Shock drives a highly coordinated transcriptional and DNA methylation response in the endothelium
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. Overall design: Severe, acute inflammation was induced by a single IP injection of a bolus of 250 µg/250 µl LPS. Control mice were given 250 µl of sterile saline via IP injection. Disease severity was scored 14 hours after the LPS challenge as described before17. Immediately after scoring, body weight and temperature were measured. Mice were then euthanized with an overdose of pentobarbital. No mice were excluded from the studies. Assignment to the saline or LPS groups was performed through randomization of mice within each genotype and sex for each litter. All handling, measurements, and scoring were performed by a researcher masked to treatment and genotype groups and based on mouse ID (ear tags). Experimental groups were unmasked at the end of each experiment. After euthanasia, the animals were cannulated on the left ventricle, and the right atrium was nicked to allow perfusion with 5 ml/min PBS for 3 min. Kidneys were collected and minced into small pieces (~1 mm3). The tissues were transferred to 50 ml conical tubes containing 5 ml of PBS containing calcium and magnesium plus 100 mg of type 1 collagenase and 100 mg dispase II for 10 minutes at 37°C. The slurry of tissue and buffer was triturated by passage through a 14-gauge cannula attached to a 10 ml syringe approximately 10 times, followed by a 20-gauge cannula attached to a 10 ml syringe. The cell suspensions were filtered through a 70 µm cell strainer, resulting in single cell suspensions prior to centrifugation at 400×g for 8 min at 4 °C. Pellets were resuspended with 1.5ml of ice-cold PBS + 0.1% BSA.   Cell suspensions were transferred to RNAse free polystyrene tubes containing 60 µl of streptavidin-conjugated dynabeads bound to biotinylated EpCAM and biotinylated CD45 antibodies. The cell suspensions were incubated with these beads for 10 min at RT with rotation. Following this, tubes were placed on a Dynal MPC-S magnet for 5 minutes. The supernatants were transferred to RNAse free polystyrene tubes containing 45ul of streptavidin-conjugated dynabeads bound to biotinylated CD31 antibodies and incubated for 10 min at RT with rotation. Following this, tubes were placed on a Dynal MPC-S magnet for 5 minutes and the supernatant was discarded. The remaining cells bound to the beads were washed six times with PBS containing 0.1% BSA to remove any contaminating cells. After removal of the final wash, the cells were resuspended in 200 µl PBS. 200 µl were transferred to an RNAse-free tube and proceeded to DNA isolation using the DNeasy Blood & Tissue Kits (Qiagen, USA).   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 Infinium Mouse Methylation BeadChip arrays for mouse genomes (>285K sites) by the Epigenomic Services from Diagenode.