Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
Arthritis Rheumatol. 2015 May;67(5):1182-92. doi: 10.1002/art.39031.
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.
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.
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.
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.
我们之前在一组未经治疗的类风湿关节炎（RA）患者中显示，Treg 细胞的抑制功能存在缺陷。然而，其他在已确诊 RA 患者队列中的研究表明，Treg 细胞功能正常。我们假设治疗可能会恢复 Treg 细胞功能并导致疾病活动减少。本研究旨在探讨甲氨蝶呤（MTX）治疗是否会导致表观遗传变化，从而恢复 RA 中 Treg 细胞的抑制功能。
使用（3）H-胸腺嘧啶掺入来测量 Treg 细胞对 T 细胞增殖的抑制作用，并通过酶联免疫吸附测定法来确定 Treg 细胞对干扰素-γ产生的抑制作用，评估 RA 患者外周血样本。通过流式细胞术和定量聚合酶链反应（qPCR）测量健康个体和 RA 患者 Treg 细胞中的 CTLA-4 和 FoxP3 表达。从健康个体中分离出 CD4+T 细胞，在存在或不存在 MTX 的情况下，用白细胞介素-2（IL-2）、IL-6 和肿瘤坏死因子-α进行培养，并使用 qPCR 和流式细胞术确定 FoxP3 表达。通过亚硫酸氢盐测序 PCR 分析 FOXP3 上游增强子的甲基化。
仅在未接受 MTX 治疗的 RA 患者中观察到 Treg 细胞功能缺陷，而 MTX 暴露的 RA 患者的 Treg 细胞已恢复抑制功能。这种恢复的抑制作用与 Treg 细胞中 FoxP3 和 CTLA-4 的表达增加有关。在 MTX 培养的 Treg 细胞中进行的亚硫酸氢盐测序 PCR 显示，FOXP3 上游增强子的甲基化显著降低。
这项研究确定了 MTX 的一种新的作用机制，即 MTX 治疗 RA 患者通过 FOXP3 基因座的去甲基化恢复缺陷的 Treg 细胞功能，从而导致 FoxP3 和 CTLA-4 表达的随后增加。
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