FOXP3Demethylation Of Circulating Regulatory T Cells In Patients With Acute Coronary Syndrome

Part ⅠFOXP3demethylation as a reliable marker of identifying regulatory T cells in patients with acute coronary syndromeObjective:The contribution of regulatory T cells (Tregs) to the pathogenesis of acute coronary syndrome (ACS) remains poorly understood. One core obstacle is the lack of Treg-specific markers. A highly conserved CpG enriched element in forkhead box P3intron1(FOXP3i1) is unmethylated only in Tregs, and measuring the unmethylation of FOXP3i1can be used to identify the role of Tregs in clinical diseases. This study investigated whether analyzing the demethylation status of FOXP3i1is a more reliable means than using Treg-specific surface markers in ACS.Methods:We evaluated circulating Tregs percentages on different levels including cell frequencies (CD4+CD25highFOXP3+Tregs and CD4+CD25highCD45+naive Tregs), FOXP3i1demethylation status and related cytokine secretion in89patients with ACS and35controls. Results:FOXP3i1demethylation assay showed that the amount of Tregs in ACS patients was significantly reduced than that in controls (p=0.0005). However, flow cytometry analysis did not identify any reduction of CD4+CD25highFOXP3+Tregs in ACS patients. Notably, younger patients had higher percentage of CD4+CD25highFOXP3+Tregs but decreased percentage of CD4+CD25highCD45+naive Tregs Tregs than either controls or older patients. There were no significant difference in plasma IL-10amounts among four groups, ACS subiects in young group had statistically higher levels of TGF-β1than ACS subjects in old group (p=0.004), and had the highest levels of interferon-y among four groups (p=0.002).Conclusions:A quantitative defect of Tregs, suggestive of decreased peripheral tolerance, could be a potential hallmark of ACS disease. Part ⅡDNA hypomethylation agent regulate regulatory T cells in patients with acute coronary syndromeObjective:Several experimental studies have shown that a DNA hypomethylation agent, DAC, can inhibit or reverse graft-versus-host disease or diabetes by inducing the expression of regulatory T cells. However, the role of DAC in atherosclerosis has never been characteristic. We aimed to investigate whether DNA hypomethylation can regulate the immune balance of ACS patients by inducing the expression of Tregs.Methods:Peripheral blood samples were collected from20patients with acute coronary syndrome. CD4+CD25+T cells were isolated from PBMCs, and then cultured with2and5 um DAC, controlled with blank, and collected after72h. Percentages of CD4+CD25+FOXP3+Tregs were evaluated by flow cytometric anaylsis; FOXP3mRNA was determined by real-time RT-PCR; the level of FOXP3demethylation was measured by pyrosequencing; the amounts of TGF-β1, IL-10, and IFN-y were detected by ELS A.Results:DAC increased the amounts of CD4+CD25highFOXP3+Tregs, FOXP3mRNA and Tregs related cytokine IL-10, but suppressed the production of pro-inflammatory cytokine IFN-y by inducing FOXP3i1demethylation in vitro in a dose-dependent manner.Conclusions:DAC can significantly induce the expression of Tregs in vitro, which contribute to the immune homeostasis of ACS.