Eicosanoid Signaling in Insect Immunology: New Genes and Unresolved Issues.

This paper is focused on eicosanoid signaling in insect immunology. We begin with eicosanoid biosynthesis through the actions of phospholipase A2, responsible for hydrolyzing the C18 polyunsaturated fatty acid, linoleic acid (18:2n-6), from cellular phospholipids, which is subsequently converted into arachidonic acid (AA; 20:4n-6) via elongases and desaturases. The synthesized AA is then oxygenated into one of three groups of eicosanoids, prostaglandins (PGs), epoxyeicosatrienoic acids (EETs) and lipoxygenase products. We mark the distinction between mammalian cyclooxygenases and insect peroxynectins, both of which convert AA into PGs. One PG, PGI2 (also called prostacyclin), is newly discovered in insects, as a negative regulator of immune reactions and a positive signal in juvenile development. Two new elements of insect PG biology are a PG dehydrogenase and a PG reductase, both of which enact necessary PG catabolism. EETs, which are produced from AA via cytochrome P450s, also act in immune signaling, acting as pro-inflammatory signals. Eicosanoids signal a wide range of cellular immune reactions to infections, invasions and wounding, including nodulation, cell spreading, hemocyte migration and releasing prophenoloxidase from oenocytoids, a class of lepidopteran hemocytes. We briefly review the relatively scant knowledge on insect PG receptors and note PGs also act in gut immunity and in humoral immunity. Detailed new information on PG actions in mosquito immunity against the malarial agent, Plasmodium berghei, has recently emerged and we treat this exciting new work. The new findings on eicosanoid actions in insect immunity have emerged from a very broad range of research at the genetic, cellular and organismal levels, all taking place at the international level.

GGPP depletion initiates metaflammation through disequilibrating CYB5R3-dependent eicosanoid metabolism.

Metaflammation is a primary inflammatory complication of metabolic disorders characterized by altered production of many inflammatory cytokines, adipokines, and lipid mediators. Whereas multiple inflammation networks have been identified, the mechanisms by which metaflammation is initiated have long been controversial. As the mevalonate pathway (MVA) produces abundant bioactive isoprenoids and abnormal MVA has a phenotypic association with inflammation/immunity, we speculate that isoprenoids from the MVA may provide a causal link between metaflammation and metabolic disorders. Using a line with the MVA isoprenoid producer geranylgeranyl diphosphate synthase (GGPPS) deleted, we find that geranylgeranyl pyrophosphate (GGPP) depletion causes an apparent metaflammation as evidenced by abnormal accumulation of fatty acids, eicosanoid intermediates, and proinflammatory cytokines. We also find that GGPP prenylate cytochrome b5 reductase 3 (CYB5R3) and the prenylated CYB5R3 then translocate from the mitochondrial to the endoplasmic reticulum (ER) pool. As CYB5R3 is a critical NADH-dependent reductase necessary for eicosanoid metabolism in ER, we thus suggest that GGPP-mediated CYB5R3 prenylation is necessary for metabolism. In addition, we observe that pharmacological inhibition of the MVA pathway by simvastatin is sufficient to inhibit CYB5R3 translocation and induces smooth muscle death. Therefore, we conclude that the dysregulation of MVA intermediates is an essential mechanism for metaflammation initiation, in which the imbalanced production of eicosanoid intermediates in the ER serve as an important pathogenic factor. Moreover, the interplay of MVA and eicosanoid metabolism as we reported here illustrates a model for the coordinating regulation among metabolite pathways.

The enzymology of human eicosanoid pathways: the lipoxygenase branches.

Eicosanoids are short-lived derivatives of polyunsaturated fatty acids that serve as autocrine and paracrine signaling molecules. They are involved numerous biological processes of both the well state and disease states. A thorough understanding of the progression the disease state and homeostasis of the well state requires a complete evaluation of the systems involved. This review examines the enzymology for the enzymes involved in the production of eicosanoids along the lipoxygenase branches of the eicosanoid pathways with particular emphasis on those derived from arachidonic acid. The enzymatic parameters, protocols to measure them, and proposed catalytic mechanisms are presented in detail.

CYP-derived eicosanoids: Implications for rheumatoid arthritis.

Today the role of cytochrome P450 metabolites in inflammatory rheumatic disease, such as rheumatoid arthritis (RA) is still poorly understood. In this review we survey the current knowledge on cytochrome P450 metabolites in rheumatoid arthritis. The balance between CYP epoxygenase- and CYP ω- hydroxylase is correlated to the regulation of NF-κB. In RA patients synovial fluid there are higher levels of IL-6, which suppresses activities of CYP enzymes, such as CYP3A, CYP2C19, CYP2C9, and CYP1A2. EETs have anti-inflammatory effects, probably attributed to the PPARγ activation. EETs inhibit bone resorption and osteoclastogenesis, and can be considered as an innovative therapeutic strategy for rheumatoid arthritis. In reference to the CYP É·-hydroxylase pathway, 20-HETE is a pro-inflammatory mediator. While there is scarce information on the role of 20-HETE inhibitors and its antagonists in rheumatoid arthritis, the elevation of EETs levels by sEH inhibitors is a promising therapeutic strategy for rheumatoid arthritis patients. In addition, hybrid compounds, such as sEH inhibitors/FLAP inhibitors, or sEHI combined with NSAIDs/COXIBs are also important therapeutic target. However, studies investigating the effects of inflammation and rheumatic disease on CYP-mediated eicosanoid metabolism are necessary. Obtaining a better understanding of the complex role of CYP-derived eicosanoids in inflammatory rheumatic disease, such as rheumatoid arthritis will provide valuable insight for basic and clinical researchers investigation.

Effects of anti-inflammatory and adaptogenic herbal extracts on gene expression of eicosanoids signaling pathways in isolated brain cells.

INTRODUCTION: The adaptogens modulate expression of genes playing key roles in development of aging-related disorders, which are considered as low-grade systemic inflammatory conditions characterized by an imbalance between pro-and anti-inflammatory eicosanoids. AIM OF THE STUDY: We compared the effects of anti-inflammatory and adaptogenic plant extracts on the expression of genes involved in biosynthesis of eicosanoids with the purpose to find those plants, which selectively upregulated the expression of anti-inflammatory lipoxins signaling pathways and inhibited pro-inflammatory signaling pathways associated with biosynthesis of leukotrienes, prostaglandins and thromboxanes. MATERIALS AND METHODS: We conducted transcriptome-wide RNA sequencing to profile gene expression alterations in T98G neuroglia cells upon treatment of plant extract and analyzed the relevance of deregulated genes to eicosanoids signaling pathways using in silico models. RESULTS: For the first time, we demonstrated that Rhodiola rosea, Withania somnifera and Eleutherococcus senticosus downregulate the expression of key genes (ALOX5AP, DPEP2, LTC4S) involved biosynthesis of leukotrienes A, B, C, D and E, resulting in inhibition of leukotriene signaling pathway suggesting their potential benefits in Alzheimer disease. The common feature for all tested anti-inflammatory plants extracts was related to downregulation of ALOX12, which was also associated with neuroprotective action of these medicinal plants as well as their potential benefits in neurodegenerative diseases. None of tested anti-inflammatory and adaptogenic plants selectively activated the ALOX15-mediated signaling pathway, which is associated with generation anti-inflammatory lipoxins. Almost all tested plants upregulated the expression of the prostaglandin E receptor 3 gene (PTGER3) suggesting their potential benefits in the treatment of cancer. CONCLUSION: Every single plant tested in this study revealed a specific "signature" on eicosanoid signaling-related gene expression, regardless of their common features as anti-inflammatory or adaptogenic activity. Further studies of the combination of Rhodiola with Withania (Adaptra) for the treatment of Alzheimer disease are required.

A combined computational strategy of sequence and structural analysis predicts the existence of a functional eicosanoid pathway in Drosophila melanogaster.

This study reports on a putative eicosanoid biosynthesis pathway in Drosophila melanogaster and challenges the currently held view that mechanistic routes to synthesize eicosanoid or eicosanoid-like biolipids do not exist in insects, since to date, putative fly homologs of most mammalian enzymes have not been identified. Here we use systematic and comprehensive bioinformatics approaches to identify most of the mammalian eicosanoid synthesis enzymes. Sensitive sequence analysis techniques identified candidate Drosophila enzymes that share low global sequence identities with their human counterparts. Twenty Drosophila candidates were selected based upon (a) sequence identity with human enzymes of the cyclooxygenase and lipoxygenase branches, (b) similar domain architecture and structural conservation of the catalytic domain, and (c) presence of potentially equivalent functional residues. Evaluation of full-length structural models for these 20 top-scoring Drosophila candidates revealed a surprising degree of conservation in their overall folds and potential analogs for functional residues in all 20 enzymes. Although we were unable to identify any suitable candidate for lipoxygenase enzymes, we report structural homology models of three fly cyclooxygenases. Our findings predict that the D. melanogaster genome likely codes for one or more pathways for eicosanoid or eicosanoid-like biolipid synthesis. Our study suggests that classical and/or novel eicosanoids mediators must regulate biological functions in insects-predictions that can be tested with the power of Drosophila genetics. Such experimental analysis of eicosanoid biology in a simple model organism will have high relevance to human development and health.

Eicosanoids via CYP450 and cardiovascular disease: Hints from genetic and nutrition studies.

Metabolites of arachidonic acid via CYP450 such as epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), have vasoactive and natriuretic properties and have been implicated in BP homeostasis and the incidence of cardio- and cerebrovascular diseases in animal studies. In humans, genetic studies considering genes implicated in arachidonic acids metabolism (CYP4F2, CYP4A11, CYP2J2, CYP2C8, CYP2C9, CYP2A1/2, EPHX2) can offer a hint to understand their role, if any, in hypertension development and its deleterious cardiovascular effects. Candidate genes studies and successive meta-analyses have shown that specific single nucleotide polymorphisms (SNPs), often functional, and haplotypes in these genes were associated with one or more cardiovascular endpoints. Nevertheless, genome wide association studies (GWAS) have never detected any SNPs nearby these genes (the only exception being the CYP2A1/2 locus) as associated with either BP, hypertension, coronary artery disease or stroke questioning their real importance for cardiovascular health in humans. Nutrition studies exploring the effects of specific foods on the formation of these compounds or others through the same pathway can offer new insights on this field.

The Cytochrome P450 Slow Metabolizers CYP2C9*2 and CYP2C9*3 Directly Regulate Tumorigenesis via Reduced Epoxyeicosatrienoic Acid Production.

Increased expression of cytochrome P450 CYP2C9, together with elevated levels of its products epoxyeicosatrienoic acids (EET), is associated with aggressiveness in cancer. Cytochrome P450 variants CYP2C9*2 and CYP2C9*3 encode proteins with reduced enzymatic activity, and individuals carrying these variants metabolize drugs more slowly than individuals with wild-type CYP2C9*1, potentially affecting their response to drugs and altering their risk of disease. Although genetic differences in CYP2C9-dependent oxidation of arachidonic acid (AA) have been reported, the roles of CYP2C9*2 and CYP2C9*3 in EET biosynthesis and their relevance to disease are unknown. Here, we report that CYP2C9*2 and CYP2C9*3 metabolize AA less efficiently than CYP2C9*1 and that they play a role in the progression of non-small cell lung cancer (NSCLC) via impaired EET biosynthesis. When injected into mice, NSCLC cells expressing CYP2C9*2 and CYP2C9*3 produced lower levels of EETs and developed fewer, smaller, and less vascularized tumors than cells expressing CYP2C9*1. Moreover, endothelial cells expressing these two variants proliferated and migrated less than cells expressing CYP2C*1. Purified CYP2C9*2 and CYP2C9*3 exhibited attenuated catalytic efficiency in producing EETs, primarily due to impaired reduction of these two variants by NADPH-P450 reductase. Loss-of-function SNPs within CYP2C9*2 and CYP2C9*3 were associated with improved survival in female cases of NSCLC. Thus, decreased EET biosynthesis represents a novel mechanism whereby CYPC29*2 and CYP2C9*3 exert a direct protective role in NSCLC development.Significance: These findings report single nucleotide polymorphisms in the human CYP2C9 genes, CYP2C9*2 and CYP2C9*3, exert a direct protective role in tumorigenesis by impairing EET biosynthesis. Cancer Res; 78(17); 4865-77. ©2018 AACR.

A Serum Small Molecule Biosignature of Radiation Exposure from Total Body Irradiated Patients.

The potential for radiological accidents and nuclear terrorism has increased the need for the development of new rapid biodosimetry methods. In addition, in a clinical setting the issue of an individual's radiosensitivity should be taken into consideration during radiotherapy. We utilized metabolomics and lipidomics to investigate changes of metabolites in serum samples following exposure to total body ionizing radiation in humans. Serum was collected prior to irradiation, at 3-8 h after a single dose of 1.25-2 Gy, and at 24 h with a total delivered dose of 2-3.75 Gy. Metabolomics revealed perturbations in glycerophosphocholine, phenylalanine, ubiquinone Q2, and oxalic acid. Alterations were observed in circulating levels of lipids from monoacylglycerol, triacylglycerol, phosphatidylcholine, and phosphatidylglycerol lipid classes. Polyunsaturated fatty acids were some of the most dysregulated lipids, with increased levels linked to proinflammatory processes. A targeted metabolomics approach for eicosanoids was also employed. The results showed a rapid response for proinflammatory eicosanoids, with a dampening of the signal at the later time point. Sex differences were observed in the markers from the untargeted approach but not the targeted method. The ability to identify and quantify small molecules in blood can therefore be utilized to monitor radiation exposure in human populations.

High dietary arachidonic acid levels induce changes in complex lipids and immune-related eicosanoids and increase levels of oxidised metabolites in zebrafish (Danio rerio).

This study explores the effect of high dietary arachidonic acid (ARA) levels (high ARA) compared with low dietary ARA levels (control) on the general metabolism using zebrafish as the model organism. The fatty acid composition of today's 'modern diet' tends towards higher n-6 PUFA levels in relation to n-3 PUFA. Low dietary n-3:n-6 PUFA ratio is a health concern, as n-6 PUFA give rise to eicosanoids and PG, which are traditionally considered pro-inflammatory, especially when derived from ARA. Juvenile zebrafish fed a high-ARA diet for 17 d had a lower whole-body n-3:n-6 PUFA ratio compared with zebrafish fed a low-ARA (control) diet (0·6 in the control group v. 0·2 in the high-ARA group). Metabolic profiling revealed altered levels of eicosanoids, PUFA, dicarboxylic acids and complex lipids such as glycerophospholipids and lysophospholipids as the most significant differences compared with the control group. ARA-derived hydroxylated eicosanoids, such as hydroxy-eicosatetraenoic acids, were elevated in response to high-ARA feed. In addition, increased levels of oxidised lipids and amino acids indicated an oxidised environment due to n-6 PUFA excess in the fish. To conclude, our results indicate that an ARA-enriched diet induces changes in complex lipids and immune-related eicosanoids and increases levels of oxidised lipids and amino acids, suggesting oxidative stress and lipid peroxidation.

Interactions among Variants in Eicosanoid Genes Increase Risk of Atherothrombotic Stroke in Chinese Populations.

BACKGROUND: Eicosanoids are lipid mediators that may play a role in ischemic stroke (IS). However, the association of variants in eicosanoid genes and these interactions with IS risk has not been investigated. The aim of the present study was to investigate the association of 11 variants in eicosanoid genes with IS and to determine whether these gene-gene interactions increase the risk of IS. METHODS: Eleven variants in prostaglandin H synthase-1 (PTGS1), PTGS2, thromboxane A2 synthase (TBXAS1), prostacyclin synthase (PTGIS), and prostaglandin E synthase (PTGES) genes were examined using mass spectrometry method in 297 patients with atherothrombotic stroke and 291 controls. Gene-gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) method. Platelet aggregation and platelet-leukocyte aggregates were measured on admission. RESULTS: There were no significant differences in the genotype distributions of the 11 variants between patients and controls. However, GMDR analysis showed a significant gene-gene interaction among rs20417, rs5602, and rs41708, which scored 10 for cross-validation consistency and 9 for the sign test (P = .014). Logistic regression analysis showed that high-risk interaction among rs20417, rs5602, and rs41708 was an independent risk factor for atherothrombotic stroke (OR = 2.45, 95% CI: 1.33-3.27, P = .019). The high-risk interactive genotypes were associated with higher platelet aggregation and platelet-leukocyte aggregates. CONCLUSIONS: PTGS2 rs20417, PTGIS rs5602, and TBXAS1 rs41708 three-locus interactions may confer a higher risk for atherothrombotic stroke. The combinatorial analysis used in this study may be helpful to elucidate complex genetic risk for IS.

Effects of Almond- and Olive Oil-Based Docosahexaenoic- and Vitamin E-Enriched Beverage Dietary Supplementation on Inflammation Associated to Exercise and Age.

n-3-polyunsaturated fatty acids and polyphenols are potential key factors for the treatment and prevention of chronic inflammation associated to ageing and non-communicable diseases. The aim was to analyse effects of an almond and olive oil beverage enriched with α-tocopherol and docosahexaenoic, exercise and age on inflammatory plasma markers, and immune gene expression in peripheral blood mononuclear cells (PBMCs). Five young and five senior athletes who were supplemented for five weeks with a functional beverage performed a stress test under controlled conditions before and after beverage supplementation. Blood samples were taken immediately before and 1 h after each test. Plasma, erythrocytes and PBMCs were isolated. Beverage supplementation increased plasmatic Tumour Necrosis Factor α (TNFα) levels depending on age and exercise. Exercise increased plasma non esterified fatty acids (NEFAs), soluble Intercellular adhesion molecule 3 (sICAM3) and soluble L-selectin (sL-Selectin), and this increase was attenuated by the supplementation. Exercise increased PGE2 plasma levels in supplemented young and in senior placebo athletes. Exercise increased NFkß-activated levels in PBMCs, which are primed to a pro-inflammatory response increasing pro-inflammatory genes expression after the exercise mainly in the young group after the supplementation. The functional beverage supplementation to young athletes enhances a pro-inflammatory circulating environment in response to the exercise that was less evident in the senior group.

Coffee consumption modulates inflammatory processes in an individual fashion.

SCOPE: Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. METHODS AND RESULTS: We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines, and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, prostaglandin E2 (PGE2), LTC4, LTE4, and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant downregulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. CONCLUSION: Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion.

Alcohol produces distinct hepatic lipidome and eicosanoid signature in lean and obese.

Alcohol- and obesity-related liver diseases often coexist. The hepatic lipidomics due to alcohol and obesity interaction is unknown. We characterized the hepatic lipidome due to 1) alcohol consumption in lean and obese mice and 2) obesity and alcohol interactions. In the French-Tsukamoto mouse model, intragastric alcohol or isocaloric dextrose were fed with either chow (lean) or high-fat, high-cholesterol diet (obese). Four groups (lean, lean alcohol, obese, and obese alcohol) were studied. MS was performed for hepatic lipidomics, and data were analyzed. Alcohol significantly increased hepatic cholesteryl esters and diacyl-glycerol in lean and obese but was more pronounced in obese. Alcohol produced contrasting changes in hepatic phospholipids with significant enrichment in lean mice versus significant decrease in obese mice, except phosphatidylglycerol, which was increased in both lean and obese alcohol groups. Most lysophospholipids were increased in lean alcohol and obese mice without alcohol use only. Prostaglandin E2; 5-, 8-, and 11-hydroxyeicosatetraenoic acids; and 9- and 13-hydroxyoctadecadienoic acids were considerably increased in obese mice with alcohol use. Alcohol consumption produced distinct changes in lean and obese with profound effects of obesity and alcohol interaction on proinflammatory and oxidative stress-related eicosanoids.

Plasma Vasoprotective Eicosanoid Concentrations in Healthy Greyhounds and Non-Greyhound Dogs.

BACKGROUND: Hypertension and albuminuria often coexist in Greyhounds, suggesting generalized vascular dysfunction that could contribute to the development of a variety of diseases in this breed. Eicosanoid metabolites of arachidonic acid (AA) mediate endothelial function, vascular reactivity, and proteinuria in humans and in rodent models. HYPOTHESIS: The eicosanoid profile of Greyhounds is shifted toward metabolites that promote vascular dysfunction, hypertension, and proteinuria. ANIMALS: Healthy Greyhounds (n = 20) and non-Greyhound (n = 20) dogs that were consecutively enrolled in a blood donor program. METHODS: Prospective study. Plasma eicosanoid metabolites were assayed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) and compared to systolic blood pressure (SP) measurements and urine albumin concentration. RESULTS: Isomers of hydroxyeicosatetraenoic acid (HETE) were higher in Greyhounds than non-Greyhounds (median, range in pmol/mL: 5(S)HETE 19.82, 8.55-32.95 versus 13.54, 4.33-26.27, P = .033; 8(S)HETE 9.39, 3.28-19.84 versus 5.80, 2.25-17.66, P = .002; 9(S)HETE 9.46, 2.43-13.79 versus 5.82, 1.50-17.16, P = .026; 12(S)HETE 10.17, 3.81-40.06 versus 7.24, 2.9-16.16, P = .022). Dihydroxyeicosatrienoic acid (DHET) isomers also were higher in Greyhounds compared to non-Greyhounds (mean ± SD in pmol/mL: 8,9DHET 5.78 ± 2.13 versus 4.03 ± 1.36, P = .004; 11,12DHET 11.98 ± 2.86 versus 8.90 ± 3.48, P = .004; 14,15DHET 7.23 ± 2.19 versus 5.76 ± 1.87, P = .028). Albuminuria correlated with total DHET (rs = 0.46, P = .003). SP was positively correlated with 11,12EET (rs = 0.42, P = .006) and 20(S)HETE (rs = 0.38, P = .017). SP and 8,9EET were inversely correlated (rs = -0.49, P = .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma eicosanoid profile in Greyhounds was consistent with activation of metabolic pathways known to promote vascular dysfunction and might contribute to higher blood pressures and albuminuria. Inhibition of these eicosanoid pathways should be evaluated as therapeutic targets in Greyhounds.

Shiga Toxin 2a-Induced Endothelial Injury in Hemolytic Uremic Syndrome: A Metabolomic Analysis.

BACKGROUND: Endothelial dysfunction plays a pivotal role in the pathogenesis of postenteropathic hemolytic uremic syndrome (HUS), most commonly caused by Shiga toxin (Stx)-producing strains of Escherichia coli. METHODS: To identify new treatment targets, we performed a metabolomic high-throughput screening to analyze the effect of Stx2a, the major Stx type associated with HUS, on human renal glomerular endothelial cells (HRGEC) and umbilical vein endothelial cells (HUVEC). Cells were treated either with sensitizing tumor necrosis factor α (TNF-α) or Stx2a, a sequence of both or remained untreated. RESULTS: We identified 341 metabolites by combined liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry. Both cell lines exhibited distinct metabolic reaction profiles but shared elevated levels of free fatty acids. Stx2a predominantly altered the nicotinamide adenine dinucleotide (NAD) cofactor pathway and the inflammation-modulating eicosanoid pathway, which are associated with lipid metabolism. In HRGEC, Stx2a strongly diminished NAD derivatives, leading to depletion of the energy substrate acetyl coenzyme A and the antioxidant glutathione. HUVEC responded to TNF-α and Stx2a by increasing production of the counteracting eicosanoids prostaglandin I2, E1, E2, and A2, while in HRGEC only more prostaglandin I2 was detected. CONCLUSIONS: We conclude that disruption of energy metabolism and depletion of glutathione contributes to Stx-induced injury of the renal endothelium and that the inflammatory response to Stx is highly cell-type specific.

Prepartal dietary energy level affects peripartal bovine blood neutrophil metabolic, antioxidant, and inflammatory gene expression.

During the dry period, cows can easily overconsume higher-grain diets, a scenario that could impair immune function during the peripartal period. Objectives were to investigate the effects of energy overfeeding on expression profile of genes associated with inflammation, lipid metabolism, and neutrophil function, in 12 multiparous Holstein cows (n=6/dietary group) fed control [CON, 1.34 Mcal/kg of dry matter (DM)] or higher-energy (HE, 1.62 Mcal/kg of DM) diets during the last 45 d of pregnancy. Blood was collected to evaluate 43 genes in polymorphonuclear neutrophil leukocytes (PMNL) isolated at -14, 7, and 14 d relative to parturition. We detected greater expression of inflammatory-related cytokines (IL1B, STAT3, NFKB1) and eicosanoid synthesis (ALOX5AP and PLA2G4A) in HE cows than in CON cows. Around parturition, all cows had a close balance in mRNA expression of the pro-inflammatory IL1B and the anti-inflammatory IL10, with greater expression of both in cows fed HE than CON. The expression of CCL2, LEPR, TLR4, IL6, and LTC4S was undetectable. Cows in the HE group had greater expression of genes involved in PMNL adhesion, motility, migration, and phagocytosis, which was similar to expression of genes related to the pro-inflammatory cytokine. This response suggests that HE cows experienced a chronic state of inflammation. The greater expression of G6PD in HE cows could have been associated with the greater plasma insulin, which would have diverted glucose to other tissues. Cows fed the HE diet also had greater expression of transcription factors involved in metabolism of long-chain fatty acids (PPARD, RXRA), suggesting that immune cells might be predisposed to use endogenous ligands such as nonesterified fatty acids available in the circulation when glucose is in high demand for milk synthesis. The lower overall expression of SLC2A1 postpartum than prepartum supports this suggestion. Targeting interleukin-1ß signaling might be of value in terms of controlling the inflammatory response around calving. The present study revealed that overfeeding cows during late pregnancy results in activation, ahead of parturition, of PMNL responses associated with stress and inflammation. These adaptations observed in PMNL did not seem to be detrimental for production.

Systematic metabolomic analysis of eicosanoids after omega-3 polyunsaturated fatty acid supplementation by a highly specific liquid chromatography-tandem mass spectrometry-based method.

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) have beneficial effects in many pathological processes, especially cardiovascular disease, and their protective eicosanoid metabolites are thought to play important roles. However, how ω-3 PUFAs affect the eicosanoid profile has not been elucidated comprehensively. Here, we systematically analyzed the eicosanoid metabolites induced by ω-3 PUFA supplementation. We developed an LC-MS/MS-based method covering 32 arachidonic acid (ARA) metabolites and 37 ω-3 PUFA-derived products. The limits of detection for eicosanoids were between 0.0625 and 1 pg and the detection specificity was optimized. We then quantified eicosanoids in mouse and human plasma and mouse aorta samples after ω-3 PUFA supplementation. Levels of EPA hydroxyl products, 4-HDoHE, 17,18-EEQ, 17,18-DiHETE, TXB2, and LXA4 were significantly changed in both mouse samples, and those of 2-series PGs, EDPs and DHA hydroxyl products were changed in aorta samples. Correlation network analysis of mouse plasma data revealed that some eicosanoids had higher connection degree or betweenness centrality score than others after ω-3 PUFA supplementation. Eicosanoids in human plasma were profiled across five time points after ω-3 PUFA supplementation. Fuzzy c-mean clustering algorithm suggested that the time curves of eicosanoid activity could be described with three kinetic patterns: sustained upregulation, short-term upregulation, and downregulation. This is the first systematic profiling of eicosanoids with ω-3 PUFA supplementation. The highly specific eicosanoid metabolomic and related data analysis methods would be powerful tools for comprehensive eicosanoid study.

Low molecular weight hyaluronan activates cytosolic phospholipase A2α and eicosanoid production in monocytes and macrophages.

Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation, there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from the cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release, and subsequent eicosanoid production and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time- and dose-dependent manner, induced cPLA2α, ERK1/2, p38, and JNK phosphorylation, as well as activated COX2 expression and prostaglandin (PG) E2 production in primary human monocytes, murine RAW 264.7, and wild-type bone marrow-derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MYD88, RHAMM or STAB2 siRNA-transfected macrophages and monocytes, we found that AA release, cPLA2α, ERK1/2, p38, and JNK phosphorylation, COX2 expression, and PGE2 production were activated by LMW HA through a TLR4/MYD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4(-/-) and Myd88(-/-) mice, but not in Cd44(-/-) mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated the M1 macrophage phenotype with the unique cPLA2α/COX2(high) and COX1/ALOX15/ALOX5/LTA4H(low) gene and PGE2/PGD2/15-HETE(high) and LXA4(low) eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.

Eicosanoid modulation by the short-chain fatty acid n-butyrate in human monocytes.

n-Butyrate deriving from bacterial fermentation in the mammalian intestine is a key determinant in gastrointestinal homeostasis. We examined the effects of this short-chain fatty acid and Toll-like receptor 2 (TLR) and TLR4 engagement on inflammatory/immunity-associated genes, cyclo-oxygenases (COXs), prostaglandins (PGs) and leukotrienes (LTs) in human monocytes. Before RNA isolation, freshly isolated human monocytes were co-incubated for different time-points with 1 mm n-butyrate alone or in combination with bacterial stimuli. Based on a knowledge-driven approach, a signature of 180 immunity/inflammation-associated genes was picked and real-time PCR analysis was performed. Pathway analysis was carried out using a web-based database analysing program. Based on these gene expression studies the findings were evaluated at the protein/mediator level by Western blot analysis, FACS and ELISA. Following co-incubation with n-butyrate and lipopolysaccharide, key enzymes of the eicosanoid pathway, like PTGS2 (COX-2), TXS, ALOX5, LTA4H and LTC4S, were significantly up-regulated compared with stimulation with lipopolysaccharide alone. Furthermore, release of the lipid mediators PGE(2), 15d-PGJ(2), LTB(4) and thromboxane B(2) was increased by n-butyrate. Regarding signalling, n-butyrate had no additional effect on mitogen-activated protein kinase and interfered differently with early and late phases of nuclear factor-κB signalling. Our results suggest that among many other mediators of eicosanoid signalling n-butyrate massively induces PGE(2) production by increasing the expression of PTGS2 (COX-2) in monocytes following TLR4 and TLR2 activation and induces secretion of LTB(4) and thromboxane B(2). This underscores the role of n-butyrate as a crucial mediator of gut-specific immunity.