Alterations of monocyte subsets in chronic heart failure patients in association with changes in epigenetic regulation

Chronic heart failure (CHF) is closely with associated altered inflammatory responses. Their mechanisms has not been fully elucidated, but there is support for the notion that the specific subsets of monocytes may play a crucial role. Here we aimed to investigate whether CHF is associated with alterations of distribution of different monocyte subsets, namely classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++. Classical monocytes are involved in phagocytosis and resolution of inflammation, a minor subset of macrophage-like CD16-positive monocytes (mostly non-classical) is capable of driving and modulating inflammatory processes by secretion of TNFα, tissue factor or ACE. Moreover, we attempted to explain these changes by investigating epigenetic regulation.

We performed a clinical and immunologic analysis of 29 CHF patients and 34 healthy age and gender matched controls. Abovementioned monocyte subsets were analyzed by flow cytometry. Methylation of 94 proinflammatory genes in nuclear blood cells was assessed using restrictase digestion based assay. In 8 CHF and 14 control serum samples, circulating microRNA regulating inflammatory reactions were assessed (miR-20a. miR-21, miR146a, miR199), using RT-PCR.

We demonstrated that monocytes of CHF patients are significantly enriched in non-classical subset as compared to healthy subjects [6.76% (4.16-9.87) vs. 3.57% (2.48-5.27), respectively; p<0.001]. Similarly, although to a lesser extent, CHF patients presented with greater frequencies of intermediate monocytes [13.2% (6.76-36.1) vs. 8.01% (4.63-15.1), p=0.013]. In contrast, percentages of classical monocytes were significantly less in CHF patients than in healthy controls [70.6% (42.4-80.1) vs. 83.1% (77.0-87.1), respectively; p<0.001]. Median level of methylation of proinflammatory genes was higher in patients with CHF 0.4% vs 0.09%, with particular methylation increase of Mapk1 (100% vs 0), IL17 receptor A (25% vs 2%) and NFATc3 (10.1% vs 0.6%) genes. Interestingly, CD14 was severely hypermethylated in controls (99.8%) and much less in CHF (50%). CHF patients had lower serum expression of miR-146a (55.8% of controls) and miR-20a (10.5% of controls) and higher of miR199a (221% of controls) and miR-21 (344% of controls).

Our data indicate that CHF is associated with accumulation of more mature immunomodulatory monocyte subsets. The observed phenotype may be result of epigenetic effects of blood cells and altered expression of circulation miRNA. Based on our results we could hypothesize that all monocyte subsets are closely implicated in inflammatory processes due to CHF