The Association of Myostatin on the Expression of Inflammatory Cytokines and Chemokines in Rheumatoid Arthritis Synovial Fibroblasts

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease initiated and progressed by RA synovial fibroblast cells (RASFs) of the synovium. Inflammatory cytokines/chemokines play a crucial role in disease development. Myostatin (MSTN) is widely comprehended as a significant regulator of pro-inflammatory cytokines/chemokines in many other autoimmune diseases. However, its role during the progression of RA is still unclear. We hypothesized that MSTN regulates the inflammatory cytokines/chemokines expression in RASFs. The immortalized RASF cells (MH7A) were treated with MSTN or MSTN inhibitor (0, 10, and 20 ng/mL) for 0, 24, and 48 h. Then, the secretion and expression of inflammatory cytokines (IL-8, IL-17, TNF-α, IL-6, IL-23, IFN-γ, IFN-β) and chemokines (CCL-2, CCL-20, CXCL-13, CXCL-1) were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, MH7A cells were treated with 20 ng/mL of TNF-α, IL-17, IFN-γ IFN-β, CCL-2, and CXCL-1 for 24 h to examine their effects on MSTN expression and secretion. Data were analyzed by Kruskal-Wallis rank sum test, followed by Dunn’s multiple comparison test. The MSTN expression and secretion were measured by RT-qPCR, ELISA, and Western blot. Results showed that 20 ng/mL MSTN treatment significantly (p<0.05) increased the secretion of most inflammatory cytokines/chemokines at 24 or 48 h. CXCL-1 showed the highest secretion (30-fold in average) in response to MSTN treatment, followed by CCL-2 (13-fold in average) at 24 h and IL-23 (3-fold in average) at 48 h. Similar to ELISA results, MSTN significantly upregulated the mRNA expression of most inflammatory cytokines/chemokines at 24 or 48 h. The highest expression was TNF-α (60-fold in average) at 48 h, followed by CXCL-1 (15-fold in average) and CCL-2 (13-fold in average) at 24 h. The effect of MSTN inhibitor on cytokines/chemokines secretion and mRNA expression was inconsistent. Among the six cytokines/chemokines used to investigate their effects on MSTN expression in MH7A cells, IFN-γ significantly increased MSTN secretion (13-fold in average) and mRNA expression (100-fold in average) but other cytokine/chemokines showed no significant response. Western blot results showed that MSTN secretion was significantly increased by IFN-γ treatment, supporting the ELISA results. Current results demonstrated that MSTN upregulated the mRNA expression and secretion of various inflammatory cytokines/chemokines and IFN-γ significantly increased MSTN expression and secretion, indicating a cross-stimulation between MSTN and inflammatory cytokines/chemokines in RA. This study was funded by NIH 5R01AR043521-29. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.