimetic. Expression of interferon beta (IFN-β), MDA5, and CXCL10 was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay. A neutralizing antibody of IFN-β and siRNA-mediated MDA5 knockdown were used to determine the role of these molecules in regulating CXCL10 expression downstream of TLR3 signaling in RFLS. Poly I:C induced IFN-β, MDA5, and CXCL10 expression in a concentration- and time-dependent manner. IFN-β neutralizing antibody suppressed the expression of MDA5 and CXCL10, and knockdown of MDA5 decreased a part of CXCL10 expression (p < 0.001). The TLR3/IFN-β/CXCL10 axis may play a crucial role in the inflammatory responses in RA synovium, and MDA5 may be partially involved in this axis.

Inflammation-Driven Secretion Potential Is Upregulated in Osteoarthritic Fibroblast-Like Synoviocytes

“…Elevated levels of CCL3 are detected in the plasma of OA patients [ 39 ]; furthermore, overexpression of CCL3 and CCL4 has been detected in pro-inflammation-stimulated chondrocytes [ 40 ]. Other researchers have demonstrated chemokine expression from the (C-X-C motif) ligand (CXCL) family in rheumatoid fibroblast-like synoviocytes (CXCL6 [ 41 ]; CXCL10 [ 42 ]; CXCL11 [ 43 ]; CXCL16 [ 44 ]). Based on this result, we chose three of them (CXCL6, CXCL10, and CXCL16, which are also known as granulocyte chemotactic protein 2 (GCP-2), interferon gamma-induced protein 10 (IP-10), and phosphatidylserine and oxidized lipoprotein (SR-PSOX), respectively) whose expression was higher in HFLS-OAs than in HFLS after pro-inflammatory stimulation.…”

TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

“…These findings suggest that CXCL10 plays an important role in the pathogenesis of RA and that it is important to clarify the mechanisms underlying the role of CXCL10 in RFLS. In RFLS treated with poly I:C, CXCL10 is produced via the TLR3/type I IFN/CXCL10 axis; a reaction that also involves MDA5 (another ISG) [10]. In this study, we found that ISG56 knockdown decreases the expression of CXCL10, but not MDA5.…”

“…Published by Frontiers Society for Experimental Biology and Medicine study was approved by the Hirosaki University Graduate School of Medicine Ethics Committee (2018-1117) [10]. The sequences of siRNAs are as follows.…”

“…MDA5 is a DExH group RNA helicase; it functions as an ATPase and a signaling molecule that regulates inflammatory responses [9]. Saruga et al [10] showed that the TLR3/type Ⅰ IFN/CXCL10 axis plays an important role in the RFLS inflammatory response. In addition, MDA5 may be associated with this axis.…”