Increased circulating levels of soluble interleukin (IL)-6 receptor α (sIL-6Rα) are commonly observed during inflammatory responses, allowing for IL-6 signaling in cells that express the ubiquitous receptor subunit gp130 but not IL-6Rα, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-κB-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6Rα-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6Rα in LPS-treated human umbilical vein and dermal blood microvascular endothelial cells. We show that co-treatment with sIL-6Rα dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This co-treatment led to a strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-κB signaling. Similar results were obtained when sIL-6Rα was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, while SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-kB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

Increased circulating levels of soluble interleukin (IL)-6 receptor α (sIL-6Rα) are commonly observed during inflammatory responses, allowing for IL-6 signaling to occur in cells that express the ubiquitous receptor subunit gp130 but not IL-6Rα, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-κB-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6Rα-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6Rα in LPS-treated human umbilical vein endothelial cells. We show that cotreatment with sIL-6Rα dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This co-treatment led to a strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-κB signaling. Similar results were obtained when sIL-6Rα was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, while SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-kB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

Interleukin 6 (IL-6) is a major mediator of the cytokine storm during shock. Its actions on the endothelium lead to pathological vascular leakage, leukocyte adhesion, and coagulopathy, which may result in multiorgan dysfunction and potentially death. Many shock survivors suffer long-term sequelae. We hypothesize that shock-induced endothelial epigenetic changes are involved in orchestrating these chronic pathological changes. Here, we interrogated the DNA methylation changes in the kidney endothelium after a challenge with lipopolysaccharides (LPS) in wilt type (WT) mice and in mice lacking endothelial SOCS3 (SOCS3iEKO). We identified 2596 differentially methylated positions (DMP) after an LPS challenge in WT mice. Notably, lack of endothelial SOCS3 leads to a highly divergent response, with 3564 DMP in response to LPS in SOCS3iEKO vs WT mice. Consistent with the hyper-inflammatory response in SOCS3iEKO mice, gene ontology analysis shows an enrichment in genes associated with an inflammatory response. To assess whether endothelial cells respond to IL-6 signaling directly by inducing DNA methylation changes, we challenged human umbilical vein endothelial cells in culture with IL-6 for 72 hours prior to methylomics assays. DNA methylation modifications were observed in sites associated with pathways regulating the regulation of cell adhesion, proliferation, and cytokine signaling. To determine the stability of these changes, we treated cells with IL-6 for 72h, followed by 96h in the absence of IL-6. We found that 40% of the DMP remained modified after the cytokine wash with a mean difference of less than 2% when comparing washed (72+96h) vs. non-washed cells (72h alone), suggesting that many changes in the endothelial methylome may remain in place for prolonged periods. In summary, persistent activation of endothelial IL-6 signaling leads to endothelial DNA methylation changes. Together, our data demonstrate that in response to pro-inflammatory stimuli, the endothelium elicits epigenetic changes that are at least in part due to IL-6/STAT3/SOCS3 signaling. These changes may regulate pathways associated with reduced long-term survival and chronic organ damage after shock.

Interleukin‐6 (IL‐6) is a major mediator of the cytokine storm in response to severe systemic inflammation and sepsis. This cytokine promotes the activation of the transcription factor STAT3 via Janus kinases (JAK)‐dependent phosphorylation on tyrosine 705. Prior work in our lab demonstrated that overactivation of this pathway within the endothelium via an endothelial‐specific deletion of the negative regulator SOCS3 (SOCS3iEKO) leads to kidney failure and acute mortality after a single lipopolysaccharides (LPS) challenge that was non‐lethal in wild‐type mice. This mortality was associated with increased systemic IL‐6 levels and vascular leak in lungs and brain of SOCS3iEKO mice. Furthermore, translating ribosome affinity purification studies showed that in response to LPS, the kidney and brain endothelia expressed high levels of IL‐6. These findings suggested that IL‐6 signaling within the endothelium might be required to promote vascular leak. To sought to test this hypothesis in vitro. We found that LPS promoted a mild increase in monolayer permeability in HUVEC that was abrogated by pretreatment with the JAK inhibitor ruxolitinib. This loss of barrier function by LPS was associated with an increase in IL‐6 expression and a modest increase in STAT3 phosphorylation. Endothelial cells express very low levels of the gp80 subunit of the IL‐6 receptor. Thus, to test whether an IL‐6 autocrine loop induced barrier function loss, we treated cells with LPS in the presence or absence of a soluble form of gp80 (herein, LPS+R). LPS+R‐treated cells showed increased STAT3 phosphorylation and severe barrier function loss. A similar increase in STAT3 phosphorylation and monolayer permeability was observed when gp80 was added to TNF‐treated cells. In contrast, addition of gp80 to LPS‐ or TNF‐treated cells did not alter the levels of p65RelA or p38 phosphorylation. Pretreatment of LPS+R‐treated cells with the p38 inhibitor SB203580 showed that activation of p38 signaling was not required for STAT3 phosphorylation or barrier function loss. In summary, our data strongly suggest that the endothelium responds to strong inflammatory challenges with an increase in IL‐6 expression that may act in an autocrine fashion to promote strong barrier function loss, potentially explaining the systemic vascular leak observed in critically ill patients with high circulating IL‐6 levels.