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ASSA13-01-13 FOXP3 Demethylation as a Means of Identifying Quantitative Defects in Regulatory T Cells in Acute Coronary Syndrome
  1. Lü Caixia,
  2. Zhang Cuntai
  1. Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Abstract

Background 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 that distinguish Tregs and activated conventional T cells. A highly conserved CpG enriched element in forkhead boxP3intron 1 (FOXP3 i l) is unmethylated only in Tregs, and measuring the unmethylation of FOXP3 i l can be used to identify the role of Tregs in clinical diseases. For this reason, we investigated whether analysing the demethylation status of FOXP3 i 1 is a more reliable means than using Treg-specific surface markers.

Methods and Results Circulating Tregs percentages, cytokine (anti-inflammatory cytokines, IL-10 and TGF-β1; pro-inflammatory cytokine, interferon-γ) production profile in subjects with acute coronary syndrome (ACS) were correlated to the amount of FOXP3 protein expression measured by flow cytometry or FOXP3 mRNA expression measured by RT-PCR or FOXP3 i 1 demethylation status measured by pyrosequencing. Activated T cells were treated in vitro with a DNA demethylation agent to study the effect of DNA methylation on FOXP3 expression, FOXP3 i l demethylation and Treg cytokine production. FOXP3 i 1 demethylation assay showed that the amount of Tregs in ACS patients was significantly reduced than that in healthy controls (p = 0.0005). This test displayed a sensitivity of 94.7% and specificity of 80% in distinguishing ACS patients from healthy controls, as indicated by analysis of the receiver operating characteristic (ROC) curve (AUC = 0.916; p < 0.001). However, flow cytometry analysis did not identify any reductions in total percentage of CD4+CD25+FOXP3+Tregs, indicating that CD4+CD25+FOXP3+Tregs in ACS patients were highly variable. Notably, younger patients with ACS had higher percentage of total CD4+CD25+FOXP3+Tregs but decreased percentage of CD4+CD25+CD45+naïve Tregs, higher amounts of interferon-γ, and lower amounts of high-density lipoprotein than either healthy controls or older patients with ACS. Furthermore, 5-aza-2′-deoxycytidine, a DNA hypomethylation agent, increased the amount of CD4+CD25+FOXP3+Tregs and IL-10 and suppressed the production of interferon-γ by inducing FOXP3 i 1 demethylation in vitro.

Conclusions Our findings showed that a quantitative defect of Tregs, suggestive of decreased peripheral tolerance, could be a potential hallmark of ACS disease. Targeting FOXP3 i l demethylation might elevate the inhibitory activity of Tregs in ACS.

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