The role of epigenetics in determining the clinical response to Methotrexate for the treatment of Rheumatoid Arthritis
Cregan S, Creevey K, McGarry T, Orr C, Veale DJ, Fearon U, Wilson AG
Centre for Arthritis and Rheumatic Diseases, Dublin Academic Medical Centre, University College Dublin, Ireland
Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease, affecting around 1% of the population, causing significant ill health, disability and increased page 44 mortality.
The precise etiology of RA is not known, but both genetic and environmental influences play a role. Methotrexate (MTX) is the most commonly used disease modifying antirheumatic drug in the management of RA, however its mechanism of action has not been extensively studied.
Primary RA synovial fibroblasts (RASFC) were isolated from synovial biopsies obtained from patients undergoing arthroscopic examination. RASFC were cultured in the presence or absence of MTX (10-100uM) for 48 hrs and cell migration, invasion, cytoskeletal rearrangement, viability, proliferation and pro-inflammatory cytokine expression were assessed by wound repair assays, transwell matrigelTM invasion chambers, F-actin immunofluorescent staining, MTT/ Crystal violet cell growth assay and ELISA respectively. In parallel, CD14+monocytes were isolated from peripheral blood mononuclear cells from RA patients both pre- and 12 weeks post-MTX therapy and global methylation was assessed.
MTX (10-100uM) inhibited RASFC repopulation of the wound margins in comparison to vehicle control where migration across the wound was clearly evident (n=7, p=0.03). In parallel, MTX significantly decreased RASFC invasion (n=7, p=0.01) and altered cytoskeletal dynamics through inhibition of lamellopodia and filopodia formation, which are indicative of cell movement. Importantly, we determined that the effect of MTX on RASFC migration and invasion were independent of both cell viability (p=0.37) and proliferation (p=0.18). Finally preliminary results demonstrated altered DNA methylation in CD14+ monocytes from RA patients (n=11) at 3 months post MTX treatment.
MTX inhibits RASFC migration, invasion and cytoskeletal re-arrangement, mechanisms which are critically involved in the pathogenesis of RA. Further preliminary data suggests that the effect of MTX on pro-inflammatory mechanisms in RA, maybe be mediated through alterations in epigenome.