Methylated FOXP3Induces Impairment Of Treg Cells In Patients With Acute Coronary Syndrome

Part Ⅰ The Study of Immunosuppressive Function of Circulating Regulatory T Cells in Patients with Acute Coronary SyndromeObjective:Atherosclerosis, the main pathophysiological condition leading to acute coronary syndromes (ACS), is now considered to be a chronic inflammatory and immune condition. Naturally occurring CD4+CD25+regulatory T cells (Tregs), specified by the transcription factor Foxp3, are a subpopulation of lymphocytes that are essential for maintaining immune system homeostasis by restraining excessive immune responses and promoting self-tolerance. We designed the study to explore whether the immunosuppressive function of CD4+CD25+Tregs are impaired in ACS patients.Methods:Sixty-two ACS patients (acute myocardial infarction and unstable angina) and52control patients were enrolled in this study. Peripheral CD4+CD25+Tregs and CD4+CD25-effector T cells (Teffs) were isolated by regents and separation of Miltenyi Biotec. We co-cultured the Tregs with Teffs at variable ratios and assessed suppressive function of Tregs by the proliferation index of CFSE-labeled Teffs in ACS patients and healthy controls respectively. Moreover, we assayed the proliferation function of CFSE-labeled effector T cells in ACS patients and healthy controls respectively.Results:①The proliferation function of Teffs were similar between ACS patients and their age-matched healthy controls (P>0.05).②We co-cultured the Tregs with autologous Teffs at variable ratios, compared with their age-matched healthy controls, patients with ACS exhibited significantly impaired immunosuppressive function of Tregs in suppression of Teffs proliferation (P<0.05).③We co-cultured the Tregs with homologous Teffs at variable ratios, compared with the Tregs from their age-matched healthy controls, Tregs in ACS patients were also significantly impaired in their ability to suppress the proliferation of CD4+CD25’effector T cells (P<0.05).Conclusions:Compared with healthy controls, the immnosuppressive function of CD4+CD25+Tregs are compromised in patients with ACS, thus, the compromised function of Tregs in ACS patients may be a factor of the happen of ACS. Part Ⅱ The Study of FOXP3Methylation of Circulating Regulatory T Cells in Patients with Acute Coronary SyndromeObjective:The epigenetic regulation of transcription factor genes is pivotal for T-cell lineage specification. A specific demethylation pattern within a conserved region of the lineage specifying transcription factor gene FOXP3, the Treg-specific demethylated region (TSDR), is restricted to regulatory T (Treg) cells and is required for stable expression of FOXP3and suppressive function of Tregs. We aimed at explore whether the methylation status of FOXP3upstream enhancer in Tregs of ACS patients is altered.Methods:Peripheral blood samples were collected from60ACS patients (acute myocardial infarction and unstable angina) and56healthy controls in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by centrifugation on Ficoll-Paque and were determined by flow cytometry to evaluate circulating CD4+CD25+ FOXP3+Tregs percentages in ACS group and control group. FOXP3upstream enhancer methylation levels of Tregs and Teffs were assayed by bisulfite sequencing in ACS group and control group, and the FOXP3mRNA levels of circulating lymphocytes were determined by real-time quantitative RT-PCR. The amounts of IL-10, TGF-β and INF-y were detected by ELISA.Results:①No differences were found in the frequency of Tregs when compared among young controls, old controls, young ACS group and old ACS group (P>0.05).②However, FOXP3upstream enhancer methylation assay of Tregs showed that the methylation levels were higher in ACS group than that in control group (P<0.05).③Real-time quantitative RT-PCR analysis of FOXP3mRNA levels showed that the amount of FOXP3mRNA were lower in ACS group than that in control group (P<0.05).④No significant differences in plasma IL-10level among young controls, old controls, young ACS group and old ACS group (P>0.05), ACS group patients had significantly higher levels of INF-y than that in control group (P<0.05), ACS group patients had significantly lower levels of TGF-β than that in control group (P<0.05), however, no significant differences in plasma INF-y or TGF-β level between young ACS and old ACS group or young control and old control group (P>0.05, respectively).Conclusions:These findings showed the increased FOXP3upstream enhancer methylation level of Tregs in ACS patients in accordance with the decreases functional suppressive capacity of Tregs, so decrease of methylation level of FOXP3upstream enhancer may be a potential strategy to treat ACS.