Recapitulation of human pathophysiology and identification of forensic biomarkers in a translational model of chlorine inhalation injury.

Chlorine gas (Cl2) has been repeatedly used as a chemical weapon, first in World War I and most recently in Syria. Life-threatening Cl2 exposures frequently occur in domestic and occupational environments, and in transportation accidents. Modeling the human etiology of Cl2-induced acute lung injury (ALI), forensic biomarkers, and targeted countermeasures development have been hampered by inadequate large animal models. The objective of this study was to develop a translational model of Cl2-induced ALI in swine to understand toxico-pathophysiology and evaluate whether it is suitable for screening potential medical countermeasures and to identify biomarkers useful for forensic analysis. Specific pathogen-free Yorkshire swine (30-40 kg) of either sex were exposed to Cl2 (≤240 ppm for 1 h) or filtered air under anesthesia and controlled mechanical ventilation. Exposure to Cl2 resulted in severe hypoxia and hypoxemia, increased airway resistance and peak inspiratory pressure, and decreased dynamic lung compliance. Cl2 exposure resulted in increased total leucocyte and neutrophil counts in bronchoalveolar lavage fluid, vascular leakage, and pulmonary edema compared with the air-exposed group. The model recapitulated all three key histopathological features of human ALI, such as neutrophilic alveolitis, deposition of hyaline membranes, and formation of microthrombi. Free and lipid-bound 2-chlorofatty acids and chlorotyrosine-modified proteins (3-chloro-l-tyrosine and 3,5-dichloro-l-tyrosine) were detected in plasma and lung tissue after Cl2 exposure. In this study, we developed a translational swine model that recapitulates key features of human Cl2 inhalation injury and is suitable for testing medical countermeasures, and validated chlorinated fatty acids and protein adducts as biomarkers of Cl2 inhalation.NEW & NOTEWORTHY We established a swine model of chlorine gas-induced acute lung injury that exhibits several features of human acute lung injury and is suitable for screening potential medical countermeasures. We validated chlorinated fatty acids and protein adducts in plasma and lung samples as forensic biomarkers of chlorine inhalation.

Membrane phospholipid remodeling modulates nonalcoholic steatohepatitis progression by regulating mitochondrial homeostasis.

BACKGROUND AND AIMS: NASH, characterized by inflammation and fibrosis, is emerging as a leading etiology of HCC. Lipidomics analyses in the liver have shown that the levels of polyunsaturated phosphatidylcholine (PC) are decreased in patients with NASH, but the roles of membrane PC composition in the pathogenesis of NASH have not been investigated. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), a phospholipid (PL) remodeling enzyme that produces polyunsaturated PLs, is a major determinant of membrane PC content in the liver. APPROACH AND RESULTS: The expression of LPCAT3 and the correlation between its expression and NASH severity were analyzed in human patient samples. We examined the effect of Lpcat3 deficiency on NASH progression using Lpcat3 liver-specific knockout (LKO) mice. RNA sequencing, lipidomics, and metabolomics were performed in liver samples. Primary hepatocytes and hepatic cell lines were used for in vitro analyses. We showed that LPCAT3 was dramatically suppressed in human NASH livers, and its expression was inversely correlated with NAFLD activity score and fibrosis stage. Loss of Lpcat3 in mouse liver promotes both spontaneous and diet-induced NASH/HCC. Mechanistically, Lpcat3 deficiency enhances reactive oxygen species production due to impaired mitochondrial homeostasis. Loss of Lpcat3 increases inner mitochondrial membrane PL saturation and elevates stress-induced autophagy, resulting in reduced mitochondrial content and increased fragmentation. Furthermore, overexpression of Lpcat3 in the liver ameliorates inflammation and fibrosis of NASH. CONCLUSIONS: These results demonstrate that membrane PL composition modulates the progression of NASH and that manipulating LPCAT3 expression could be an effective therapeutic for NASH.

Homodimeric Granzyme A Opsonizes Mycobacterium tuberculosis and Inhibits Its Intracellular Growth in Human Monocytes via Toll-Like Receptor 4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Lipid biology of plasmalogen-derived halolipids: Signature molecules of myeloperoxidase and eosinophil peroxidase activity.

Myeloperoxidase and eosinophil peroxidase exert their antimicrobial functions through the oxidative actions of their hypohalous acid products. Plasmalogen phospholipids are particularly susceptible to oxidation of their vinyl ether functional group by hypohalous acids. This produces a family of halogenated lipid products with pro-inflammatory roles and potential biomarker utility. The initial product of plasmalogen oxidation by HOCl is 2-chlorofatty aldehyde, which has been shown to play a key role at the blood-endothelium interface. In vitro and in vivo studies indicate increased endothelial barrier permeability, neutrophil chemotaxis, neutrophil and platelet adherence to endothelium, and promotion of erythrocyte lysis as some of its effects. These effects may be due to protein modification by 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde is metabolized by host dehydrogenases to 2-chlorofatty acid. While it is less chemically reactive, 2-chlorofatty acid has partial overlap of pro-inflammatory effects with 2-chlorofatty aldehyde and unique actions such as induction of neutrophil extracellular trap formation. The stability of 2-chlorofatty acid in plasma also makes it well-suited as a biomarker of HOCl generation, and its plasma levels may be predictive of disease outcomes. 2-Bromofatty aldehydes and acids are produced analogously from HOBr reaction with plasmalogens. Their functions have yet to be well-elucidated, though similarities with chlorolipids have been observed, and increased reactivity with proteins is expected through enhanced electrophilicity of the alpha carbon. Altogether, these halogenated lipids represent underexplored mediators of diseases involving excess hypohalous acid production.

Targeting phospholipid remodeling pathway improves insulin resistance in diabetic mouse models.

Previous studies have revealed that membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) is involved in the development of insulin resistance in type 2 diabetes. In this study, we aimed to investigate the therapeutic potential of targeting Lpcat3 in the treatment of insulin resistance in diabetic mouse models. Lpcat3 expression was suppressed in the whole body by antisense oligonucleotides (ASO) injection or in the liver by adeno-associated virus (AAV)-encoded Cre in high-fat diet (HFD)-induced and genetic ob/ob type 2 diabetic mouse models. Glucose tolerance test (GTT), insulin tolerance test (ITT), fasting blood glucose, and insulin levels were used to assess insulin sensitivity. Lipid levels in the liver and serum were measured. The expression of genes involved in de novo lipogenesis was analyzed by real-time RT-PCR. Metabolic rates were measured by indirect calorimetry using the Comprehensive Lab Animal Monitoring System (CLAMS). Our data demonstrate that acute knockout of hepatic Lpcat3 by AAV-Cre improves both hyperglycemia and hypertriglyceridemia in HFD-fed mice. Similarly, whole-body ablation of Lpcat3 by ASO administration improves obesity and insulin resistance in both HFD-fed and ob/ob mice. These findings demonstrate that targeting LPCAT3 could be a novel therapy for insulin resistance.

Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo.

OBJECTIVE: Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. METHODS: A whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. RESULTS: Although the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. CONCLUSION: Ube4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Polymorphisms near the membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes are associated with worsened nonalcoholic fatty liver (NASH), and nonalcoholic fatty liver disease (NAFLD)/NASH may decrease MBOAT7 expression independent of these polymorphisms. We hypothesized that enhancing MBOAT7 function would improve NASH. METHODS: Genomic and lipidomic databases were mined for MBOAT7 expression and hepatic phosphatidylinositol (PI) abundance in human NAFLD/NASH. Male C57BL6/J mice were fed either choline-deficient high-fat diet or Gubra Amylin NASH diet and subsequently infected with adeno-associated virus expressing MBOAT7 or control virus. NASH histological scoring and lipidomic analyses were performed to assess MBOAT7 activity, hepatic PI, and lysophosphatidylinositol (LPI) abundance. RESULTS: Human NAFLD/NASH decreases MBOAT7 expression and hepatic abundance of arachidonate-containing PI. Murine NASH models display subtle changes in MBOAT7 expression, but significantly decreased activity. After MBOAT7 overexpression, liver weights, triglycerides, and plasma alanine and aspartate transaminase were modestly improved by MBOAT7 overexpression, but NASH histology was not improved. Despite confirmation of increased activity with MBOAT7 overexpression, content of the main arachidonoylated PI species was not rescued by MBOAT7 although the abundance of many PI species was increased. Free arachidonic acid was elevated but the MBOAT7 substrate arachidonoyl-CoA was decreased in NASH livers compared to low-fat controls, likely due to the decreased expression of long-chain acyl-CoA synthetases. CONCLUSION: Results suggest decreased MBOAT7 activity plays a role in NASH, but MBOAT7 overexpression fails to measurably improve NASH pathology potentially due to the insufficient abundance of its arachidonoyl-CoA substrate.

Hepatic Phospholipid Remodeling Modulates Insulin Sensitivity and Systemic Metabolism.

The liver plays a central role in regulating glucose and lipid metabolism. Aberrant insulin action in the liver is a major driver of selective insulin resistance, in which insulin fails to suppress glucose production but continues to activate lipogenesis in the liver, resulting in hyperglycemia and hypertriglyceridemia. The underlying mechanisms of selective insulin resistance are not fully understood. Here It is shown that hepatic membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) regulates insulin signaling and systemic glucose and lipid metabolism. Hyperinsulinemia induced by high-fat diet (HFD) feeding augments hepatic Lpcat3 expression and membrane unsaturation. Loss of Lpcat3 in the liver improves insulin resistance and blunts lipogenesis in both HFD-fed and genetic ob/ob mouse models. Mechanistically, Lpcat3 deficiency directly facilitates insulin receptor endocytosis, signal transduction, and hepatic glucose production suppression and indirectly enhances fibroblast growth factor 21 (FGF21) secretion, energy expenditure, and glucose uptake in adipose tissue. These findings identify hepatic LPCAT3 and membrane phospholipid composition as a novel regulator of insulin sensitivity and provide insights into the pathogenesis of selective insulin resistance.

Comparison of Low-Density Lipoprotein Oxidation by Hydrophilic O(3 P)-Precursors and Lipid-O(3 P)-Precursor Conjugates.

Lipid oxidation by reactive oxygen species (ROS) provide several different oxidation products that have been implicated in inflammatory responses. Ground state atomic oxygen [O(3 P)] is produced by the photodeoxygenation of certain heterocyclic oxides and has a reactivity that is unique from other ROS. Due to the reactive nature of O(3 P), the site of O(3 P)-generation is expected to influence the products in heterogenous solutions or environments. In this work, the oxidation of low-density lipoprotein (LDL) by lipids with covalently bound O(3 P)-photoprecursors was compared to more hydrophilic O(3 P)-photoprecursors. Lipid oxidation products were quantified after Bligh-Dyer extraction and pentafluorobenzyl bromide (PFB) derivatization by GC-MS. Unlike the more hydrophilic O(3 P)-photoprecursors, the oxidation of LDL during the irradiation of lipid-(O3 P)-photoprecursor conjugates showed little quenching by the addition of the O(3 P)-scavenging sodium allyl sulfonate. This indicated that lipophilic O(3 P)-photoprecursors are expected to generate lipid oxidation products where other more hydrophilic O(3 P)-photoprecursors could be quenched by other reactive groups present in solution or the environment.

Characterization of N-Acetyl Cysteine Adducts with Exogenous and Neutrophil-Derived 2-Chlorofatty Aldehyde.

Hypochlorous acid is produced by leukocyte myeloperoxidase activity. 2-Chlorofatty aldehydes (2-ClFALDs) are formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and are thus produced following leukocyte activation as well as in the lungs of mice exposed to chlorine gas. The biological role of 2-ClFALD is largely unknown. Recently, we used an alkyne analog (2-ClHDyA) of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDA), to identify proteins covalently modified by 2-ClHDyA in endothelial cells and epithelial cells. Here, we demonstrate that 2-ClHDA reduces the metabolic activity of RAW 264.7 cells in a dose-dependent manner. 2-ClHDyA localizes to the mitochondria, endoplasmic reticulum and Golgi in RAW 264.7 cells and modifies many proteins. The thiol-containing precursor of glutathione, N-acetyl cysteine (NAC), was shown to produce an adduct with 2-ClHDA with the loss of Cl- (HDA-NAC). This adduct was characterized in both positive and negative ion modes using LC-MS/MS and electrospray ionization. NAC treatment of neutrophils reduced the 2-ClFALD levels in PMA-stimulated cells with subsequent increases in HDA-NAC. NAC treatments reduced the 2-ClHDA-elicited loss of metabolic activity in RAW 264.7 cells as well as 2-ClHDA protein modification. These studies demonstrate that 2-ClFALD toxic effects can be reduced by NAC, which reduces protein modification.

Myeloperoxidase-derived hypochlorous acid targets human airway epithelial plasmalogens liberating protein modifying electrophilic 2-chlorofatty aldehydes.

Neutrophil and airway epithelial cell interactions are critical in the inflammatory response to viral infections including respiratory syncytial virus, Sendai virus, and SARS-CoV-2. Airway epithelial cell dysfunction during viral infections is likely mediated by the interaction of virus and recruited neutrophils at the airway epithelial barrier. Neutrophils are key early responders to viral infection. Neutrophil myeloperoxidase catalyzes the conversion of hydrogen peroxide to hypochlorous acid (HOCl). Previous studies have shown HOCl targets host neutrophil and endothelial cell plasmalogen lipids, resulting in the production of the chlorinated lipid, 2-chlorofatty aldehyde (2-ClFALD). We have previously shown that the oxidation product of 2-ClFALD, 2-chlorofatty acid (2-ClFA) is present in bronchoalveolar lavage fluid of Sendai virus-infected mice, which likely results from the attack of the epithelial plasmalogen by neutrophil-derived HOCl. Herein, we demonstrate small airway epithelial cells contain plasmalogens enriched with oleic acid at the sn-2 position unlike endothelial cells which contain arachidonic acid enrichment at the sn-2 position of plasmalogen. We also show neutrophil-derived HOCl targets epithelial cell plasmalogens to produce 2-ClFALD. Further, proteomics and over-representation analysis using the ω-alkyne analog of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDyA) showed cell adhesion molecule binding and cell-cell junction enriched categories similar to that observed previously in endothelial cells. However, in contrast to endothelial cells, proteins in distinct metabolic pathways were enriched with 2-ClFALD modification, particularly pyruvate metabolism was enriched in epithelial cells and mitochondrial pyruvate respiration was reduced. Collectively, these studies demonstrate, for the first time, a novel plasmalogen molecular species distribution in airway epithelial cells that are targeted by myeloperoxidase-derived hypochlorous acid resulting in electrophilic 2-ClFALD, which potentially modifies epithelial physiology by modifying proteins.

E. coli strain-dependent lipid alterations in cocultures with endothelial cells and neutrophils modeling sepsis.

Dysregulated lipid metabolism is common in infection and inflammation and is a part of the complex milieu underlying the pathophysiological sequelae of disease. Sepsis is a major cause of mortality and morbidity in the world and is characterized by an exaggerated host response to an infection. Metabolic changes, including alterations in lipid metabolism, likely are important in sepsis pathophysiology. Here, we designed an in vitro cell culture model using endothelial cells, E. coli, and neutrophils to mimic sepsis in a simplified cell model. Lipid alterations were studied in the presence of the pathogenic E. coli strain CFT073 and non-pathogenic E. coli strain JM109. We employed untargeted lipidomics to first identify lipid changes and then targeted lipidomics to confirm changes. Both unique and shared lipid signatures were identified in cocultures with these E. coli strains. In the absence of neutrophils, the CFT073 strain elicited alterations in lysophosphatidylcholine and diglyceride molecular species during coculture while both strains led to increases in phosphatidylglycerols. Lipid alterations in these cocultures changed with the addition of neutrophils. In the presence of neutrophils with E. coli and endothelial cells, triglyceride increases were a unique response to the CFT073 strain while phosphatidylglycerol and diglyceride increases occurred in response to both strains. Phosphatidylethanolamine also increased in neutrophils, E. coli and endothelial cells cocultures, and this response was greater in the presence of the CFT073 strain. We further evaluated changes in phosphatidylethanolamine in a rat model of sepsis, which showed multiple plasma phosphatidylethanolamine molecular species were elevated shortly after the induction of sepsis. Collectively, these findings demonstrate unique lipid responses by co-cultures of E. coli with endothelial cells which are dependent on the E. coli strain as well as the presence of neutrophils. Furthermore, increases in phosphatidylethanolamine levels in CFT073 urosepsis E. coli, endothelial cell, neutrophil cocultures were similarly observed in the plasma of septic rats.

Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking.

Membrane contact sites (MCS), close membrane apposition between organelles, are platforms for interorganellar transfer of lipids including cholesterol, regulation of lipid homeostasis, and co-ordination of endocytic trafficking. Sphingosine kinases (SphKs), two isoenzymes that phosphorylate sphingosine to the bioactive sphingosine-1-phosphate (S1P), have been implicated in endocytic trafficking. However, the physiological functions of SphKs in regulation of membrane dynamics, lipid trafficking and MCS are not known. Here, we report that deletion of SphKs decreased S1P with concomitant increases in its precursors sphingosine and ceramide, and markedly reduced endoplasmic reticulum (ER) contacts with late endocytic organelles. Expression of enzymatically active SphK1, but not catalytically inactive, rescued the deficit of these MCS. Although free cholesterol accumulated in late endocytic organelles in SphK null cells, surprisingly however, cholesterol transport to the ER was not reduced. Importantly, deletion of SphKs promoted recruitment of the ER-resident cholesterol transfer protein Aster-B (also called GRAMD1B) to the plasma membrane (PM), consistent with higher accessible cholesterol and ceramide at the PM, to facilitate cholesterol transfer from the PM to the ER. In addition, ceramide enhanced in vitro binding of the Aster-B GRAM domain to phosphatidylserine and cholesterol liposomes. Our study revealed a previously unknown role for SphKs and sphingolipid metabolites in governing diverse MCS between the ER network and late endocytic organelles versus the PM to control the movement of cholesterol between distinct cell membranes.

Plasmalogen Loss in Sepsis and SARS-CoV-2 Infection.

Plasmalogens are plasma-borne antioxidant phospholipid species that provide protection as cellular lipid components during cellular oxidative stress. In this study we investigated plasma plasmalogen levels in human sepsis as well as in rodent models of infection. In humans, levels of multiple plasmenylethanolamine molecular species were decreased in septic patient plasma compared to control subject plasma as well as an age-aligned control subject cohort. Additionally, lysoplasmenylcholine levels were significantly decreased in septic patients compared to the control cohorts. In contrast, plasma diacyl phosphatidylethanolamine and phosphatidylcholine levels were elevated in septic patients. Lipid changes were also determined in rats subjected to cecal slurry sepsis. Plasma plasmenylcholine, plasmenylethanolamine, and lysoplasmenylcholine levels were decreased while diacyl phosphatidylethanolamine levels were increased in septic rats compared to control treated rats. Kidney levels of lysoplasmenylcholine as well as plasmenylethanolamine molecular species were decreased in septic rats. Interestingly, liver plasmenylcholine and plasmenylethanolamine levels were increased in septic rats. Since COVID-19 is associated with sepsis-like acute respiratory distress syndrome and oxidative stress, plasmalogen levels were also determined in a mouse model of COVID-19 (intranasal inoculation of K18 mice with SARS-CoV-2). 3 days following infection, lung infection was confirmed as well as cytokine expression in the lung. Multiple molecular species of lung plasmenylcholine and plasmenylethanolamine were decreased in infected mice. In contrast, the predominant lung phospholipid, dipalmitoyl phosphatidylcholine, was not decreased following SARS-CoV-2 infection. Additionally total plasmenylcholine levels were decreased in the plasma of SARS-CoV-2 infected mice. Collectively, these data demonstrate the loss of plasmalogens during both sepsis and SARS-CoV-2 infection. This study also indicates plasma plasmalogens should be considered in future studies as biomarkers of infection and as prognostic indicators for sepsis and COVID-19 outcomes.

Endothelial Cell Protein Targeting by Myeloperoxidase-Derived 2-Chlorofatty Aldehyde.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

Identification of novel neutrophil very long chain plasmalogen molecular species and their myeloperoxidase mediated oxidation products in human sepsis.

Plasmalogens are a class of phospholipids containing vinyl ether linked aliphatic groups at the sn-1 position. Plasmalogens are known to contain 16- and 18-carbon aliphatic groups at the sn-1 position. Here, we reveal that the human neutrophil plasmenylethanolamine pool uniquely includes molecular species with very long carbon chain (VLC) aliphatic groups, including 20-, 22- and 24-carbon vinyl ether linked aliphatic groups at the sn-1 position. We identified these novel VLC plasmalogen species by electrospray ionization mass spectrometry methods. VLC plasmalogens were only found in the neutrophil plasmenylethanolamine pool. During neutrophil activation, VLC plasmenylethanolamines undergo myeloperoxidase-dependent oxidation to produce VLC 2-chlorofatty aldehyde and its oxidation product, 2-chlorofatty acid (2-ClFA). Furthermore, plasma concentrations of VLC 2-ClFA are elevated in human sepsis. These studies demonstrate for the first time VLC plasmenylethanolamine molecular species, their myeloperoxidase-mediated chlorolipid products and the presence of these chlorolipids in human sepsis.

Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis.

OBJECTIVE: Obesity and insulin resistance greatly increase the risk of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). We have previously discovered that whole-body and adipocyte-specific Ip6k1deletion protects mice from high-fat-diet-induced obesity and insulin resistance due to improved adipocyte thermogenesis and insulin signaling. Here, we aimed to determine the impact of hepatocyte-specific and whole-body Ip6k1 deletion (HKO and Ip6k1-KO or KO) on liver metabolism and NAFLD/NASH. METHODS: Body weight and composition; energy expenditure; glycemic profiles; and serum and liver metabolic, inflammatory, fibrotic and toxicity parameters were assessed in mice fed Western and high-fructose diet (HFrD) (WD: 40% kcal fat, 1.25% cholesterol, no added choline and HFrD: 60% kcal fructose). Mitochondrial oxidative capacity was evaluated in isolated hepatocytes. RNA-Seq was performed in liver samples. Livers from human NASH patients were analyzed by immunoblotting and mass spectrometry. RESULTS: HKO mice displayed increased hepatocyte mitochondrial oxidative capacity and improved insulin sensitivity but were not resistant to body weight gain. Improved hepatocyte metabolism partially protected HKO mice from NAFLD/NASH. In contrast, enhanced whole-body metabolism and reduced body fat accumulation significantly protected whole-body Ip6k1-KO mice from NAFLD/NASH. Mitochondrial oxidative pathways were upregulated, whereas gluconeogenic and fibrogenic pathways were downregulated in Ip6k1-KO livers. Furthermore, IP6K1 was upregulated in human NASH livers and interacted with the enzyme O-GlcNAcase that reduces protein O-GlcNAcylation. Protein O-GlcNAcylation was found to be reduced in Ip6k1-KO and HKO mouse livers. CONCLUSION: Pleiotropic actions of IP6K1 in the liver and other metabolic tissues mediate hepatic metabolic dysfunction and NAFLD/NASH, and thus IP6K1 deletion may be a potential treatment target for this disease.

Neutrophil Myeloperoxidase Derived Chlorolipid Production During Bacteria Exposure.

Neutrophils are the most abundant white blood cells recruited to the sites of infection and inflammation. During neutrophil activation, myeloperoxidase (MPO) is released and converts hydrogen peroxide to hypochlorous acid (HOCl). HOCl reacts with plasmalogen phospholipids to liberate 2-chlorofatty aldehyde (2-ClFALD), which is metabolized to 2-chlorofatty acid (2-ClFA). 2-ClFA and 2-ClFALD are linked with inflammatory diseases and induce endothelial dysfunction, neutrophil extracellular trap formation (NETosis) and neutrophil chemotaxis. Here we examine the neutrophil-derived chlorolipid production in the presence of pathogenic E. coli strain CFT073 and non-pathogenic E. coli strain JM109. Neutrophils cocultured with CFT073 E. coli strain and JM109 E. coli strain resulted in 2-ClFALD production. 2-ClFA was elevated only in CFT073 coculture. NETosis is more prevalent in CFT073 cocultures with neutrophils compared to JM109 cocultures. 2-ClFA and 2-ClFALD were both shown to have significant bactericidal activity, which is more severe in JM109 E. coli. 2-ClFALD metabolic capacity was 1000-fold greater in neutrophils compared to either strain of E. coli. MPO inhibition reduced chlorolipid production as well as bacterial killing capacity. These findings indicate the chlorolipid profile is different in response to these two different strains of E. coli bacteria.

The lipid biology of sepsis.

Sepsis, defined as the dysregulated immune response to an infection leading to organ dysfunction, is one of the leading causes of mortality around the globe. Despite the significant progress in delineating the underlying mechanisms of sepsis pathogenesis, there are currently no effective treatments or specific diagnostic biomarkers in the clinical setting. The perturbation of cell signaling mechanisms, inadequate inflammation resolution, and energy imbalance, all of which are altered during sepsis, are also known to lead to defective lipid metabolism. The use of lipids as biomarkers with high specificity and sensitivity may aid in early diagnosis and guide clinical decision making. In addition, identifying the link between specific lipid signatures and their role in sepsis pathology may lead to novel therapeutics. In this review, we discuss the recent evidence on dysregulated lipid metabolism both in experimental and human sepsis focused on bioactive lipids, fatty acids, and cholesterol as well as the enzymes regulating their levels during sepsis. We highlight not only their potential roles in sepsis pathogenesis but also the possibility of using these respective lipid compounds as diagnostic and prognostic biomarkers of sepsis.

Chlorine inhalation induces acute chest syndrome in humanized sickle cell mouse model and ameliorated by postexposure hemopexin.

Triggering factors of Acute Chest Syndrome (ACS) is a leading cause of death in patients with Sickle Cell Disease (SCD) and targeted therapies are limited. Chlorine (Cl2) inhalation happens frequently, but its role as a potential trigger of ACS has not been determined. In this study, we hypothesized that Cl2 exposure resembling that in the vicinity of industrial accidents induces acute hemolysis with acute lung injury, reminiscent of ACS in humanized SCD mice. When exposed to Cl2 (500 ppm for 30 min), 64% of SCD mice succumbed within 6 h while none of the control mice expressing normal human hemoglobin died (p<0.01). Surviving SCD mice had evidence of acute hemolysis, respiratory acidosis, acute lung injury, and high concentrations of chlorinated palmitic and stearic acids (p<0.05) in their plasmas and RBCs compared to controls. Treatment with a single intraperitoneal dose of human hemopexin 30 min after Cl2 inhalation reduced mortality to around 15% (p<0.01) with reduced hemolysis (decreased RBCs fragility (p<0.001) and returned plasma heme to normal levels (p<0.0001)), improved oxygenation (p<0.0001) and reduced acute lung injury scores (p<0.0001). RBCs from SCD mice had significant levels of carbonylation (which predisposes RBCs to hemolysis) 6 h post-Cl2 exposure which were absent in RBCs of mice treated with hemopexin. To understand the mechanisms leading to carbonylation, we incubated RBCs from SCD mice with chlorinated lipids and identified sickling and increased hemolysis compared to RBCs obtained from control mice and treated similarly. Our study indicates that Cl2 inhalation induces ACS in SCD mice via induction of acute hemolysis, and that post exposure administration of hemopexin reduces mortality and lung injury. Our data suggest that SCD patients are vulnerable in Cl2 exposure incidents and that hemopexin is a potential therapeutic agent.