Biosynthesis Of Lipid Mediators Research Articles

Altered eicosanoid production and phospholipid remodeling during cell culture

The remodeling of PUFAs by the Lands cycle is responsible for the diversity of phospholipid molecular species found in cells. There have not been detailed studies of the alteration of phospholipid molecular species as a result of serum starvation or depletion of PUFAs that typically occurs during tissue culture. The time-dependent effect of cell culture on phospholipid molecular species in RAW 264.7 cells cultured for 24, 48, or 72 h was examined by lipidomic strategies. These cells were then stimulated to produce arachidonate metabolites derived from the cyclooxygenase pathway, thromboxane B2, PGE2, and PGD2, and the 5-lipoxygenase pathway, leukotriene (LT)B4, LTC4, and 5-HETE, which decreased with increasing time in culture. However, the 5-lipoxygenase metabolites of a 20:3 fatty acid, LTB3, all trans-LTB3, LTC3, and 5-hydroxyeicosatrienoic acid, time-dependently increased. Molecular species of arachidonate containing phospholipids were drastically remodeled during cell culture, with a new 20:3 acyl group being populated into phospholipids to replace increasingly scarce arachidonate. In addition, the amount of TNFα induced by lipopolysaccharide stimulation was significantly increased in the cells cultured for 72 h compared with 24 h, suggesting that the remodeling of PUFAs enhanced inflammatory response. These studies supported the rapid operation of the Lands cycle to maintain cell growth and viability by populating PUFA species; however, without sufficient n-6 fatty acids, 20:3 n-9 accumulated, resulting in altered lipid mediator biosynthesis and inflammatory response.

Evolution and Expansion of the Prokaryote-Like Lipoxygenase Family in the Brown Alga Saccharina japonica.

Lipoxygenase (LOX) plays important roles in fatty acid oxidation and lipid mediator biosynthesis. In this study, we give first insights into brown algal LOX evolution. Whole genome searches revealed four, three, and eleven LOXs in Ectocarpus siliculosus, Cladosiphon okamuranus, and Saccharina japonica, respectively. In phylogenetic analyses, LOXs from brown algae form a robust clade with those from prokaryotes, suggesting an ancestral origin and slow evolution. Brown algal LOXs were divided into two clades, C1 and C2 in a phylogenetic tree. Compared to the two species of Ectocarpales, LOX gene expansion occurred in the kelp S. japonica through tandem duplication and segmental duplication. Selection pressure analysis showed that LOX genes in brown algae have undergone strong purifying selection, while the selective constraint in the C2 clade was more relaxed than that in the C1 clade. Furthermore, within each clade, LOXs of S. japonica evolved under more relaxed selection constraints than E. siliculosus and C. okamuranus. Structural modeling showed that unlike LOXs of plants and animals, which contain a β barrel in the N-terminal part of the protein, LOXs in brown algae fold into a single domain. Analysis of previously published transcriptomic data showed that LOXs in E. siliculosus are responsive to hyposaline, hypersaline, oxidative, and copper stresses. Moreover, clear divergence of expression patterns was observed among different life stages, as well as between duplicate gene pairs. In E. siliculosus, all four LOXs are male-biased in immature gametophytes, and mature gametophytes showed significantly higher LOX mRNA levels than immature gametophytes and sporophytes. In S. japonica, however, our RNA-Seq data showed that most LOXs are highly expressed in sporophytes. Even the most recently duplicated gene pairs showed divergent expression patterns, suggesting that functional divergence has likely occurred since LOX genes duplicated, which potentially contributes to the production of various oxylipins in brown algae.

Omega-3 fatty acids in atherosclerosis and coronary artery disease.

Omega-3 polyunsaturated fatty acids have emerged as possible protective factors associated with a decreased risk for myocardial infarction in populations with a high marine food intake, which may relate to effects on lipid metabolism, thrombosis and inflammation. Omega-3 fatty acids decrease triglyceride levels and also compete as substrates for enzymes involved in the biosynthesis of lipid mediators. The balance between omega-3-derived specialized proresolving mediators and pro-inflammatory lipid mediators from arachidonic acid metabolism can be measured as the resolvin-to-leukotriene ratio, which has been shown to predict subclinical atherosclerosis. The results of experimental, observational and randomized studies of omega-3 fatty acids are somewhat variable and should be interpreted in view of the models used and the populations studied.

Supplementation of linoleic acid (C18:2n-6) or α-linolenic acid (C18:3n-3) changes microbial agonist-induced oxylipid biosynthesis

Oxylipids are derived from polyunsaturated fatty acids (PUFA) in cellular membranes and the relative abundance or balance may contribute to disease pathogenesis. Previous studies documented unique oxylipid profiles from cows with either coliform or Streptococcus uberis mastitis, suggesting that lipid mediator biosynthesis may be dependent on the type of microbial-derived agonist. Changing the fatty acid content of peripheral blood leukocytes also may be critical to the relative expression of oxylipid profiles and the outcome of bacterial infection. No information is available in dairy cows describing how changing cellular PUFA content will modify oxylipids in the context of a microbial agonist challenge. Therefore, the hypothesis for the current study was that PUFA supplementation would change bovine leukocyte fatty acid content and respective oxylipid profiles from ex vivo microbial agonist-challenged leukocytes. Fatty acid content of leukocytes and plasma was quantified in (1) samples from cows not supplemented with PUFA, (2) cows supplemented with linoleic acid (LnA), and (3) cows supplemented with α-linolenic acid (ALA). Plasma oxylipids were assessed after S. uberis or lipopolysaccharide exposure and was compared with unstimulated oxylipid profiles. Fatty acid supplementation with ALA significantly increased ALA content of blood leukocytes and plasma relative to LnA. Fatty acid supplementation affected several S. uberis-induced oxylipids, but only S. uberis-induced 15-oxoETE was greater with ALA supplementation compared with LnA. Notably, only LPS-induced 5,6 LXA4 was altered with fatty acid supplementation, but no significant effect of LnA vs. ALA treatment was identified. Future studies are needed to understand how leukocyte activation and membrane PUFA availability collectively contribute to differential oxylipid profiles.

Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase.

Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.

Carbon Monoxide Improves Efficacy of Mesenchymal Stromal Cells During Sepsis by Production of Specialized Proresolving Lipid Mediators.

Mesenchymal stromal cells are being investigated as a cell-based therapy for a number of disease processes, with promising results in animal models of systemic inflammation and sepsis. Studies are ongoing to determine ways to further improve the therapeutic potential of mesenchymal stromal cells. A gas molecule that improves outcome in experimental sepsis is carbon monoxide. We hypothesized that preconditioning of mesenchymal stromal cells with carbon monoxide ex vivo would promote further therapeutic benefit when cells are administered in vivo after the onset of polymicrobial sepsis in mice. Animal study and primary cell culture. Laboratory investigation. BALB/c mice. Polymicrobial sepsis was induced by cecal ligation and puncture. Mesenchymal stromal cells, mesenchymal stromal cells-conditioned with carbon monoxide, fibroblasts, or fibroblasts-conditioned with carbon monoxide were delivered by tail vein injections to septic mice. The mice were assessed for survival, bacterial clearance, and the inflammatory response during sepsis in each of the groups. Mesenchymal stromal cells were also assessed for their ability to promote bacterial phagocytosis by neutrophils, the production of specialized proresolving lipid mediators, and their importance for mesenchymal stromal cells function using gene silencing. Ex vivo preconditioning with carbon monoxide allowed mesenchymal stromal cells to be administered later after the onset of sepsis (6 hr), and yet maintain their therapeutic effect with increased survival. Carbon monoxide preconditioned mesenchymal stromal cells were also able to alleviate organ injury, improve bacterial clearance, and promote the resolution of inflammation. Mesenchymal stromal cells exposed to carbon monoxide, with docosahexaenoic acid substrate, produced specialized proresolving lipid mediators, particularly D-series resolvins, which promoted survival. Silencing of lipoxygenase pathways (5-lipoxygenase and 12/15-lipoxygenase), which are important enzymes for specialized proresolving lipid mediator biosynthesis, resulted in a loss of therapeutic benefit bestowed on mesenchymal stromal cells by carbon monoxide. Taken together, these data suggest that production of specialized proresolving lipid mediators contribute to improved mesenchymal stromal cell efficacy when exposed to carbon monoxide, resulting in an improved therapeutic response during sepsis.

Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease

In 1970, it was well accepted that the central role of lipids was in energy storage and metabolism, and it was assumed that amphipathic lipids simply served a passive structural role as the backbone of biological membranes. As a result, the scientific community was focused on nucleic acids, proteins, and carbohydrates as information-containing molecules. It took considerable effort until scientists accepted that lipids also “encode” specific and unique biological information and play a central role in cell signaling. Along with this realization came the recognition that the enzymes that act on lipid substrates residing in or on membranes and micelles must also have important signaling roles, spurring curiosity into their potentially unique modes of action differing from those acting on water-soluble substrates. This led to the creation of the concept of “surface dilution kinetics” for describing the mechanism of enzymes acting on lipid substrates, as well as the demonstration that lipid enzymes such as phospholipase A2 (PLA2) contain allosteric activator sites for specific phospholipids as well as for membranes. As our understanding of phospholipases advanced, so did the understanding that many of the lipids released by these enzymes are chiral information-containing signaling molecules; for example, PLA2 regulates the generation of precursors for the biosynthesis of eicosanoids and other bioactive lipid mediators of inflammation and resolution underlying disease progression. The creation of the LIPID MAPS initiative in 2003 and the ensuing development of the lipidomics field have revealed that lipid metabolites are central to human metabolism. Today lipids are recognized as key mediators of health and disease as we enter a new era of biomarkers and personalized medicine. This article is my personal “reflection” on these scientific advances.

Structure–activity relationship studies on 1-(2-oxopropyl)indole-5-carboxylic acids acting as inhibitors of cytosolic phospholipase A2α: Effect of substituents at the indole 3-position on activity, solubility, and metabolic stability

Cytosolic phospholipase A2α (cPLA2α) is a key enzyme in the biosynthesis of pro-inflammatory lipid mediators and therefore represents an attractive target for the development of new anti-inflammatory drugs. Recently, we have found that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (4) is a potent inhibitor of the enzyme. In this work, we evaluate the effect of butanoyl- and hexanoyl-substituents in position 3 of the indole scaffold of this compound bearing terminal groups of varying polarity. As a result, inhibitory potency was not affected considerably in most cases, while metabolic phase I and phase II in vitro stability and aqueous solubility could be influenced and modulated by the structural modifications performed.

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome

Clinical chorioamnionitis at term (TCC) is the most common obstetrical infliction diagnosed in labor and delivery units worldwide and is associated with a substantial increase in maternal and neonatal morbidity and mortality. This obstetrical complication is a heterogeneous condition, as only half of patients have detectable microorganisms in the amniotic cavity. Because bioactive lipids play a key role in the initiation and resolution of an inflammatory response, we aimed to characterize the amniotic fluid lipidome in patients with TCC. We studied the amniotic fluid of patients in the following groups: 1) spontaneous labor at term without clinical chorioamnionitis (TLB) and 2) spontaneous labor at term with clinical chorioamnionitis (TCC). The TCC group was subdivided into a) those with microbial invasion of the amniotic cavity (TCC-MIAC) and b) those without microbial invasion of the amniotic cavity (TCC-noMIAC). The amniotic fluid concentration of proinflammatory lipid mediators did not differ between patients in TLB with TCC. In contrast, concentration of lipids with anti-inflammatory/proresolution properties was significantly lower in all patients with TCC than in those with TLB. These results suggest that while proinflammatory lipid mediators are involved in infection-driven intra-amniotic inflammation, a relative deficiency of anti-inflammatory/proresolution lipid mediator biosynthesis is a characteristic of TCC.

Phytomedicine polypharmacology: Cancer therapy through modulating the tumor microenvironment and oxylipin dynamics.

Integrative approaches in cancer therapy have recently been extended beyond the induction of cytotoxicity to controlling the tumor microenvironment and modulating inflammatory cascades and pathways such as lipid mediator biosynthesis and their dynamics. Profiling of important lipid messengers, such as oxylipins, produced as part of the physiological response to pharmacological stimuli, provides a unique opportunity to explore drug pharmacology and the possibilities for molecular management of cancer physiopathology. Whereas single targeted chemotherapeutic drugs commonly lack efficacy and invoke drug resistance and/or adverse effects in cancer patients, traditional herbal medicines are seen as bright prospects for treating complex diseases, such as cancers, in a systematic and holistic manner. Understanding the molecular mechanisms of traditional medicine and its bioactive chemical constituents may aid the modernization of herbal remedies and the discovery of novel phytoagents for cancer management. In this review, systems-based polypharmacology and studies to develop multi-target drugs or leads from phytomedicines and their derived natural products that may overcome the problems of current anti-cancer drugs, are proposed and summarized.

Understanding the Mechanism of the Hydrogen Abstraction from Arachidonic Acid Catalyzed by the Human Enzyme 15-Lipoxygenase-2. A Quantum Mechanics/Molecular Mechanics Free Energy Simulation.

Lipoxygenases (LOXs) are a family of enzymes involved in the biosynthesis of several lipid mediators. In the case of human 15-LOX, the 15-LOX-1 and 15-LOX-2 isoforms show slightly different reaction regiospecificity and substrate specificity, indicating that substrate binding and recognition may be different, a fact that could be related to their different biological role. Here, we have used long molecular dynamics simulations, QM(DFT)/MM potential energy and free energy calculations (using the newly developed DHAM method), to investigate the binding mode of the arachidonic acid (AA) substrate into 15-LOX-2 and the rate-limiting hydrogen-abstraction reaction 15-LOX-2 catalyzes. Our results strongly indicate that hydrogen abstraction from C13 in 15-LOX-2 is only consistent with the "tail-first" orientation of AA, with its carboxylate group interacting with Arg429, and that only the pro-S H13 hydrogen will be abstracted (being the pro-R H13 and H10 too far from the acceptor oxygen atom). At the B3LYP/6-31G(d) level the potential and free energy barriers for the pro-S H13 abstraction of AA by 15-LOX-2 are 18.0 and 18.6 kcal/mol, respectively. To analyze the kinetics of the hydrogen abstraction process, we determined a Markov model corresponding to the unbiased simulations along the state-discretized reaction coordinate. The calculated rates based on the second largest eigenvalue of the Markov matrices agree well with experimental measurements, and also provide the means to directly determine the pre-exponential factor for the reaction by comparing with the free energy barrier height. Our calculated pre-exponential factor is close to the value of kBT/h. On the other hand, our results suggest that the spin inversion of the complete system (including the O2 molecule) that is required to happen at some point along the full process to lead to the final hydroperoxide product, is likely to take place during the hydrogen transfer, which is a proton coupled electron transfer. Overall, a different binding mode from the one accepted for 15-LOX-1 is proposed, which provides a molecular basis for 15-LOX-2 exclusive 15-HPETE production in front of the double (although highly 15-) 12/15 regiospecificity of 15-LOX-1. Understanding how these different isoenzymes achieve their regiospecificity is expected to help in specific inhibitor design.

Gene Expression of Proresolving Lipid Mediator Pathways Is Associated With Clinical Outcomes in Trauma Patients.

Specialized proresolving lipid mediators have emerged as powerful modulators of inflammation and activators of resolution. Animal models show significant benefits of specialized proresolving lipid mediators on survival and wound healing after major burn trauma. To date, no studies have investigated specialized proresolving lipid mediators and their relation to other lipid mediator pathways in humans after trauma. Here we determine if patients with poor outcomes after trauma have dysregulated lipid mediator pathways. We studied blood leukocyte expression of 18 genes critical to the synthesis, signaling, and metabolism of specialized proresolving lipid mediators and proinflammatory lipid mediators, and we correlated these expression patterns with clinical outcomes in trauma patients from the Inflammation and the Host Response to Injury study. Seven U.S. medical trauma centers. Ninety-six patients enrolled in the Inflammation and Host Response to Injury study, after blunt trauma and unambiguously classified as having uncomplicated or complicated recoveries. Twenty-eight healthy volunteers were enrolled as controls. None. Within each patient, the 18 genes of interest were used to calculate scores for distinct families of lipid mediators, including resolvins, lipoxins, prostaglandins, and leukotrienes, as well as leukotriene to resolvin score ratios. Scores were built using a simple weighting scheme, taking into consideration both dependent and independent activities of enzymes and receptors responsible for lipid mediator biosynthesis and function. Individually, ALOX12, PTGS2, PTGES, PTGDS, ALOX5AP, LTA4H, FPR2, PTGER2, LTB4R, HPGD, PTGR1, and CYP4F3 were expressed differentially over 28 days posttrauma between patients with uncomplicated and complicated recoveries (p < 0.05). When all genes were combined into scores, patients with uncomplicated recoveries had differential and higher resolvin scores (p < 0.001) and lower leukotriene scores (p < 0.001). A final combined ratio was calculated for each patient, and posttrauma leukotriene score to resolvin score ratios were significantly lower in patients with uncomplicated clinical courses (p < 0.001). proresolving lipid mediator lipidomics and/or protein expression, and identifying associated therapeutic targets, may influence the clinical management of trauma patients.

Polyunsaturated fatty acids influence differential biosynthesis of oxylipids and other lipid mediators during bovine coliform mastitis

Coliform mastitis is a severe and sometimes fatal disease characterized by an unregulated inflammatory response. The initiation, progression, and resolution of inflammatory responses are regulated, in part, by potent oxylipid metabolites derived from polyunsaturated fatty acids. The purpose of this study was to characterize the biosynthesis and diversity of oxylipid metabolites during acute bovine coliform mastitis. Eleven cows diagnosed with naturally occurring acute systemic coliform mastitis and 13 healthy control cows, matched for lactation number and days in milk, were selected for comparison of oxylipid and free fatty acid concentrations in both milk and plasma. Oxylipids and free fatty acids were quantified using liquid chromatography-tandem mass spectrometry. All polyunsaturated fatty acids quantified in milk were elevated during coliform mastitis with linoleic acid being the most abundant. Oxylipids synthesized through the lipoxygenase and cytochrome P450 pathways accounted for the majority of the oxylipid biosynthesis. This study demonstrated a complex and diverse oxylipid network, most pronounced at the level of the mammary gland. Substrate availability, biosynthetic pathways, and degree of metabolism influence the biosynthesis of oxylipids during bovine coliform mastitis. Further studies are required to identify targets for novel interventions that modulate oxylipid biosynthesis during coliform mastitis to optimize inflammation.

Polyunsaturated fatty acid-phospholipid remodeling and inflammation.

To highlight some of the recent advances related to the control of polyunsaturated fatty acid (PUFA) incorporation and remodeling in membrane glycerophospholipids in inflammatory cells. Several enzymes have recently been identified that are associated with the control of PUFA incorporation and remodeling into membrane phospholipids and their release. The functional roles of the different enzyme isotypes in the control of PUFA availability for lipid mediator biosynthesis and cell signaling are only now being established. The expression of specific acyl-CoA synthetase, lysophospholipid acyltransferase and phospholipase A2 isotypes has recently been shown to have an impact on membrane PUFA content, on the production of lipid mediators and on inflammation. A better understanding of the complex processes associated with the control PUFA remodeling in membrane phospholipids may lead to the discovery of new therapeutic targets for the treatment of inflammatory diseases.

Effect of pulsed electromagnetic field treatment on programmed resolution of inflammation pathway markers in human cells in culture.

Inflammation is a complex process involving distinct but overlapping biochemical and molecular events that are highly regulated. Pulsed electromagnetic field (PEMF) therapy is increasingly used to treat pain and edema associated with inflammation following surgery involving soft tissue. However, the molecular and cellular effects of PEMF therapy on pathways involved in the resolution of inflammation are poorly understood. Using cell culture lines relevant to trauma-induced inflammation of the skin (human dermal fibroblasts, human epidermal keratinocytes, and human mononuclear cells), we investigated the effect of PEMF on gene expression involved in the acute and resolution phases of inflammation. We found that PEMF treatment was followed by changes in the relative amount of messenger (m)RNAs encoding enzymes involved in heme catabolism and removal of reactive oxygen species, including an increase in heme oxygenase 1 and superoxide dismutase 3 mRNAs, in all cell types examined 2 hours after PEMF treatment. A relative increase in mRNAs encoding enzymes involved in lipid mediator biosynthesis was also observed, including an increase in arachidonate 12- and 15-lipoxygenase mRNAs in dermal fibroblasts and epidermal keratinocytes, respectively. The relative amount of both of these lipoxygenase mRNAs was elevated in mononuclear cells following PEMF treatment relative to nontreated cells. PEMF treatment was also followed by changes in the mRNA levels of several cytokines. A decrease in the relative amount of interleukin 1 beta mRNA was observed in mononuclear cells, similar to that previously reported for epidermal keratinocytes and dermal fibroblasts. Based on our results, we propose a model in which PEMF therapy may promote chronic inflammation resolution by mediating gene expression changes important for inhibiting and resolving inflammation.

Deciphering of mitochondrial cardiolipin oxidative signaling in cerebral ischemia-reperfusion.

It is believed that biosynthesis of lipid mediators in the central nervous system after cerebral ischemia-reperfusion starts with phospholipid hydrolysis by calcium-dependent phospholipases and is followed by oxygenation of released fatty acids (FAs). Here, we report an alternative pathway whereby cereberal ischemia-reperfusion triggered oxygenation of a mitochondria-specific phospholipid, cardiolipin (CL), is followed by its hydrolysis to yield monolyso-CLs and oxygenated derivatives of fatty (linoleic) acids. We used a model of global cerebral ischemia-reperfusion characterized by 9 minutes of asphyxia leading to asystole followed by cardiopulmonary resuscitation in postnatal day 17 rats. Global ischemia and cardiopulmonary resuscitation resulted in: (1) selective oxidation and hydrolysis of CLs, (2) accumulation of lyso-CLs and oxygenated free FAs, (3) activation of caspase 3/7 in the brain, and (4) motor and cognitive dysfunction. On the basis of these findings, we used a mitochondria targeted nitroxide electron scavenger, which prevented CL oxidation and subsequent hydrolysis, attenuated caspase activation, and improved neurocognitive outcome when administered after cardiac arrest. These data show that calcium-independent CL oxidation and subsequent hydrolysis represent a previously unidentified pathogenic mechanism of brain injury incurred by ischemia-reperfusion and a clinically relevant therapeutic target.

A mitochondrial pathway for biosynthesis of lipid mediators.

The central role of mitochondria in metabolic pathways and in cell death mechanisms requires sophisticated signaling systems. Essential in this signaling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin, are oxidized by the intermembrane space hemoprotein, cytochrome c. We show that an assortment of oxygenated cardiolipin species undergoes phospholipase A2-catalyzed hydrolysis thus generating multiple oxygenated fatty acids, including well known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway including oxidation of polyunsaturated cardiolipins and accumulation of their hydrolysis products – oxygenated linoleic, arachidonic acids and monolyso-cardiolipins – is activated in vivo after acute tissue injury.

Mechanisms of human eosinophil migration induced by the combination of IL-5 and the endocannabinoid 2-arachidonoyl-glycerol

Mechanisms of human eosinophil migration induced by the combination of IL-5 and the endocannabinoid 2-arachidonoyl-glycerol

Dietary Polyunsaturated Fatty Acids and Inflammation: The Role of Phospholipid Biosynthesis

The composition of fatty acids in the diets of both human and domestic animal species can regulate inflammation through the biosynthesis of potent lipid mediators. The substrates for lipid mediator biosynthesis are derived primarily from membrane phospholipids and reflect dietary fatty acid intake. Inflammation can be exacerbated with intake of certain dietary fatty acids, such as some ω-6 polyunsaturated fatty acids (PUFA), and subsequent incorporation into membrane phospholipids. Inflammation, however, can be resolved with ingestion of other fatty acids, such as ω-3 PUFA. The influence of dietary PUFA on phospholipid composition is influenced by factors that control phospholipid biosynthesis within cellular membranes, such as preferential incorporation of some fatty acids, competition between newly ingested PUFA and fatty acids released from stores such as adipose, and the impacts of carbohydrate metabolism and physiological state. The objective of this review is to explain these factors as potential obstacles to manipulating PUFA composition of tissue phospholipids by specific dietary fatty acids. A better understanding of the factors that influence how dietary fatty acids can be incorporated into phospholipids may lead to nutritional intervention strategies that optimize health.

Contributions of the Three CYP1 Monooxygenases to Pro-Inflammatory and Inflammation-Resolution Lipid Mediator Pathways

All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry-based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(⁻/⁻) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid-derived leukotriene B₄ (LTB₄) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid-derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid-derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB₄ levels were shown to parallel increased neutrophil numbers, and 20-hydroxy-LTB₄ formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.