Lipidomic changes in a novel sepsis outcome-based analysis reveals potent pro-inflammatory and pro-resolving signaling lipids.

The purpose of this study was to investigate changes in the lipidome of patients with sepsis to identify signaling lipids associated with poor outcomes that could be linked to future therapies. Adult patients with sepsis were enrolled within 24h of sepsis recognition. Patients meeting Sepsis-3 criteria were enrolled from the emergency department or intensive care unit and blood samples were obtained. Clinical data were collected and outcomes of rapid recovery, chronic critical illness (CCI), or early death were adjudicated by clinicians. Lipidomic analysis was performed on two platforms, the Sciex™ 5500 device to perform a lipidomic screen of 1450 lipid species and a targeted signaling lipid panel using liquid-chromatography tandem mass spectrometry. For the lipidomic screen, there were 274 patients with sepsis: 192 with rapid recovery, 47 with CCI, and 35 with early deaths. CCI and early death patients were grouped together for analysis. Fatty acid (FA) 12:0 was decreased in CCI/early death, whereas FA 17:0 and 20:1 were elevated in CCI/early death, compared to rapid recovery patients. For the signaling lipid panel analysis, there were 262 patients with sepsis: 189 with rapid recovery, 45 with CCI, and 28 with early death. Pro-inflammatory signaling lipids from ω-6 poly-unsaturated fatty acids (PUFAs), including 15-hydroxyeicosatetraenoic (HETE), 12-HETE, and 11-HETE (oxidation products of arachidonic acid [AA]) were elevated in CCI/early death patients compared to rapid recovery. The pro-resolving lipid mediator from ω-3 PUFAs, 14(S)-hydroxy docosahexaenoic acid (14S-HDHA), was also elevated in CCI/early death compared to rapid recovery. Signaling lipids of the AA pathway were elevated in poor-outcome patients with sepsis and may serve as targets for future therapies.

DHCR7 Expression Predicts Poor Outcomes and Mortality From Sepsis.

This is a study of lipid metabolic gene expression patterns to discover precision medicine for sepsis. OBJECTIVES: Sepsis patients experience poor outcomes including chronic critical illness (CCI) or early death (within 14 d). We investigated lipid metabolic gene expression differences by outcome to discover therapeutic targets. DESIGN SETTING AND PARTICITPANTS: Secondary analysis of samples from prospectively enrolled sepsis patients (first 24 hr) and a zebrafish endotoxemia model for drug discovery. Patients were enrolled from the emergency department or ICU at an urban teaching hospital. Enrollment samples from sepsis patients were analyzed. Clinical data and cholesterol levels were recorded. Leukocytes were processed for RNA sequencing and reverse transcriptase polymerase chain reaction. A lipopolysaccharide zebrafish endotoxemia model was used for confirmation of human transcriptomic findings and drug discovery. MAIN OUTCOMES AND MEASURES: The derivation cohort included 96 patients and controls (12 early death, 13 CCI, 51 rapid recovery, and 20 controls) and the validation cohort had 52 patients (6 early death, 8 CCI, and 38 rapid recovery). RESULTS: The cholesterol metabolism gene 7-dehydrocholesterol reductase (DHCR7) was significantly up-regulated in both derivation and validation cohorts in poor outcome sepsis compared with rapid recovery patients and in 90-day nonsurvivors (validation only) and validated using RT-qPCR analysis. Our zebrafish sepsis model showed up-regulation of dhcr7 and several of the same lipid genes up-regulated in poor outcome human sepsis (dhcr24, sqlea, cyp51, msmo1, and ldlra) compared with controls. We then tested six lipid-based drugs in the zebrafish endotoxemia model. Of these, only the Dhcr7 inhibitor AY9944 completely rescued zebrafish from lipopolysaccharide death in a model with 100% lethality. CONCLUSIONS: DHCR7, an important cholesterol metabolism gene, was up-regulated in poor outcome sepsis patients warranting external validation. This pathway may serve as a potential therapeutic target to improve sepsis outcomes.

Vutiglabridin Modulates Paraoxonase 1 and Ameliorates Diet-Induced Obesity in Hyperlipidemic Mice.

Vutiglabridin is a clinical-stage synthetic small molecule that is being developed for the treatment of obesity and its target proteins have not been fully identified. Paraoxonase-1 (PON1) is an HDL-associated plasma enzyme that hydrolyzes diverse substrates including oxidized low-density lipoprotein (LDL). Furthermore, PON1 harbors anti-inflammatory and antioxidant capacities and has been implicated as a potential therapeutic target for treating various metabolic diseases. In this study, we performed a non-biased target deconvolution of vutiglabridin using Nematic Protein Organisation Technique (NPOT) and identified PON1 as an interacting protein. We examined this interaction in detail and demonstrate that vutiglabridin binds to PON1 with high affinity and protects PON1 against oxidative damage. Vutiglabridin treatment significantly increased plasma PON1 levels and enzyme activity but not PON1 mRNA in wild-type C57BL/6J mice, suggesting that vutiglabridin modulates PON1 post-transcriptionally. We further investigated the effects of vutiglabridin in obese and hyperlipidemic LDLR-/- mice and found that it significantly increases plasma PON1 levels, while decreasing body weight, total fat mass, and plasma cholesterol levels. Overall, our results demonstrate that PON1 is a direct, interacting target of vutiglabridin, and that the modulation of PON1 by vutiglabridin may provide benefits for the treatment of hyperlipidemia and obesity.

Role of enterocyte Enpp2 and autotaxin in regulating lipopolysaccharide levels, systemic inflammation, and atherosclerosis.

Conversion of lysophosphatidylcholine to lysophosphatidic acid (LPA) by autotaxin, a secreted phospholipase D, is a major pathway for producing LPA. We previously reported that feeding Ldlr-/- mice standard mouse chow supplemented with unsaturated LPA or lysophosphatidylcholine qualitatively mimicked the dyslipidemia and atherosclerosis induced by feeding a Western diet (WD). Here, we report that adding unsaturated LPA to standard mouse chow also increased the content of reactive oxygen species and oxidized phospholipids (OxPLs) in jejunum mucus. To determine the role of intestinal autotaxin, enterocyte-specific Ldlr-/-/Enpp2 KO (intestinal KO) mice were generated. In control mice, the WD increased enterocyte Enpp2 expression and raised autotaxin levels. Ex vivo, addition of OxPL to jejunum from Ldlr-/- mice on a chow diet induced expression of Enpp2. In control mice, the WD raised OxPL levels in jejunum mucus and decreased gene expression in enterocytes for a number of peptides and proteins that affect antimicrobial activity. On the WD, the control mice developed elevated levels of lipopolysaccharide in jejunum mucus and plasma, with increased dyslipidemia and increased atherosclerosis. All these changes were reduced in the intestinal KO mice. We conclude that the WD increases the formation of intestinal OxPL, which i) induce enterocyte Enpp2 and autotaxin resulting in higher enterocyte LPA levels; that ii) contribute to the formation of reactive oxygen species that help to maintain the high OxPL levels; iii) decrease intestinal antimicrobial activity; and iv) raise plasma lipopolysaccharide levels that promote systemic inflammation and enhance atherosclerosis.

DHCR7 Expression Predicts Poor Outcomes and Mortality from Sepsis.

Objective: Sepsis patients experience poor outcomes including chronic critical illness (CCI) or early death (within 14 days). We investigated lipid metabolic gene expression differences by outcome to discover therapeutic targets. Design: Secondary analysis of samples from prospectively enrolled sepsis patients and a zebrafish sepsis model for drug discovery. Setting: Emergency department or ICU at an urban teaching hospital. Patients: Sepsis patients presenting within 24 hours. Methods: Enrollment samples from sepsis patients were analyzed. Clinical data and cholesterol levels were recorded. Leukocytes were processed for RNA sequencing (RNA-seq) and reverse transcriptase polymerase chain reaction (RT-qPCR). A lipopolysaccharide (LPS) zebrafish sepsis model was used for confirmation of human transcriptomic findings and drug discovery. Measurements and Main Results: There were 96 samples in the derivation (76 sepsis, 20 controls) and 52 in the validation cohort (sepsis only). The cholesterol metabolism gene 7-Dehydrocholesterol Reductase ( DHCR7) was significantly upregulated in both derivation and validation cohorts in poor outcome sepsis compared to rapid recovery patients and in 90-day non-survivors (validation only) and validated using RT-qPCR analysis. Our zebrafish sepsis model showed upregulation of dhcr7 and several of the same lipid genes upregulated in poor outcome human sepsis (dhcr24, sqlea, cyp51, msmo1 , ldlra) compared to controls. We then tested six lipid-based drugs in the zebrafish sepsis model. Of these, only the Dhcr7 inhibitor AY9944 completely rescued zebrafish from LPS death in a model with 100% lethality. Conclusions: DHCR7, an important cholesterol metabolism gene, was upregulated in poor outcome sepsis patients warranting external validation. This pathway may serve as a potential therapeutic target to improve sepsis outcomes.

Apolipoprotein A-I mimetics mitigate intestinal inflammation in COX2-dependent inflammatory bowel disease model.

Cyclooxygenase 2 (Cox2) total knockout and myeloid knockout (MKO) mice develop Crohn's-like intestinal inflammation when fed cholate-containing high fat diet (CCHF). We demonstrated that CCHF impaired intestinal barrier function and increased translocation of endotoxin, initiating TLR/MyD88-dependent inflammation in Cox2 KO but not WT mice. Cox2 MKO increased pro-inflammatory mediators in LPS-activated macrophages, and in the intestinal tissue and plasma upon CCHF challenge. Cox2 MKO also reduced inflammation resolving lipoxin A4 (LXA4) in intestinal tissue, while administration of an LXA4 analog rescued disease in Cox2 MKO mice fed CCHF. The apolipoprotein A-I (APOA1) mimetic 4F mitigated disease in both the Cox2 MKO/CCHF and piroxicam-accelerated Il10-/- models of inflammatory bowel disease (IBD) and reduced elevated levels of pro-inflammatory mediators in tissue and plasma. APOA1 mimetic Tg6F therapy was also effective in reducing intestinal inflammation in the Cox2 MKO/CCHF model. We further demonstrated that APOA1 mimetic peptides: i) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (oxPAPC) dependent pro-inflammatory responses in human macrophages and intestinal epithelium; and ii) directly cleared pro-inflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for pro-inflammatory and inflammation resolving lipids in IBD pathology and a translational potential for APOA1 mimetic peptides for the treatment of IBD.

Paraoxonase 2 protects against acute myocardial ischemia-reperfusion injury by modulating mitochondrial function and oxidative stress via the PI3K/Akt/GSK-3ß RISK pathway.

OBJECTIVE: To investigate the novel role of Paraoxonase 2 (PON2) in modulating acute myocardial ischemia-reperfusion injury (IRI). APPROACH: IRI was induced both in vivo and ex vivo in male, C57BL6/J (WT) and PON2-deficient (PON-def) mice. In addition, in vitro hypoxia-reoxygenation injury (HRI) was induced in H9c2 cells expressing empty vector (H9c2-EV) or human PON2 (H9c2-hPON2) ±â€¯LY294002 (a potent PI3K inhibitor). Infarct size, PON2 gene expression, mitochondrial calcium retention capacity (CRC), reactive oxygen species (ROS) generation, mitochondrial membrane potential, CHOP and pGSK-3ß protein levels, and cell apoptosis were evaluated. RESULTS: PON2 gene expression is upregulated in WT mice following in vivo IRI. PON2-def mice exhibit a 2-fold larger infarct, increased CHOP levels, and reduced pGSK-3ß levels compared to WT controls. Global cardiac mitochondria isolated from PON2-def mice exhibit reduced CRC and increased ROS production. Cardiomyocytes isolated from PON2-def mice subjected to ex vivo IRI have mitochondria with reduced CRC (also seen under non-IRI conditions), and increased ROS generation and apoptosis compared to WT controls. PON2 knockdown in H9c2 cells subjected to HRI leads to an increase in mitochondrial membrane depolarization. H9c2-hPON2 cells exhibit i) improvement in mitochondrial membrane potential, pGSK-3ß levels and mitochondrial CRC, and ii) decrease in CHOP levels, mitochondrial ROS generation and cell apoptosis, when compared to H9c2-EV controls. Treatment with LY294002 resulted in a decrease of mitochondrial CRC and increase in mitochondrial ROS production and cell apoptosis in the H9c2-hPON2 group versus H9c2-EV controls. CONCLUSION: PON2 protects against acute myocardial IRI by reducing mitochondrial dysfunction and oxidative stress in cardiomyocytes via activation of the PI3K/Akt/GSK-3ß RISK pathway.

Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion injury.

Macrophages polarize into heterogeneous proinflammatory M1 and antiinflammatory M2 subtypes. Heme oxygenase 1 (HO-1) protects against inflammatory processes such as ischemia-reperfusion injury (IRI), organ transplantation, and atherosclerosis. To test our hypothesis that HO-1 regulates macrophage polarization and protects against IRI, we generated myeloid-specific HO-1-knockout (mHO-1-KO) and -transgenic (mHO-1-Tg) mice, with deletion or overexpression of HO-1, in various macrophage populations. Bone marrow-derived macrophages (BMDMs) from mHO-1-KO mice, treated with M1-inducing LPS or M2-inducing IL-4, exhibited increased mRNA expression of M1 (CXCL10, IL-1ß, MCP1) and decreased expression of M2 (Arg1 and CD163) markers as compared with controls, while BMDMs from mHO-1-Tg mice displayed the opposite. A similar pattern was observed in the hepatic M1/M2 expression profile in a mouse model of liver IRI. mHO-1-KO mice displayed increased hepatocellular damage, serum AST/ALT levels, Suzuki's histological score of liver IRI, and neutrophil and macrophage infiltration, while mHO-1-Tg mice exhibited the opposite. In human liver transplant biopsies, subjects with higher HO-1 levels showed lower expression of M1 markers together with decreased hepatocellular damage and improved outcomes. In conclusion, myeloid HO-1 expression modulates macrophage polarization, and protects against liver IRI, at least in part by favoring an M2 phenotype.

Treating the Intestine with Oral ApoA-I Mimetic Tg6F Reduces Tumor Burden in Mouse Models of Metastatic Lung Cancer.

Having demonstrated that apolipoprotein A-I (apoA-I) mimetic peptides ameliorate cancer in mouse models, we sought to determine the mechanism for the anti-tumorigenic function of these peptides. CT-26 cells (colon cancer cells that implant and grow into tumors in the lungs) were injected into wild-type BALB/c mice. The day after injection, mice were either continued on chow or switched to chow containing 0.06% of a concentrate of transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F). After four weeks, the number of lung tumors was significantly lower in Tg6F-fed mice. Gene expression array analyses of jejunum and lung identified Notch pathway genes significantly upregulated, whereas osteopontin (Spp1) was significantly downregulated by Tg6F in both jejunum and lung. In jejunum, Tg6F increased protein levels for Notch1, Notch2, Dll1, and Dll4. In lung, Tg6F increased protein levels for Notch1 and Dll4 and decreased Spp1. Tg6F reduced oxidized phospholipid levels (E06 immunoreactivity) and reduced 25-hydroxycholesterol (25-OHC) levels, which are known to inhibit Notch1 and induce Spp1, respectively. Notch pathway promotes anti-tumorigenic patrolling monocytes, while Spp1 facilitates pro-tumorigenic myeloid derived suppressor cells (MDSCs) formation. Tg6F-fed mice had higher numbers of patrolling monocytes in jejunum and in lung (p < 0.02), and lower plasma levels of Spp1 with reduced numbers of MDSCs in jejunum and in lung (p < 0.03). We conclude that Tg6F alters levels of specific oxidized lipids and 25-OHC to modulate Notch pathways and Spp1, which alter small intestine immune cells, leading to similar changes in lung that reduce tumor burden.

Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux.

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol.

Effect of exposure to atmospheric ultrafine particles on production of free fatty acids and lipid metabolites in the mouse small intestine.

BACKGROUND: Exposure to ambient ultrafine particulate matter (UFP) is a well-recognized risk factor for cardiovascular and respiratory diseases. However, little is known about the effects of air pollution on gastrointestinal disorders. OBJECTIVE: We sought to assess whether exposure to ambient UFP (diameter < 180 nm) increased free fatty acids and lipid metabolites in the mouse small intestine. METHODS: Ldlr-null mice were exposed to filtered air (FA) or UFP collected at an urban Los Angeles, California, site that was heavily affected by vehicular emissions; the exposure was carried out for 10 weeks in the presence or absence of D-4F, an apolipoprotein A-I mimetic peptide with antioxidant and anti-inflammation properties on a high-fat or normal chow diet. RESULTS: Compared with FA, exposure to UFP significantly increased intestinal hydroxyeicosatetraenoic acids (HETEs), including 15-HETE, 12-HETE, 5-HETE, as well as hydroxyoctadecadienoic acids (HODEs), including 13-HODE and 9-HODE. Arachidonic acid (AA) and prostaglandin D2 (PGD2) as well as some of the lysophosphatidic acids (LPA) in the small intestine were also increased in response to UFP exposure. Administration of D-4F significantly reduced UFP-mediated increase in HETEs, HODEs, AA, PGD2, and LPA. Although exposure to UFP further led to shortened villus length accompanied by prominent macrophage and neutrophil infiltration into the intestinal villi, administration of D-4F mitigated macrophage infiltration. CONCLUSIONS: Exposure to UFP promotes lipid metabolism, villus shortening, and inflammatory responses in mouse small intestine, whereas administration of D-4F attenuated these effects. Our findings provide a basis to further assess the mechanisms underlying UFP-mediated lipid metabolism in the digestive system with clinical relevance to gut homeostasis and diseases.