A 2D inflammatory co-culture model for investigating synovial fibroblast and macrophage interactions in rheumatoid arthritis

Rheumatoid arthritis (RA), a chronic autoimmune disease, marked by sustained inflammation and joint destruction, largely driven by pathological interactions between fibroblast-like synoviocytes (FLS) and macrophages. Current in vitro and in vivo models fall short in replicating the complex synovial microenvironment of RA, hindering translational research. In this study, we developed a simplified yet physiologically relevant 2D co-culture system that models both acute and chronic RA phases by modulating the ratio of healthy FLS to M1-polarized macrophages, simulating the intimal synovial layer. Direct exposure of FLS to inflammatory macrophages led to marked activation, with significant upregulation of pro-inflammatory cytokines-TNF-α (1.57-fold), IL-1β (6.30-fold), and IL-6 (4.94-fold)-and elevated expression of matrix metalloproteinases MMP2 (2.05-fold) and MMP9 (37.61-fold), key mediators of joint degradation. Using a transwell system, indirect exposure further induced FLS invasion, reflecting chronic inflammatory progression. Therapeutic validation using methotrexate (MTX) demonstrated its anti-invasive effect on activated FLS, supporting the model's utility in drug screening. This co-culture platform effectively captures RA-specific cellular responses and inflammatory dynamics, offering a scalable, high-throughput-compatible system for mechanistic studies and therapeutic evaluation.