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Sepsis is defined as the systemic, dysregulated host immune response to an infection that leads to injury to host organ systems and, often, death. Complex interactions between pathogens and their hosts elicit microcirculatory dysfunction. Neutrophil myeloperoxidase (MPO) is critical for combating pathogens, but MPO-derived hypochlorous acid (HOCl) can react with host molecular species as well. Plasmalogens are targeted by HOCl, leading to the production of 2-chlorofatty acids (2-CLFAs). 2-CLFAs are associated with human sepsis mortality, decrease in vitro endothelial barrier function, and activate human neutrophil extracellular trap formation. Here, we sought to examine 2-CLFAs in an in vivo rat sepsis model. Intraperitoneal cecal slurry sepsis with clinically relevant rescue therapies led to ∼73% mortality and evidence of microcirculatory dysfunction. Plasma concentrations of 2-CLFAs assessed 8 h after sepsis induction were lower in rats that survived sepsis than in nonsurvivors. 2-CLFA levels were elevated in kidney, liver, spleen, lung, colon, and ileum in septic animals. In vivo, exogenous 2-CLFA treatments increased kidney permeability, and in in vitro experiments, 2-CLFA also increased epithelial surface expression of vascular cell adhesion molecule 1 and decreased epithelial barrier function. Collectively, these studies support a role of free 2-CLFAs as biomarkers of sepsis mortality, potentially mediated, in part, by 2-CLFA-elicited endothelial and epithelial barrier dysfunction.

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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.

Platelet-activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre-analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography-mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally-activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn-2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed-phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.

Neutrophilic inflammation is a component of sepsis‐associated acute respiratory distress syndrome (ARDS). Neutrophil myeloperoxidase (MPO) activity produces HOCl, which leads to increased plasma 2‐chlorofatty acid (2‐ClFA) levels due to HOCl targeting plasmalogens. Thus, these studies were designed to investigate the role of 2‐ClFA in ARDS during sepsis. In patients with sepsis (n=198), free 2‐ClFA levels were significantly associated with ARDS (odds ratio (OR) 1.87 (95% CI 1.41, 2.49); p < 0.001), and with 30‐day mortality (OR 1.38 (95% CI 1.11, 1.72); p=0.004), for each log increase in free 2‐chlorostearic acid. Of considerable interest, plasma 2‐ClFA levels correlated with plasma levels of angiopoietin‐2, E‐selectin, and soluble thrombomodulin suggesting 2‐ClFAs may mediate endothelial dysfunction in human sepsis. Further studies with human lung microvascular endothelial cells (HMVEC‐L) showed 2‐ClFA treatment resulted in increased adhesion molecule surface expression; increased angiopoietin‐2 release and dose‐dependent endothelial permeability. In contrast, these changes were not observed in endothelial cells treated with non‐chlorinated palmitic acid. Next, the cecal slurry model of rat sepsis was investigated. Injecting rats with 15ml of cecal slurry/kg resulted in mortality within 26h accompanied by significant increases in 2‐ClFA levels in the plasma and lung in comparison to rats injected with vehicle. The ultimate goal of these studies will be to test therapeutic agents that prevent chlorinated lipid‐caused organ injury including ARDS during sepsis. Collectively, these studies suggest that 2‐ClFAs derived from neutrophil MPO‐catalyzed oxidation contribute to pulmonary endothelial injury and ARDS, and have prognostic utility in sepsis‐associated ARDS.Support or Funding InformationThis study was supported by NIH grants R01GM115553 (to DAF and JM). and R01HL122474 (to NJM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sepsis is a serious cause of morbidity and mortality worldwide. Sepsis is the life‐threatening organ dysfunction caused by the dysregulation of the inflammatory response to an infection. One aspect of this immune response is the systemic activation of neutrophils. Intended to combat microbes through phagocytosis, production of reactive oxygen species, and the release of antimicrobial enzymes through degranulation, neutrophil defense mechanisms can also damage host tissue. Neutrophil myeloperoxidase (MPO) catalyzes the production of hypochlorous acid (HOCl). In addition to microbial targets, HOCl can attack the vinyl ether bond of host membrane plasmalogens, releasing 2‐chlorofatty aldehyde (2‐ClFALD), which is oxidized to 2‐chlorofatty acid (2‐ClFA). Collectively, these plasmalogen oxidation products are members of the chlorolipid family. Human plasma levels of the chlorolipid, 2‐ClFA, predict 30‐day mortality and acute respiratory distress syndrome (ARDS) in sepsis patients. Similarly, septic rats have time‐dependent increases in the plasma, lung, kidney, and intestine levels of 2‐ClFA. For these studies rat sepsis was caused by intraperitoneal injections of 15mL of cecal slurry (isolated from donor rats)/kg body weight. In vitro studies using a click chemistry analog of 2‐ClFA have shown 2‐ClFA localizes to the Weibel‐Palade bodies of human coronary artery endothelial cells and induce the release of their contents. Release of von Willebrand factor, angiopoietin‐2, and P‐ and E‐selectin surface expression were caused by 2‐ClFA exposure to endothelial cells from several vascular beds including human coronary artery endothelial cells and human lung microvascular endothelial cells. The release of these molecules elicits endothelial activation/dysfunction including neutrophil and platelet adherence as well as loss of the endothelial permeability barrier. Additionally, 2‐ClFA induces human neutrophils to release their DNA as neutrophil extracellular traps (NETs) in the process known as NETosis, also implicated in organ failure in sepsis. These in vitro studies with human endothelial cells and human neutrophils suggest 2‐ClFA may mediate the microvascular failure associated with sepsis including endothelial dysfunction and microvascular plugging by NETs. Overall, these studies indicate the importance of chlorolipids in sepsis as prognostic biomarkers of human sepsis outcomes and potential mediators of multi‐organ failure associated with sepsis mortality.Support or Funding InformationFord, PI, Multi‐PI R01 grant (GM115553), Chlorinated lipids in sepsis, NIH Ford, PI, Multi‐PI R01 grant (GM129508), Neutrophil‐dependent mediators of sepsis, NIH Pike, PI, F30 Fellowship grant (F30HL142193), Plasmalogen‐Derived Chlorinated Lipids: Mediators of Acute Lung Injury in Sepsis, NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.