Treg Cell Function in Rheumatoid Arthritis Is Compromised by CTLA-4 Promoter Methylation Resulting in a Failure to Activate the Indoleamine 2,3-Dioxygenase Pathway
Treg Cell Function in Rheumatoid Arthritis Is Compromised by CTLA-4 Promoter Methylation Resulting in a Failure to Activate the Indoleamine 2,3-Dioxygenase Pathway
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DOI：
10.1002/art.38715
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发表时间：
2014-09-01
影响因子：
13.3
通讯作者：
Williams, Richard O.
中科院分区：
文献类型：
--
作者：
Cribbs, Adam P.;Kennedy, Alan;Williams, Richard O.
目标。功能受损的Treg细胞表达异常低水平的CTLA-4已在类风湿性关节炎（RA）中得到充分证实。然而，这种表达减少背后的分子缺陷是未知的。本研究的目的是评估DNA甲基化在调节RA患者Treg细胞中CTLA-4表达中的作用，并阐明其抑制功能降低的机制。采用定量聚合酶链反应（PCR）和流式细胞术检测RA患者和健康对照Treg细胞中CTLA-4的表达。通过亚硫酸酯特异性PCR分析CTLA-4基因启动子的甲基化，然后进行测序。通过荧光素酶测定CTLA-4基因启动子的甲基化依赖性转录活性，并通过染色质免疫沉淀测定NF-AT与CTLA-4基因启动子的结合。采用自体混合淋巴细胞反应分析CTLA-4表达在Teff细胞调控中的作用。RA患者Treg细胞中CTLA-4表达下调是由CTLA-4基因启动子中先前未识别的NF-AT结合位点甲基化引起的。结果，Treg细胞不能诱导色氨酸降解酶吲哚胺2,3-双加氧酶（IDO）的表达和激活，从而导致免疫调节犬尿氨酸途径的激活失败。我们首次表明，表观遗传修饰通过无法激活IDO通路导致RA中Treg细胞功能缺陷。因此，本研究为研究旨在加强RA患者IDO通路的潜在治疗策略开创了先例。
Objective. Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function.Methods. CTLA-4 expression in Treg cells from RA patients and healthy controls was measured by quantitative polymerase chain reaction (PCR) and flow cytometry. Methylation of the CTLA-4 gene promoter was analyzed by bisulfite-specific PCR, followed by sequencing. Methylation-dependent transcriptional activity of the CTLA-4 gene promoter was measured by luciferase assay, and NF-AT binding to the CTLA-4 gene promoter was determined by chromatin immunoprecipitation. The role of CTLA-4 expression in controlling Teff cells was analyzed using an autologous mixed lymphocyte reaction.Results. Down-regulation of CTLA-4 expression in Treg cells from RA patients was caused by methylation of a previously unidentified NF-AT binding site within the CTLA-4 gene promoter. As a consequence, Treg cells were unable to induce expression and activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which in turn resulted in a failure to activate the immunomodulatory kynurenine pathway.Conclusion. We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients.