Synovial fibroblast activation in rheumatoid arthritis - Role of disease-associated risk genes and effects of methotrexate

Abstract

The overall aim of this thesis was to increase the knowledge of molecular mechanisms shaping the pathogenic phenotype of activated fibroblast-like synoviocytes (FLS), which are drivers of chronic inflammation and destruction of the joints in rheumatoid arthritis (RA). As a main approach, we studied the role of previously identified multi-evidence, RA-associated risk genes in these cells. Moreover, we investigated effects of anti-rheumatic drugs, in particular methotrexate (MTX), on the expression of RA risk genes and pathogenic behaviors of activated RA-FLS. Cellular and molecular studies were primarily performed on joint tissue and cultured FLS from patients with RA and controls. For in vitro experiments, cytokines or growth factors known to activate FLS were added to simulate the inflammatory environment of the RA joint. Paper I demonstrated that the RA risk gene AIRE (autoimmune regulator), mostly known as a transcriptional regulator in the thymus, is induced in RA-FLS by pro-inflammatory cytokines. Using AIRE silencing and RNA sequencing, we found that AIRE promotes the expression of a set of chemokines in activated RA-FLS. Paper II showed that MTX increases the transcription of the RA risk gene LBH (limb-bud and heart development) and other tumor suppressor genes in activated RA-FLS, possibly via epigenetic changes. Also, cell cycle analysis demonstrated that MTX inhibits proliferation of activated RA-FLS. Paper III applied transcriptomic analysis to further explore the influence of MTX and of a Janus kinase inhibitor on these cells. Therapeutic concentrations of MTX altered the expression of a vast number of genes in activated RA-FLS, and unexpectedly upregulated pro-inflammatory mediators like IL1A (interleukin-1a) and the RA risk gene CSF2 (granulocyte-macrophage colony-stimulating factor, GM-CSF). We propose a mechanism whereby MTX increases the production of GM-CSF via autocrine IL-1a signaling, with potential to enhance inflammatory interactions between FLS and macrophages in the RA synovium. In conclusion, our results support the biological relevance of multi-evidence RA risk genes in activated RA-FLS and add new knowledge on the pharmacodynamics of MTX in RA. The findings emphasize that RA-FLS are important targets in future treatment strategies to improve outcome in RA.