SOCS3 limits TNF and endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin-6 signal in human endothelial cells.

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 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 cotreatment led to 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, whereas SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-κB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

Resolvin D1 Enhances Necroptotic Cell Clearance Through Promoting Macrophage Fatty Acid Oxidation and Oxidative Phosphorylation.

OBJECTIVE: Plaque necrosis is a key feature of defective resolution in atherosclerosis. Recent evidence suggests that necroptosis promotes plaque necrosis; therefore, we sought to determine how necroptotic cells (NCs) impact resolution programs in plaques. Approach and Results: To investigate the role(s) of necroptosis in advanced atherosclerosis, we used mice deficient of Mlkl, an effector of necroptosis. Mlkl-/- mice that were injected with a gain-of-function mutant PCSK9 (AAV8-gof-PCSK9) and fed a Western diet for 16 weeks, showed significantly less plaque necrosis, increased fibrous caps and improved efferocytosis compared with AAV8-gof-PCSK9 injected wt controls. Additionally, hypercholesterolemic Mlkl-/- mice had a significant increase in proresolving mediators including resolvin D1 (RvD1) and a decrease in prostanoids including thromboxane in plaques and in vitro. We found that exuberant thromboxane released by NCs impaired the clearance of both apoptotic cells and NCs through disruption of oxidative phosphorylation in macrophages. Moreover, we found that NCs did not readily synthesize RvD1 and that exogenous administration of RvD1 to macrophages rescued NC-induced defective efferocytosis. RvD1 also enhanced the uptake of NCs via the activation of p-AMPK (AMP-activated protein kinase), increased fatty acid oxidation, and enhanced oxidative phosphorylation in macrophages. CONCLUSIONS: These results suggest that NCs derange resolution by limiting key SPMs and impairing the efferocytic repertoire of macrophages. Moreover, these findings provide a molecular mechanism for RvD1 in directing proresolving metabolic programs in macrophages and further suggests RvD1 as a potential therapeutic strategy to limit NCs in tissues. Graphic Abstract: A graphic abstract is available for this article.

Unique inflammatory profile is associated with higher SARS-CoV-2 acute respiratory distress syndrome (ARDS) mortality.

The COVID19 pandemic has caused more than a million of deaths worldwide, primarily due to complications from COVID19-associated acute respiratory distress syndrome (ARDS). Controversy surrounds the circulating cytokine/chemokine profile of COVID19-associated ARDS, with some groups suggesting that it is similar to patients without COVID19 ARDS and others observing substantial differences. Moreover, although a hyperinflammatory phenotype associates with higher mortality in non-COVID19 ARDS, there is little information on the inflammatory landscape's association with mortality in patients with COVID19 ARDS. Even though the circulating leukocytes' transcriptomic signature has been associated with distinct phenotypes and outcomes in critical illness including ARDS, it is unclear whether the mortality-associated inflammatory mediators from patients with COVID19 are transcriptionally regulated in the leukocyte compartment. Here, we conducted a prospective cohort study of 41 mechanically ventilated patients with COVID19 infection using highly calibrated methods to define the levels of plasma cytokines/chemokines and their gene expressions in circulating leukocytes. Plasma IL1RA and IL8 were found positively associated with mortality, whereas RANTES and EGF negatively associated with that outcome. However, the leukocyte gene expression of these proteins had no statistically significant correlation with mortality. These data suggest a unique inflammatory signature associated with severe COVID19.

Molecular Mechanisms of Vascular Damage During Lung Injury.

A variety of pulmonary and systemic insults promote an inflammatory response causing increased vascular permeability, leading to the development of acute lung injury (ALI), a condition necessitating hospitalization and intensive care, or the more severe acute respiratory distress syndrome (ARDS), a disease with a high mortality rate. Further, COVID-19 pandemic-associated ARDS is now a major cause of mortality worldwide. The pathogenesis of ALI is explained by injury to both the vascular endothelium and the alveolar epithelium. The disruption of the lung endothelial and epithelial barriers occurs in response to both systemic and local production of pro-inflammatory cytokines. Studies that evaluate the association of genetic polymorphisms with disease risk did not yield many potential therapeutic targets to treat and revert lung injury. This failure is probably due in part to the phenotypic complexity of ALI/ARDS, and genetic predisposition may be obscured by the multiple environmental and behavioral risk factors. In the last decade, new research has uncovered novel epigenetic mechanisms that control ALI/ARDS pathogenesis, including histone modifications and DNA methylation. Enzyme inhibitors such as DNMTi and HDACi may offer new alternative strategies to prevent or reverse the vascular damage that occurs during lung injury. This review will focus on the latest findings on the molecular mechanisms of vascular damage in ALI/ARDS, the genetic factors that might contribute to the susceptibility for developing this disease, and the epigenetic changes observed in humans, as well as in experimental models of ALI/ADRS.

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

Resolvin D1 suppresses macrophage senescence and splenic fibrosis in aged mice.

Aging is associated with systemic, non-resolving inflammation and the accumulation of senescent cells. The resolution of inflammation (or inflammation-resolution) is in part mediated by the balance between specialized pro-resolving mediators (SPMs) and pro-inflammatory leukotrienes (LTs). Aged mice (i.e. 2 years of age) exhibit a significant decrease in the SPM:LT ratio in specific organs including the spleen, which suggests that this organ may exhibit heightened inflammation and may be particularly amenable to SPM therapy. Previous studies have shown that resolvin D1 (RvD1) is decreased in spleens of aged mice compared with young controls. Therefore, we asked whether treatment of RvD1 in aged mice would impact markers of cellular senescence in splenic macrophages, and downstream effects on splenic fibrosis, a hallmark of splenic aging. We found that in aged mice, both zymosan-elicited and splenic macrophages showed an increase in mRNA expression of inflammatory and eicosanoid biosynthesis genes and a dysregulation of genes involved in the cell cycle. Injections with RvD1 reversed these changes. Importantly, RvD1 also decreased splenic fibrosis, a hallmark of splenic aging. Our findings suggest that RvD1 treatment may limit several features of aging, including senescence and fibrosis in spleens from aged mice. Summary Aging is associated with systemic, low grade, non-resolving inflammation. The resolution of inflammation is in part mediated by the balance between specialized pro-resolving mediators (SPMs) and pro-inflammatory lipid mediators, like leukotrienes (LTs). A hallmark of aging is the accumulation of senescent cells that promote low grade inflammation by secreting pro-inflammatory cytokines and lipid mediators. Splenic macrophages contribute to systemic aging, and spleens of aged mice demonstrate decreased levels of the SPM called resolvin D1 (RvD1). Whether addition of RvD1 is protective in spleens of aged mice is unknown and is focus of this study. RvD1 treatment to aged mice led to decreased mRNA expression of markers of cellular senescence and inflammation in splenic macrophages compared with age-matched vehicle controls. Moreover, RvD1 decreased splenic fibrosis, which occurs due to persistent low-grade inflammation in aging. Promoting inflammation resolution with RvD1 thus limits macrophage senescence, pro-inflammatory signals and established splenic fibrosis in aging.

A transient brain endothelial translatome response to endotoxin is associated with mild cognitive changes post-shock in young mice.

Sepsis-associated encephalopathy (SAE) is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. LPS induced a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a crucial role for IL-6, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge, but reductions in gene expression regulating protein translation and respiratory electron transport remained. We observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock.

Effects of low-dose versus placebo or conventional-dose postmenopausal hormone therapy on variables related to cardiovascular risk: a systematic review and meta-analyses of randomized clinical trials.

CONTEXT: Hormone therapy (HT), the most efficient treatment for menopausal symptoms, might have deleterious cardiovascular (CV) effects. OBJECTIVE: This study aimed to evaluate the effects of low-dose estrogen HT on CV risk factors vs conventional-dose HT and placebo in postmenopausal women with no established CV disease. DATA SOURCES: MEDLINE, Cochrane Central, and EMBASE were searched for trials published in 1990-2013; a hand search of reference lists of selected articles was performed; and ClinicalTrials.gov was searched for unpublished trials. STUDY SELECTION: Within randomized controlled trials of healthy postmenopausal women comparing low-dose HT to placebo or conventional-dose HT, 11 418 studies were initially identified. DATA EXTRACTION: Data were independently extracted by two investigators. Disagreements were resolved by a third author. DATA SYNTHESIS: Twenty-eight trials (3360 patients) were included. Low-dose HT vs placebo or conventional-dose HT did not effect weight, body mass index (BMI), blood pressure, C-reactive protein, or high-density lipoprotein cholesterol (HDL-C). Low-dose HT was associated with lower levels of total cholesterol (-12.16 mg/dL, 95% confidence interval [CI], -17.41 - -6.92) and low-density lipoprotein cholesterol (LDL-C) (-12.16 mg/dL; 95% CI, -16.55 - -7.77) vs placebo. Compared with conventional-dose HT, low-dose HT was associated with higher total cholesterol (5.05 mg/dL; 95% CI, 0.88-9.21) and LDL-C (4.49 mg/dL; 95% CI, 0.59-8.39). Low-dose HT was not associated with differences in triglycerides vs placebo. Oral, low-dose HT was associated with lower triglycerides vs conventional-dose HT (-14.09 mg/dL; 95% CI, -24.2 - -3.93). CONCLUSION: In this population of apparently healthy postmenopausal women, the effect of low-dose HT did not differ from that of placebo or conventional-dose HT regarding weight, BMI, blood pressure, CRP, or HDL-C. In contrast, low-dose HT was associated with better lipid profile vs placebo, and induced higher total and LDL-C and lower triglycerides vs conventional-dose HT.