A Quantitative in Vitro Framework Reveals State-Dependent Pharmacological Responses to Repeated Inflammatory Stimulation in Synovial Fibroblasts

Rheumatoid arthritis is characterized by chronic synovial inflammation and recurrent flares, which are thought to involve joint-specific inflammatory memory. Synovial fibroblasts (SFs) contribute to this process through altered responses to repeated inflammatory stimulation. However, pharmacological targeting of these state-dependent responses remains limited, partly due to the lack of screening systems that distinguish responses under initial and repeated stimulation conditions. Here, we established a quantitative in vitro system using primary murine SFs. Repeated tumor necrosis factor-α stimulation enhanced interleukin-6 (IL-6) production, recapitulating key features previously reported in human SFs. This system enabled direct comparison of drug effects under single and repeated stimulation conditions, revealing distinct response patterns and identifying compounds that selectively modulate the enhanced response induced by repeated stimulation. Notably, a complement receptor antagonist attenuated the additional increase in IL-6 production observed after repeated stimulation, accompanied by the attenuation of glycolysis-related gene expression, while showing no significant effect under single stimulation conditions. These findings highlight the importance of state-dependent pharmacological evaluation and demonstrate the utility of this experimental framework for analyzing differential responses associated with repeated inflammatory stimulation.