Regulation of endothelial DNA methylation by IL‐6 signaling

Abstract

Interleukin 6 (IL‐6) is a major mediator of the septic cytokine storm. It activates the transcription factor STAT3, resulting in pathological vascular leakage, leukocyte adhesion, and coagulation cascade activation, leading to acute multiorgan dysfunction. Septic shock survivors have a high risk of developing post‐intensive care syndrome (PICS), a condition of chronic cardiovascular disease, cognitive impairment, and reduced overall survival. The causes for this syndrome are not fully understood. We hypothesize that acute STAT3‐mediated endotheliopathy promotes changes in the endothelial DNA methylation landscape, leading to the long‐lasting effects of PICS. To test this in vitro, HUVEC were treated with a combination of IL‐6 and sIL‐6Rα (IL‐6+R) or saline for 6h, 24h, or 72h, and DNA methylation was profiled using Infinium Methylation EPIC arrays after bisulfite conversion. Unbiased clustering showed a separation between treated and control cells only after 72h. 360 CpG sites were significantly modified (FDR‐adjusted p=<0.05). Of these, 329 CpG sites were hypomethylated and 31 were hypermethylated. Gene ontology analysis of the differentially methylated genes showed enrichment of pathways directly linked with the platelet and leukocyte interaction, cell proliferation and angiogenesis, and signaling. Consistent with the demethylation of the LAMP3 locus, a 72h treatment with IL‐6+R induced a dramatic increase in LAMP3 expression. The same treatment induced an inhibition of ADAM19 and MYO10 gene expression, consistent with their hypermethylation status. These changes were not associated with differences in the expression of DNA methyltransferases and TET enzymes. The IL‐6+R‐induced increase in LAMP3 expression was further increased in cells that had been previously treated with IL‐6+R for 72h and then incubated in the absence of IL‐6+R for another 96h, compared to saline‐treated cells, suggesting a long‐lasting effect of a prior IL‐6+R treatment via demethylation of the promoter. In contrast, SOCS3 expression (a gene directly activated by STAT3) was similar in cells that were pretreated with IL‐6+R or not. In summary, persistent activation of IL‐6 leads to endothelial DNA methylation changes and corresponding gene expression differences. These differences may regulate pathways that can be associated with reduced long‐term survival and prolonged organ damage as observed in PICS.