br Materials and Methods br Results

Materials and Methods
Results
Discussion In the present population-based study, we assessed the relationship between pre-diagnostic Treg to tTL ratios and future cardiovascular events using a highly specific epigenetic assay for the quantification of T cell subsets. After a median follow-up time of about 7years, lower Treg/tTL ratios were not associated with either an increased incidence of myocardial infarction or ischemic stroke in multivariable models accounting for traditional risk factors of atherosclerotic CVD. These findings do not support a potential role of altered Treg-mediated immune tolerance as a risk factor for human CVD. Previous studies in humans were centered almost exclusively on patients with established cardiovascular disease and therefore it remained uncertain whether reduced circulating levels of Tregs are an important etiological factor, or rather reflect facilitated Treg trafficking to ischemic tissues as part of regulatory mechanisms involved in resolution of post-infarction buy Quinacrine Dihydrochloride (Frangogiannis, 2012). In accordance with a protective role of Tregs in CVD development, the Malmö Diet and Cancer Study cardiovascular cohort (MDCS-CV) study showed that low circulating Treg levels, defined as CD4+FoxP3+ T cells, were associated with an increased risk for acute myocardial infarction (hazard ratio 1.9 for the lowest Treg tertile) during a 15-year follow-up. On the contrary, no association of Treg frequencies with overall stroke risk was found (Wigren et al., 2012). Our results for ischemic stroke are in agreement with these findings by Wigren and colleagues, whereas absence of an association between low Treg/tTL ratios and MI risk contrasts with their results. However, the present study differs from the MDCS-CV study in several respects. We used an epigenetic approach for immune cell quantification as compared with a cytokine release assay combined with flow cytometry in the previous study. This difference may be relevant because Tregs form only a minor fraction of lymphocytes in blood and epigenetic signatures involving the FOXP3 TSDR demethylation status are currently considered to be the most specific marker of stable Tregs (Floess et al., 2007; Polansky et al., 2008; Baron et al., 2007). An earlier comparison between quantification of Treg/tTL ratios in cord and adult blood samples using epigenetic qPCR and flow cytometry assays showed reasonable method agreement, even though it has to be noted that differences to some degree between the two methods can be expected as the epigenetic tool allows a more specific detection of functionally stable FOXP3+ Tregs compared to surface marker-based flow cytometry (Nettenstrom et al., 2013). Besides the different assays applied in the present study and the study by Wigren et al., deviating denominators used to determine relative Treg frequencies (CD3+ T cells in EPIC-Heidelberg vs. CD4+ T cells in the MDCS-CV study) must be considered when making a direct comparison of results from the two studies. In fact, there may be differences in the directions of associations between distinct T cell subpopulations, such as CD4+ and CD8+ T cells, with inflammatory and autoimmune-mediated pathogenesis of atherosclerotic CVD (Ammirati et al., 2015), and CD3+CD8+ T cells were positively associated with MI risk in the MDCS-CV study (Kolbus et al., 2013). Moreover, EPIC-Heidelberg cohort participants included here were free of diabetes (0% vs. 21% and 11% among cases and controls from the MDCS-CV), younger (mean baseline age 55years vs. 65years), and incident MI cases had less frequently reported hypertension at baseline (46% vs. 87%) compared to the MDCS-CV study. In contrast, MI cases occurred more frequently in men as compared with those cases identified in the MDCS-CV (79% vs. 53%). Of note, male gender, increasing age as well as other established CVD risk factors such as diabetes and arterial hypertension have all been proposed to be inversely associated with absolute Treg numbers (Kornete et al., 2013; Idris-Khodja et al., 2014). Inconsistency of results between both studies may thus be due to heterogeneity concerning the cardiovascular risk profile, and could imply that the role of circulating Tregs in CVD development depends on the extent or type of pre-existing arterial remodeling. Further clarification of this issue will require verification in other cohorts including atherosclerosis imaging approaches.