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.