Methotrexate promotes the release of granulocyte–macrophage colony-stimulating factor from rheumatoid arthritis fibroblast-like synoviocytes via autocrine interleukin-1 signaling

Activated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs, only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor, tofacitinib (TOFA), on gene expression in RA-FLS, in order to identify untargeted disease mediators. Primary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA, with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR, and supernatants were analyzed for protein concentrations. Six thousand three hundred fifty genes were differentially expressed, the majority being upregulated, in MTX-treated activated RA-FLS and 970 genes, the majority being downregulated, in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on ‘Molecular mechanisms of cancer’ and TOFA on ‘Interferon signaling’. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte–macrophage colony-stimulating factor, GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h, could be mediated via either NF-κB or AP-1 transcription factors, and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting, MTX-treatment of activated RA-FLS induced IL1B expression in macrophages. MTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis.