title: Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Cribbs
authors.forename: Alan
authors.initials: A
authors.lastname: Kennedy
authors.forename: Henry
authors.initials: H
authors.lastname: Penn
authors.forename: Parisa
authors.initials: P
authors.lastname: Amjadi
authors.forename: Patricia
authors.initials: P
authors.lastname: Green
authors.forename: Jordan E
authors.initials: JE
authors.lastname: Read
authors.forename: Fionula
authors.initials: F
authors.lastname: Brennan
authors.forename: Bernard
authors.initials: B
authors.lastname: Gregory
authors.forename: Richard O
authors.initials: RO
authors.lastname: Williams
mesh_terms: D000328:Adult; D000368:Aged; D018501:Antirheumatic Agents / Q000494:pharmacology*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000276:immunology*; D060908:CTLA-4 Antigen / Q000187:drug effects / Q000378:metabolism; D016022:Case-Control Studies; D049109:Cell Proliferation / Q000187:drug effects*; D019175:DNA Methylation / Q000187:drug effects*; D004742:Enhancer Elements, Genetic / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D051858:Forkhead Transcription Factors / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000187:drug effects / Q000378:metabolism; D007963:Leukocytes, Mononuclear; D008297:Male; D008727:Methotrexate / Q000494:pharmacology*; D008875:Middle Aged; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000378:metabolism
chemical_list: D018501:Antirheumatic Agents; D060908:CTLA-4 Antigen; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D012333:RNA, Messenger; D007371:Interferon-gamma; D008727:Methotrexate