Shock drives a highly coordinated transcriptional and DNA methylation response in the endothelium_DNA methylation

Endothelial dysfunction is a critical factor in promoting organ failure during septic shock. Organ dysfunction during shock increases the risk of long-term sequelae in survivors through mechanisms that remain unknown. We postulated that vascular dysfunction during shock contributes to long-term morbidity post-shock through transcriptional and epigenetic changes within the endothelium. We performed cross-omics analyses on kidney endothelium from acute endotoxin-challenged mice lacking or not the JAK/STAT3 inhibitor SOCS3. This analysis revealed significant DNA methylation changes upon proinflammatory signaling associated with transcriptional activity through AP1, STAT, and IRF families, suggesting a mechanism driving transcription-induced gene-specific methylation changes. In vitro, we demonstrated that IL-6 induces similar changes in DNA methylation. Specific genes showed DNA methylation changes in response to an IL-6+R and consistently changes in their expression levels within 72 hours of IL-6+R treatment. Further, changes in the endothelial methylome remain in place for prolonged periods without IL-6, suggesting that this cytokine may elicit transcriptional changes long after the resolution of inflammation. Also, demonstrated that DNA methylation changes could directly alter the expression of these genes and that STAT3 activation had a causal role in this transcriptional response. Our findings provide evidence for a critical role of IL-6 signaling in regulating shock-induced epigenetic changes and sustained endothelial activation, offering a new therapeutic target to limit vascular dysfunction. Human Umbilical Vein Endothelial Cells (HUVECs) were isolated in-house. The identity and purity of the HUVEC isolations were confirmed for each isolation by more than 99% positive immunostaining with endothelial cell markers (FITC-Ulex europaeus lectin, VE-cadherin) and more than 99.9% negative for α–smooth muscle actin. Cells were assayed between passages 3 and 8. To induce IL-6 signaling, cells were plated at full confluence at a density of 8 × 104 cells/cm2 on plates precoated for 30 min with 0.1% gelatin and incubated at least 48 h prior to the start of experiments. Cells were then treated with a combination of 200 ng/mL recombinant human IL-6 and 100 ng/mL sIL-6Rα (IL-6+R) or PBS (control) for 72 hours. After 72 hours, a subset of control and IL-6+R-treated cells were washed and incubated for 96 hours in EGM-2 Growth Medium. Treated HUVECs and controls were immediately centrifuged and stored until DNA extraction. DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, USA) according to the manufacturer's instructions, including optional treatment with 100 mg/ml RNase A. The concentration of DNA was measured by the Qubit dsDNA BR Assay Kit (Molecular Probes). The DNA samples were stored at -80°C. Bisulfite conversion of 500 ng of genomic DNA was performed using an EZ DNA Methylation-Gold™ Kit (Zymo Research) following manufacturer’s instructions. Analysis of DNA methylation was carried out using Illumina Infinium MethylationEPIC BeadChip arrays33 for human genomes (>850K sites)