BACKGROUND The genetic risk associated with rheumatoid arthritis (RA) includes genes regulating DNA methylation, one of the hallmarks of epigenetic re-programing, as well as many T-cell genes, with a strong MHC association, pointing to immunogenetic mechanisms as disease triggers leading to chronicity. The aim of our study was to explore DNA methylation in early, drug-naïve RA patients, towards a better understanding of early events in pathogenesis. RESULT Monocytes, naïve and memory CD4+ T-cells were sorted from 6 healthy controls and 10 RA patients. DNA methylation was assessed using a genome-wide Illumina 450K CpG promoter array. Differential methylation was confirmed using bisulfite sequencing for a specific gene promoter, ELISA for several cytokines and flow cytometry for cell surface markers. Differentially methylated (DM) CpGs were observed in 1047 genes in naïve CD4+ T-cells, 913 in memory cells and was minimal in monocytes with only 177 genes. Naive CD4+ T-cells were further investigated as presenting differential methylation in the promoter of > 500 genes associated with several disease-relevant pathways, including many cytokines and their receptors. We confirmed hypomethylation of a region of the TNF-alpha gene in early RA and differential expression of 3 cytokines (IL21, IL34 and RANKL). Using a bioinformatics package (DMRcate) and an in-house analysis based on differences in β values, we established lists of DM genes between health and RA. Publicly available gene expression data were interrogated to confirm differential expression of over 70 DM genes. The lists of DM genes were further investigated based on a functional relationship database analysis, which pointed to an IL6/JAK1/STAT3 node, related to TNF-signalling and engagement in Th17 cell differentiation amongst many pathways. Five DM genes for cell surface markers (CD4, IL6R, IL2RA/CD25, CD62L, CXCR4) were investigated towards identifying subpopulations of CD4+ T-cells undergoing these modifications and pointed to a subset of naïve T-cells, with high levels of CD4, IL2R, and CXCR4, but reduction and loss of IL6R and CD62L, respectively. CONCLUSION Our data provided novel conceptual advances in the understanding of early RA pathogenesis, with implications for early treatment and prevention.

中文翻译：

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早期类风湿关节炎患者外周血CD4 + T细胞的差异CpG DNA甲基化。

背景与类风湿性关节炎（RA）相关的遗传风险包括调节DNA甲基化的基因（表观遗传重编程的标志之一）以及许多T细胞基因，它们与MHC的关联性很强，指出免疫遗传机制是疾病触发前瞻性慢性病。我们研究的目的是探索无药物治疗的早期RA患者的DNA甲基化，以更好地了解发病机理中的早期事件。结果从6名健康对照者和10名RA患者中筛选出单核细胞，幼稚和记忆CD4 + T细胞。使用全基因组的Illumina 450K CpG启动子阵列评估DNA甲基化。使用亚硫酸氢盐测序确定特定的基因启动子，ELISA鉴定几种细胞因子，流式细胞术鉴定细胞表面标志物，从而确认差异甲基化。在幼稚的CD4 + T细胞的1047个基因中观察到差异化的甲基化（DM）CpGs，在记忆细胞中观察到913个，在仅有177个基因的单核细胞中观察到最小的CpGs。进一步研究了幼稚的CD4 + T细胞，发现在与多种疾病相关途径（包括许多细胞因子及其受体）相关的> 500个基因的启动子中存在差异甲基化。我们证实了早期RA中TNF-α基因区域的甲基化不足和3种细胞因子（IL21，IL34和RANKL）的差异表达。使用生物信息学软件包（DMRcate）和基于β值差异的内部分析，我们建立了健康与RA之间的DM基因列表。询问公开可用的基因表达数据，以确认70多个DM基因的差异表达。在功能关系数据库分析的基础上进一步研究了DM基因的列表，该关系分析指出IL6 / JAK1 / STAT3节点与TNF信号转导和参与Th17细胞分化的许多途径有关。研究了五个用于细胞表面标记的DM基因（CD4，IL6R，IL2RA / CD25，CD62L，CXCR4），以鉴定经历了这些修饰的CD4 + T细胞的亚群，并指出了一些幼稚的T细胞，其中CD4含量高， IL2R和CXCR4，但分别减少和减少IL6R和CD62L。结论我们的数据为了解早期RA发病机理提供了新的概念性进展，对早期治疗和预防具有重要意义。与TNF信号通路和Th17细胞分化过程中的多种途径有关。研究了五个用于细胞表面标记的DM基因（CD4，IL6R，IL2RA / CD25，CD62L，CXCR4），以鉴定经历了这些修饰的CD4 + T细胞亚群，并指出了一些幼稚的T细胞，其中CD4含量高， IL2R和CXCR4，但分别减少和减少IL6R和CD62L。结论我们的数据为了解早期RA发病机理提供了新的概念性进展，对早期治疗和预防具有重要意义。与TNF信号通路和Th17细胞分化过程中的多种途径有关。研究了五个用于细胞表面标记的DM基因（CD4，IL6R，IL2RA / CD25，CD62L，CXCR4），以鉴定经历了这些修饰的CD4 + T细胞的亚群，并指出了一些幼稚的T细胞，其中CD4含量高， IL2R和CXCR4，但分别减少和减少IL6R和CD62L。结论我们的数据为早期RA发病机理的理解提供了新的概念性进展，对早期治疗和预防具有重要意义。但IL6R和CD62L分别减少和丢失。结论我们的数据为了解早期RA发病机理提供了新的概念性进展，对早期治疗和预防具有重要意义。但IL6R和CD62L分别减少和丢失。结论我们的数据为了解早期RA发病机理提供了新的概念性进展，对早期治疗和预防具有重要意义。