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Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis
Author(s) -
Cribbs Adam P.,
Kennedy Alan,
Penn Henry,
Amjadi Parisa,
Green Patricia,
Read Jordan E.,
Brennan Fionula,
Gregory Bernard,
Williams Richard O.
Publication year - 2015
Publication title -
arthritis and rheumatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.106
H-Index - 314
eISSN - 2326-5205
pISSN - 2326-5191
DOI - 10.1002/art.39031
Subject(s) - foxp3 , flow cytometry , immunology , bisulfite sequencing , rheumatoid arthritis , regulatory t cell , t cell , peripheral blood mononuclear cell , methotrexate , medicine , dna methylation , cancer research , biology , il 2 receptor , gene expression , immune system , in vitro , gene , biochemistry
Objective We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA. Methods Peripheral blood samples from RA patients were assessed using 3 H‐thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme‐linked immunosorbent assay to determine Treg cell suppression of interferon‐γ production. CTLA‐4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin‐2 (IL‐2), IL‐6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR. Results Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX‐exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA‐4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer. Conclusion This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA‐4 expression.