A novel fatty acid analogue triggers CD36-GPR120 interaction and exerts anti-inflammatory action in endotoxemia.

Inflammation is a mediator of a number of chronic pathologies. We synthesized the diethyl (9Z,12Z)-octadeca-9,12-dien-1-ylphosphonate, called NKS3, which decreased lipopolysaccharide (LPS)-induced mRNA upregulation of proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) not only in primary intraperitoneal and lung alveolar macrophages, but also in freshly isolated mice lung slices. The in-silico studies suggested that NKS3, being CD36 agonist, will bind to GPR120. Co-immunoprecipitation and proximity ligation assays demonstrated that NKS3 induced protein-protein interaction of CD36 with GPR120in RAW 264.7 macrophage cell line. Furthermore, NKS3, via GPR120, decreased LPS-induced activation of TAB1/TAK1/JNK pathway and the LPS-induced mRNA expression of inflammatory markers in RAW 264.7 cells. In the acute lung injury model, NKS3 decreased lung fibrosis and inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and nitric oxide (NO) production in broncho-alveolar lavage fluid. NKS3 exerted a protective effect on LPS-induced remodeling of kidney and liver, and reduced circulating IL-1ß, IL-6 and TNF-α concentrations. In a septic shock model, NKS3 gavage decreased significantly the LPS-induced mortality in mice. In the last, NKS3 decreased neuroinflammation in diet-induced obese mice. Altogether, these results suggest that NKS3 is a novel anti-inflammatory agent that could be used, in the future, for the treatment of inflammation-associated pathologies.

Thromboelastographic evaluation of the effectiveness of choline or CDP-choline treatment on endotoxin-induced hemostatic alterations in dogs.

Sepsis/endotoxemia associates with coagulation abnormalities. We showed previously that exogenous choline treatment reversed the changes in platelet count and function as well as prevented disseminated intravascular coagulation (DIC) in endotoxemic dogs. The aim of this follow-up study was to evaluate the effect of treatment with choline or cytidine-5'-diphosphocholine (CDP-choline), a choline donor, on endotoxin-induced hemostatic alterations using thromboelastography (TEG). Dogs were randomized to six groups and received intravenously (iv) saline, choline (20 mg/kg) or CDP-choline (70 mg/kg) in the control groups, whereas endotoxin (0.1 mg/kg, iv) was used alone or in combination with choline or CDP-choline at the same doses in the treatment groups. TEG variables including R- and K-time (clot formation), maximum amplitude (MA) and α-angle (clot stability), G value (clot elasticity), and EPL, A, and LY30 (fibrinolysis), as well as overall assessment of coagulation (coagulation index - CI), were measured before and at 0.5-48 h after the treatments. TEG parameters did not change significantly in the control groups, except for CI parameter after choline administration. Endotoxemia resulted in increased R-time and A value (P < 0.05), decreased K-time (P < 0.05), α-angle (P < 0.001) and CI values (P < 0.01) at different time points. Treatment with either choline or CDP-choline attenuated or prevented completely the alterations in TEG parameters in endotoxemic dogs with CDP-choline being more effective. These results confirm and extend the effectiveness of choline or CDP-choline in endotoxemia by further demonstrating their efficacy in attenuating or preventing the altered viscoelastic properties of blood clot measured by TEG.

Biomolecular Actions by Intestinal Endotoxemia in Metabolic Syndrome.

Metabolic syndrome (MetS) is a combination of metabolic disorders that concurrently act as factors promoting systemic pathologies such as atherosclerosis or diabetes mellitus. It is now believed to encompass six main interacting conditions: visceral fat, imbalance of lipids (dyslipidemia), hypertension, insulin resistance (with or without impairing both glucose tolerance and fasting blood sugar), and inflammation. In the last 10 years, there has been a progressive interest through scientific research investigations conducted in the field of metabolomics, confirming a trend to evaluate the role of the metabolome, particularly the intestinal one. The intestinal microbiota (IM) is crucial due to the diversity of microorganisms and their abundance. Consequently, IM dysbiosis and its derivate toxic metabolites have been correlated with MetS. By intervening in these two factors (dysbiosis and consequently the metabolome), we can potentially prevent or slow down the clinical effects of the MetS process. This, in turn, may mitigate dysregulations of intestinal microbiota axes, such as the lung axis, thereby potentially alleviating the negative impact on respiratory pathology, such as the chronic obstructive pulmonary disease. However, the biomolecular mechanisms through which the IM influences the host's metabolism via a dysbiosis metabolome in both normal and pathological conditions are still unclear. In this study, we seek to provide a description of the knowledge to date of the IM and its metabolome and the factors that influence it. Furthermore, we analyze the interactions between the functions of the IM and the pathophysiology of major metabolic diseases via local and systemic metabolome's relate endotoxemia.

Potential Therapeutic Strategies and Drugs That Target Vascular Permeability in Severe Infectious Diseases.

Severe infection pathogenicity is induced by processes such as pathogen exposure, immune cell activation, inflammatory cytokine production, and vascular hyperpermeability. Highly effective drugs, such as antipathogenic agents, steroids, and antibodies that suppress cytokine function, have been developed to treat the first three processes. However, these drugs cannot completely suppress severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, developing novel drugs that inhibit vascular hyperpermeability is crucial. This review summarizes the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced vascular hyperpermeability and identifies inhibitors that increase endothelial cell (EC) junction-related proteins and determines their efficacy in COVID-19 and endotoxemia models. Analyzing the effects of SARS-CoV-2 on vascular permeability revealed that SARS-CoV-2 suppresses Claudin-5 (CLDN5) expression, which is responsible for adhesion between ECs, thereby increasing vascular permeability. Inhibiting CLDN5 function in mice induced vascular hyperpermeability and pulmonary edema. In contrast, Enhancing CLDN5 expression suppressed SARS-CoV-2-induced endothelial hyperpermeability, suggesting that SARS-CoV-2-induced vascular hyperpermeability contributes to pathological progression, which can be suppressed by upregulating EC junction proteins. Based on these results, we focused on Roundabout4 (Robo4), another EC-specific protein that stabilizes EC junctions. EC-specific Robo4 overexpression suppressed vascular hyperpermeability and mortality in lipopolysaccharide-treated mice. An ALK1 inhibitor (a molecule that increases Robo4 expression), suppressed vascular hyperpermeability and mortality in lipopolysaccharide- and SARS-CoV-2-treated mice. These results indicate that Robo4 expression-increasing drugs suppress vascular permeability and pathological phenotype in COVID-19 and endotoxemia models.

The protective effects of naringenin, a citrus flavonoid, non-complexed or complexed with hydroxypropyl-ß-cyclodextrin against multiorgan damage caused by neonatal endotoxemia.

BACKGROUND: Endotoxemia is a severe and dangerous clinical syndrome that results in elevated morbidity, especially in intensive care units. Neonates are particularly susceptible to endotoxemia due to their immature immune systems. There are few effective treatments for neonatal endotoxemia. One group of compounds with potential in the treatment of neonatal inflammatory diseases such as endotoxemia is the flavonoids, mainly due to their antioxidant and anti-inflammatory properties. Among these, naringenin (NGN) is a citrus flavonoid which has already been reported to have anti-inflammatory, antioxidant, anti-nociceptive and anti-cancer effects. Unfortunately, its clinical application is limited by its low solubility and bioavailability. However, cyclodextrins (CDs) have been widely used to improve the solubility of nonpolar drugs and enhance the bioavailability of these natural products. OBJECTIVE: We, therefore, aimed to investigate the effects of NGN non-complexed and complexed with hydroxypropyl-ß-cyclodextrin (HPßCD) on neonatal endotoxemia injuries in a rodent model and describe the probable molecular mechanisms involved in NGN activities. METHOD: We used exposure to a bacterial lipopolysaccharide (LPS) to induce neonatal endotoxemia in the mice. RESULTS: It was found that NGN (100 mg/kg i.p.) exposure during the neonatal period reduced leukocyte migration and decreased pro-inflammatory cytokine (TNF-α, IL-1ß and IL-6) levels in the lungs, heart, kidneys or cerebral cortex. In addition, NGN upregulated IL-10 production in the lungs and kidneys of neonate mice. The administration of NGN also enhanced antioxidant enzyme catalase and SOD activity, reduced lipid peroxidation and protein carbonylation and increased the reduced sulfhydryl groups in an organ-dependent manner, attenuating the oxidative damage caused by LPS exposure. NGN decreased ERK1/2, p38MAPK and COX-2 activation in the lungs of neonate mice. Moreover, NGN complexed with HPßCD was able to increase the animal survival rate. CONCLUSION: NGN attenuated inflammatory and oxidative damage in the lungs, heart and kidneys caused by neonatal endotoxemia through the MAPK signaling pathways regulation. Our results show that NGN has beneficial effects against neonatal endotoxemia and could be useful in the treatment of neonatal inflammatory injuries.

The DNA-dependent protein kinase catalytic subunit exacerbates endotoxemia-induced myocardial microvascular injury by disrupting the MOTS-c/JNK pathway and inducing profilin-mediated lamellipodia degradation.

Rationale: The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) promotes pathological mitochondrial fission during septic acute kidney injury. The mitochondrial open reading frame of the 12S rRNA type-c (MOTS-c) is a mitochondria-derived peptide that exhibits anti-inflammatory properties during cardiovascular illnesses. We explored whether endotoxemia-induced myocardial microvascular injury involved DNA-PKcs and MOTS-c dysregulation. Methods: To induce endotoxemia in vivo, endothelial cell-specific DNA-PKcs-knockout mice were injected intraperitoneally with a single dose of lipopolysaccharide (10 mg/kg) and evaluated after 72 h. Results: Lipopolysaccharide exposure increased DNA-PKcs activity in cardiac microvascular endothelial cells, while pharmacological inhibition or endothelial cell-specific genetic ablation of DNA-PKcs reduced lipopolysaccharide-induced myocardial microvascular dysfunction. Proteomic analyses showed that endothelial DNA-PKcs ablation primarily altered mitochondrial protein expression. Verification assays confirmed that DNA-PKcs drastically repressed MOTS-c transcription by inducing mtDNA breaks via pathological mitochondrial fission. Inhibiting MOTS-c neutralized the endothelial protective effects of DNA-PKcs ablation, whereas MOTS-c supplementation enhanced endothelial barrier function and myocardial microvascular homeostasis under lipopolysaccharide stress. In molecular studies, MOTS-c downregulation disinhibited c-Jun N-terminal kinase (JNK), allowing JNK to phosphorylate profilin-S173. Inhibiting JNK or transfecting cells with a profilin phosphorylation-defective mutant improved endothelial barrier function by preventing F-actin depolymerization and lamellipodial degradation following lipopolysaccharide treatment. Conclusions: DNA-PKcs inactivation during endotoxemia could be a worthwhile therapeutic strategy to restore MOTS-c expression, prevent JNK-induced profilin phosphorylation, improve F-actin polymerization, and enhance lamellipodial integrity, ultimately ameliorating endothelial barrier function and reducing myocardial microvascular injury.

Influence of Escherichia coli lipopolysaccharide-induced endotoxemia on plasma and tissue disposition of florfenicol after intramuscular administration in rabbits.

To assess the effects of the acute inflammatory response (AIR) induced by Escherichia coli lipopolysaccharide (LPS) on plasma and tissue disposition of florfenicol (FFC) and its metabolite florfenicol amine (FFC-a), after its intramuscular (IM) administration, twenty-two New Zealand rabbits were randomly distributed in two experimental groups: Group 1 (LPS) was treated with three intravenous doses of 2 µg LPS/kg bw, before an intramuscular dose of 20 mg/kg FFC twenty-four h after the first LPS or SS injection; Group 2 (Control) was treated with saline solution (SS) in equivalent volumes as LPS-treated group. Blood samples were collected before (T0) and at different times after FFC administration. Acute inflammatory response was assessed in a parallel study where significant increases in body temperature, C-reactive protein concentrations and leukopenia were observed in the group treated with LPS. In another two groups of rabbits, 4 h after FFC treatment, rabbits were euthanized and tissue samples were collected for analysis of FFC and FFC-a concentrations. Pharmacokinetic parameters of FFC that showed significantly higher values in LPS-treated rabbits compared with control rabbits were absorption half-life, area under the curve, mean residence time and clearance /F (Cl/F). Elimination half-life and mean residence time of FFC-a were significantly higher in LPS-treated rabbits, whereas the metabolite ratio of FFC-a decreased significantly. Significant differences in tissue distribution of FFC and FFC-a were observed in rabbits treated with LPS. Modifications in plasma and tissue disposition of FFC and FFC-a were attributed mainly to haemodynamic modifications induced by the AIR through LPS administration.

Postprandial lipid and vascular responses following consumption of a commercially-relevant interesterified palmitic acid-rich spread in comparison to functionally-equivalent non-interesterified spread and spreadable butter: a randomised controlled trial in healthy adults.

Background: Interesterification is an industrial processing technique used widely where hard fats are essential for functionality and consumer acceptability, e.g. margarines and lower fat spreads. Objective: The aim of this study was to compare acute cardiovascular effects of functionally equivalent spreads (similar solid fat content) made with interesterified (IE) or non-IE palm-based fats, or spreadable butter. Methods: A randomised, controlled, 4-armed crossover, double-blind study (25 men, 25 women; 35-75 years; healthy; mean BMI 24.5, SD 3.8), compared effects of mixed nutrient meals containing 50 g fat from functionally equivalent products [IE spread, non-IE spread and spreadable butter (SB), with rapeseed oil (RO) as a reference treatment: with 16.7%, 27.9%, 19.3% and 4% palmitic acid, respectively] on 8 h postprandial changes in plasma triacylglycerol (TAG) and endothelial dysfunction (flow-mediated dilatation; FMD). Circulating reactive oxygen species (estimated using a neutrophil oxidative burst assay), glucose, insulin, NEFA, lipoprotein particle profiles, inflammatory markers (glycoprotein acetylation (Glyc-A) and IL-6), and biomarkers of endotoxemia were measured. Results: Postprandial plasma TAG concentrations after test meals were similar. However following RO versus the 3 spreads, there were significantly higher postprandial apolipoprotein B concentrations, and small HDL and LDL particle concentrations, and lower postprandial extra-large, large, and medium HDL particle concentrations, as well as smaller average HDL and LDL particle sizes. There were no differences following IE compared to the other spreads. Postprandial FMD% did not decrease after high-fat test meals, and there were no differences between treatments. Postprandial serum IL-6 increased similarly after test meals, but RO provoked a greater increase in postprandial concentrations of glycoprotein acetyls (GlycA), as well as 8 h sCD14, an endotoxemia marker. All other postprandial outcomes were not different between treatments. Conclusions: In healthy adults, a commercially-available IE-based spread did not evoke a different postprandial triacylglycerol, lipoprotein subclass, oxidative stress, inflammatory or endotoxemic response to functionally-equivalent, but compositionally-distinct alternative spreads. Clinical trial registry number: NCT03438084 (https://ClinicalTrials.gov).

Myeloid-Derived Suppressor-like Cells as a Prognostic Marker in Critically Ill Patients: Insights from Experimental Endotoxemia and Intensive Care Patients.

Patients admitted to the intensive care unit (ICU) often experience endotoxemia, nosocomial infections and sepsis. Polymorphonuclear and monocytic myeloid-derived suppressor cells (PMN-MDSCs and M-MDSCs) can have an important impact on the development of infectious diseases, but little is known about their potential predictive value in critically ill patients. Here, we used unsupervised flow cytometry analyses to quantify MDSC-like cells in healthy subjects challenged with endotoxin and in critically ill patients admitted to intensive care units and at risk of developing infections. Cells phenotypically similar to PMN-MDSCs and M-MDSCs increased after endotoxin challenge. Similar cells were elevated in patients at ICU admission and normalized at ICU discharge. A subpopulation of M-MDSC-like cells expressing intermediate levels of CD15 (CD15int M-MDSCs) was associated with overall mortality (p = 0.02). Interestingly, the high abundance of PMN-MDSCs and CD15int M-MDSCs was a good predictor of mortality (p = 0.0046 and 0.014), with area under the ROC curve for mortality of 0.70 (95% CI = 0.4-1.0) and 0.86 (0.62-1.0), respectively. Overall, our observations support the idea that MDSCs represent biomarkers for sepsis and that flow cytometry monitoring of MDSCs may be used to risk-stratify ICU patients for targeted therapy.

Lung single-cell RNA profiling reveals response of pulmonary capillary to sepsis-induced acute lung injury.

Background: Sepsis-induced acute lung injury (ALI) poses a significant threat to human health. Endothelial cells, especially pulmonary capillaries, are the primary barriers against sepsis in the lungs. Therefore, investigating endothelial cell function is essential to understand the pathophysiological processes of sepsis-induced ALI. Methods: We downloaded single-cell RNA-seq expression data from GEO with accession number GSE207651. The mice underwent cecal ligation and puncture (CLP) surgery, and lung tissue samples were collected at 0, 24, and 48 h. The cells were annotated using the CellMarker database and FindAllMarkers functions. GO enrichment analyses were performed using the Metascape software. Gene set enrichment Analysis (GSEA) and variation Analysis (GSVA) were performed to identify differential signaling pathways. Differential expression genes were collected with the "FindMarkers" function. The R package AUCell was used to score individual cells for pathway activities. The Cellchat package was used to explore intracellular communication. Results: Granulocytes increased significantly as the duration of endotoxemia increased. However, the number of T cells, NK cells, and B cells declined. Pulmonary capillary cells were grouped into three sub-clusters. Capillary-3 cells were enriched in the sham group, but declined sharply in the CLP.24 group. Capillary-1 cells peaked in the CLP.24 group, while Capillary-2 cells were enriched in the CLP.48 group. Furthermore, we found that Cd74+ Capillary-3 cells mainly participated in immune interactions. Plat+ Capillary-1 and Clec1a+ Capillary-2 are involved in various physiological processes. Regarding cell-cell interactions, Plat+ Capillary-1 plays the most critical role in granulocyte adherence to capillaries during ALI. Cd74+ Capillary cells expressing high levels of major histocompatibility complex (MHC) and mainly interacted with Cd8a+ T cells in the sham group. Conclusion: Plat+ capillaries are involved in the innate immune response through their interaction with neutrophils via ICAM-1 adhesion during endotoxemia, while Cd74+ capillaries epxressed high level of MHC proteins play a role in adaptive immune response through their interaction with T cells. However, it remains unclear whether the function of Cd74+ capillaries leans towards immunity or tolerance, and further studies are needed to confirm this.

Commonly disrupted pathways in brain and kidney in a pig model of systemic endotoxemia.

Sepsis is a life-threatening state that arises due to a hyperactive inflammatory response stimulated by infection and rarely other insults (e.g., non-infections tissue injury). Although changes in several proinflammatory cytokines and signals are documented in humans and small animal models, far less is known about responses within affected tissues of large animal models. We sought to understand the changes that occur during the initial stages of inflammation by administering intravenous lipopolysaccharide (LPS) to Yorkshire pigs and assessing transcriptomic alterations in the brain, kidney, and whole blood. Robust transcriptional alterations were found in the brain, with upregulated responses enriched in inflammatory pathways and downregulated responses enriched in tight junction and blood vessel functions. Comparison of the inflammatory response in the pig brain to a similar mouse model demonstrated some overlapping changes but also numerous differences, including oppositely dysregulated genes between species. Substantial changes also occurred in the kidneys following LPS with several enriched upregulated pathways (cytokines, lipids, unfolded protein response, etc.) and downregulated gene sets (tube morphogenesis, glomerulus development, GTPase signal transduction, etc.). We also found significant dysregulation of genes in whole blood that fell into several gene ontology categories (cytokines, cell cycle, neutrophil degranulation, etc.). We observed a strong correlation between the brain and kidney responses, with significantly shared upregulated pathways (cytokine signaling, cell death, VEGFA pathways) and downregulated pathways (vasculature and RAC1 GTPases). In summary, we have identified a core set of shared genes and pathways in a pig model of systemic inflammation.

Self-Cascade Redox Modulator Trilogically Renovates Intestinal Microenvironment for Mitigating Endotoxemia.

Endotoxemia is a life-threatening multiple organ failure disease caused by bacterial endotoxin infection. Unfortunately, current single-target therapy strategies have failed to prevent the progression of endotoxemia. Here, we reported that alanine fullerene redox modulator (AFRM) remodeled the intestinal microenvironment for multiple targets endotoxemia mitigation by suppressing inflammatory macrophages, inhibiting macrophage pyroptosis, and repairing epithelial cell barrier integrity. Specifically, AFRM exhibited broad-spectrum and self-cascade redox regulation properties with superoxide dismutase (SOD)-like enzyme, peroxidase (POD)-like enzyme activity, and hydroxyl radical (•OH) scavenging ability. Guided by proteomics, we demonstrated that AFRM regulated macrophage redox homeostasis and down-regulated LPS/TLR4/NF-κB and MAPK/ERK signaling pathways to suppress inflammatory hyperactivation. Of note, AFRM could attenuate inflammation-induced macrophage pyroptosis via inhibiting the activation of gasdermin D (GSDMD). In addition, our results revealed that AFRM could restore extracellular matrix and cell-tight junction proteins and protect the epithelial cell barrier integrity by regulating extracellular redox homeostasis. Consequently, AFRM inhibited systemic inflammation and potentiated intestinal epithelial barrier damage repair during endotoxemia in mice. Together, our work suggested that fullerene based self-cascade redox modulator has the potential in the management of endotoxemia through synergistically remodeling the inflammation and epithelial barriers in the intestinal microenvironment.

LncRNA 220, a newly discovered long non-conding RNA inhibiting apoptosis and autophagy in Kupffer cells in LPS-induced endotoxemic mice through the XBP1u-PI3K-AKT pathway.

Sepsis is recognized as a potentially fatal condition characterized by acute organ dysfunction resulting from an imbalanced immune response to infection. Acute liver injury (ALI) arises as an inflammatory outcome of immune response dysregulation associated with sepsis. Kupffer cells, which are liver-specific macrophages, are known to have a significant impact on ALI, although the precise regulatory mechanism remains unclear. Numerous studies have showcased the regulatory impact of long non-coding RNAs (lncRNAs) on the progression of diverse ailments, yet their precise regulatory mechanisms remain predominantly unexplored. In this study, a novel long non-coding RNA (lncRNA), referred to as lncRNA 220, was discovered using high-throughput sequencing. The expression of lncRNA 220 was found to be significantly elevated in the livers of mice with lipopolysaccharide (LPS)-induced endotoxemia, specifically during the 8-hour time period. Furthermore, in Kupffer cells treated with LPS, lncRNA 220 was observed to inhibit apoptosis and autophagy by activating the PI3K-AKT-mTORC1 pathway. This effect was achieved through the reduction of X-box protein 1 unspliced (Xbp1u) mRNA stability and suppression of its translation in the context of endoplasmic reticulum stress (ERS). Ultimately, this intervention mitigated the progression of LPS-induced ALI. To summarize, our study establishes lncRNA 220 as a newly identified regulator that suppresses apoptosis and autophagy in Kupffer cells subjected to LPS treatment, indicating its potential as a molecular target for ALI in endotoxemic mice.

Cholinergic signaling via the α7 nicotinic acetylcholine receptor regulates the migration of monocyte-derived macrophages during acute inflammation.

BACKGROUND: The involvement of the autonomic nervous system in the regulation of inflammation is an emerging concept with significant potential for clinical applications. Recent studies demonstrate that stimulating the vagus nerve activates the cholinergic anti-inflammatory pathway that inhibits pro-inflammatory cytokines and controls inflammation. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in mediating cholinergic anti-inflammatory effects through a downstream intracellular mechanism involving inhibition of NF-κB signaling, which results in suppression of pro-inflammatory cytokine production. However, the role of the α7nAChR in the regulation of other aspects of the immune response, including the recruitment of monocytes/macrophages to the site of inflammation remained poorly understood. RESULTS: We observed an increased mortality in α7nAChR-deficient mice (compared with wild-type controls) in mice with endotoxemia, which was paralleled with a significant reduction in the number of monocyte-derived macrophages in the lungs. Corroborating these results, fluorescently labeled α7nAChR-deficient monocytes adoptively transferred to WT mice showed significantly diminished recruitment to the inflamed tissue. α7nAChR deficiency did not affect monocyte 2D transmigration across an endothelial monolayer, but it significantly decreased the migration of macrophages in a 3D fibrin matrix. In vitro analysis of major adhesive receptors (L-selectin, ß1 and ß2 integrins) and chemokine receptors (CCR2 and CCR5) revealed reduced expression of integrin αM and αX on α7nAChR-deficient macrophages. Decreased expression of αMß2 was confirmed on fluorescently labeled, adoptively transferred α7nAChR-deficient macrophages in the lungs of endotoxemic mice, indicating a potential mechanism for α7nAChR-mediated migration. CONCLUSIONS: We demonstrate a novel role for the α7nAChR in mediating macrophage recruitment to inflamed tissue, which indicates an important new aspect of the cholinergic regulation of immune responses and inflammation.

Upregulation of PTPN1 aggravates endotoxemia-induced cardiac dysfunction through inhibiting mitophagy.

OBJECTIVES: To investigate the role of protein tyrosine phosphatase non-receptor type 1 (PTPN1) in mitophagy during sepsis and its underlying mechanisms and determine the therapeutic potential of PTPN1 inhibitors in endotoxemia-induced cardiac dysfunction. METHODS: A mouse model of endotoxemia was established by administering an intraperitoneal injection of lipopolysaccharide (LPS). The therapeutic effect of targeting PTPN1 was evaluated using its inhibitor Claramine (CLA). Mitochondrial structure and function as well as the expression of mitophagy-related proteins were evaluated. Rat H9c2 cardiomyocytes were exposed to mouse RAW264.7 macrophage-derived conditioned medium. Cryptotanshinone, a specific p-STAT3 (Y705) inhibitor, was used to confirm the role of STAT3 in PTPN1-mediated mitophagy following LPS exposure. Electrophoretic mobility shift and dual luciferase reporter assays were performed to discern the mechanisms by which STAT3 regulated the expression of PINK1 and PRKN. RESULTS: CLA alleviated LPS-induced myocardial damage, cardiac dysfunction, and mitochondrial injury and dysfunction in the mouse heart. PTPN1 upregulation exacerbated LPS-induced mitochondrial injury and dysfunction in H9c2 cardiomyocytes, but inhibited LPS-induced mitophagy. LPS promoted the interaction between PTPN1 and STAT3 and reduced STAT3 phosphorylation at Tyr705 (Y705), which was required to inhibit mitophagy by PTPN1. Upon LPS stimulation, PTPN1 negatively regulated the transcription of PINK1 and PRKN through dephosphorylation of STAT3 at Y705. STAT3 regulated the transcription of PINK1 and PRKN by binding to STAT3-responsive elements in their promoters. CONCLUSION: PTPN1 upregulation aggravates endotoxemia-induced cardiac dysfunction by impeding mitophagy through dephosphorylation of STAT3 at Y705 and negative regulation of PINK1 and PRKN transcription.

Assessing the effects of oxytocin in changes of core body temperature during LPS-induced endotoxemia: A novel approach using Extended Poincaré Plot Analysis.

Oxytocin has shown cardioprotective effects during inflammation and may modify the core body temperature changes in LPS-induced endotoxemia. Notably, the time series analysis of core body temperature fluctuations may indicate thermoregulation alterations. This study aims to assess the effects of oxytocin on changes in the core body temperature by analyzing the fluctuations of the temperature time series of endotoxemic rats. Twelve hours of continuous core body temperature fluctuations time series were obtained from adult male Dark Agouti rats implanted with a telemetric transmitter under the following treatment: lipopolysaccharide (LPS); oxytocin (O); lipopolysaccharide + oxytocin (LPS + O), and vehicle or control (C). The temperature fluctuations time series were analyzed using the Extended Poincaré Plot Analysis (EPPA), a novel approach for measuring nonlinear features, to compute the autocorrelation by Pearson's correlation coefficient r, the standard deviation perpendicular to the line of identity (SD1), and the standard deviation parallel to the line of identity (SD2). The autocorrelation of the temperature fluctuations assessed by Pearson's coefficient was significantly higher in the LPS group compared to control rats (C). Likewise, the co-administration of oxytocin during endotoxemia (LPS + O) significantly reduced the autocorrelation and increased the short-term variability (SD1) of temperature fluctuations compared to those recorded with a single dose of LPS. Thus, we concluded that oxytocin may introduce thermoregulatory changes under LPS-induced endotoxemia. The EPPA is a simple and powerful approach to assess physiological variability that can provide valuable insights into changes in thermoregulation.

Low-grade endotoxemia is associated with cardiovascular events in community-acquired pneumonia.

OBJECTIVES: Community-acquired pneumonia (CAP) is associated with low-grade endotoxemia but its relationship with cardiovascular events (CVE) has not been investigated. METHODS: We evaluated the incidence of CVE including myocardial infarction, stroke, and cardiovascular death in 523 adult patients hospitalized for CAP. Serum lipopolysaccharide (LPS) and zonulin, a marker of gut permeability, were analyzed in the cohort, that was followed-up during hospitalization and up to 43 months thereafter. RESULTS: During the hospital-stay, 55 patients experienced CVE with a progressive increase from the lowest (0.6%) to highest LPS tertile (23.6%, p < 0.001). Logistic regression analyses showed that higher LPS tertile was independently associated with CVE; LPS significantly correlated with age, hs-CRP and zonulin. In a sub-group of 23 CAP patients, blood E. coli DNA was higher in patients compared to 24 controls and correlated with LPS. During the long-term follow-up, 102 new CVE were registered; the highest tertile of LPS levels was associated with incident CVE; Cox regression analysis showed that LPS tertiles, age, history of CHD, and diabetes independently predicted CVE. CONCLUSIONS: In CAP low-grade endotoxemia is associated to short- and long-term risk of CVE. Further study is necessary to assess if lowering LPS by non-absorbable antibiotics may result in improved outcomes.

Protective effect of melatonin on blood-brain barrier damage caused by Endotoxemia.

OBJECTIVE: Endotoxins, products of Gram-negative bacteria, are the primary cause of blood-brain barrier (BBB) damage. In the present study, we aimed to investigate the possible neuroprotection mechanisms of melatonin on BBB damage induced by endotoxemia. METHODS: Adult, female Sprague-Dawley rats (n = 42) were separated into four random groups as a control group and three treatment groups. Lipopolysaccharide (7,5 mg/kg/day) was administrated for a single dose to generate a 24-hour sepsis model on rats. Melatonin (10 mg/kg/day) was treated a week before sepsis. Afterward, the dissected brain tissues were examined by histopathological, biochemical, and molecular analyses. RESULTS: LPS caused weight loss in the groups. As a result, degenerated neurons with cytoplasmic vacuoles and irregular pyknotic nuclei, pale stained necrotic neurons, and vascular congestion were observed in LPS-exposed rats. However, MEL decreased the number of degenerated neurons in treated groups. MEL treatment increased ZO1 and Occludin immunoreactivity while decreasing TLR4 in brain tissues. MEL effect on protein expression was recorded for ZO1 increase and TLR4 decrease in brain tissue compared to LPS groups. MEL also decreased MDA levels in brain tissue. CONCLUSIONS: MEL recovered the degenerative damage of sepsis by contributing to blood-brain barrier integrity, and by decreasing inflammation, thus the neuroprotective effects of MEL might provide an experimental basis for clinical applications.

Cytokine Adsorption Effects of a Novel Hemofiltration Column for the Treatment of Experimental Endotoxemia.

INTRODUCTION: The TKM-101 is a new hemofiltration column packed with a polymer alloy membrane consisting of polyethersulfone, polyvinylpyrrolidone, and sulfonated poly (arylene ether) copolymers. We examined the ability of the TKM-101 column to remove cytokines and humoral mediators from blood in vitro and the effects of extracorporeal treatment with the TKM-101 column on the mortality rate and inflammatory responses to endotoxic shock in vivo. METHODS: In vitro and in vivo laboratory investigations were conducted. In the in vitro experiment, the adsorption abilities of TKM-101, AN69-ST, and control columns for cytokine-related sepsis in blood were compared using human serum samples. In the in vivo experiment, male Sprague-Dawley rats were anesthetized and injected with Escherichia coli endotoxin (15 mg/kg, intravenously). Afterward, the rats were assigned (in a double-blind manner) to one of three groups (n = 17 per group): apheresis with a control column (control group), apheresis with an AN69-ST column (AN69-ST group), or apheresis with a TKM-101 column (TKM-101 group). Outcomes were compared among the groups. RESULTS: In vitro, the concentrations of all evaluated cytokines significantly decreased with the TKM-101 column compared to those with the control column; however, there were no significant differences between the TKM-101 and AN69-ST columns. In vivo, the mortality rates 8 h after endotoxin injection were 65%, 29%, and 29% for the control, AN69-ST, and TKM-101 groups, respectively. Hypotension and elevated plasma cytokine concentrations were less prominent in the TKM-101 and AN69-ST groups compared to those in the control group. CONCLUSIONS: TKM-101 effectively removed proteins of varying sizes, from small-sized proteins such as interleukin (IL)-8 to mid-sized protein such as IL-10 in vitro. Moreover, TKM-101 treatment reduced mortality and had inhibitory effects on inflammatory responses in endotoxemic rats. These findings suggest that TKM-101 treatment may be available for use in patients with sepsis and/or endotoxemia.

Endotoxemia Correlates with Kidney Function and Length of Stay in Critically Ill Patients.

INTRODUCTION: Endotoxin is a key driver of sepsis, which frequently causes acute kidney injury (AKI). However, endotoxins may also be found in non-bacteremic critically ill patients, likely from intestinal translocation. Preclinical models show that endotoxins can directly injure the kidneys, and in COVID-19 patients, endotoxemia correlated with AKI. We sought to determine correlations between endotoxemia and kidney and hospital outcomes in a broad group of critically ill patients. METHODS: In this single-center, serial prospective study, 124 predominantly Caucasian adult patients were recruited within 48 h of admission to Stony Brook University Hospital Intensive Care Unit (ICU). Demographics, vital signs, laboratory data, and outcomes were collected. Circulating endotoxin was measured on days 1, 4, and 8 using the endotoxin activity assay (EAA). The association of EAA with outcomes was examined with EAA: (1) categorized as <0.6, ≥0.6, and nonresponders (NRs); and (2) used as a continuous variable. RESULTS: Patients with EAA ≥0.6 had a higher prevalence of proteinuria, and lower arterial oxygen saturation (SaO2) to fraction of inspired oxygen (FiO2) (SaO2/FiO2) ratio versus patients with EAA <0.6. EAA levels positively correlated with serum creatinine (sCr) levels on day 1. Patients whose EAA level stayed ≥0.6 had a slower decline in sCr compared to those whose EAA started at ≥0.6 and subsequently declined. Patients with AKI stage 1 and EAA ≥0.6 on day 1 showed slower decline in sCr compared to patients with stage 1 AKI and EAA <0.6. EAA ≥0.6 and NR patients had longer hospital stay and delayed ICU discharge versus EAA <0.6. CONCLUSIONS: High EAA levels correlated with worse kidney function and outcomes. Patients whose EAA levels fell, and those with AKI stage I and day 1 EAA <0.6 recovered more quickly compared to those with EAA ≥0.6, suggesting that removal of circulating endotoxins may be beneficial in critically ill patients.