Endogenous Bioactive Lipids Research Articles

Vaccination with DHA-derived specialized proresolving mediators increases the antibody-mediated immune response against influenza virus (VAC2P.925)

Abstract Influenza viruses remain a critical global health concern. More efficacious vaccines are needed to protect against influenza virus, yet few adjuvants are approved for routine use. Specialized proresolving mediators (SPMs) are powerful endogenous bioactive lipid regulators of inflammation, with great clinical translational properties. We previously identified endogenous levels of the SPM resolvin D1, protectin D1 and 17-hydroxydocosahexaenoic acid (17-HDHA) in the mouse spleen. Here, we investigated the ability of 17-HDHA to enhance the adaptive immune response using a pre-clinical influenza vaccination mouse model. Our findings revealed that mice immunized with influenza H1N1-derived HA protein plus 17-HDHA increased antigen specific antibody titers. 17-HDHA increased the number of plasma cells and HA-specific antibody-secreting cells in the bone marrow, but it did not affect the frequency of GC B cells or Tfh cells in the draining lymph node. Importantly, the 17-HDHA-mediated increased antibody production was more protective against live pH1N1 influenza virus infection in mice. This is the first report on the immuno-stimulatory effects of omega-3-derived SPMs on the humoral immune response. These findings illustrate a previously unknown biological relation between proresolution signals and the adaptive immune system. In addition, 17-HDHA as well as other immuno-stimulatory SPM have the potential to be used as novel endogenous adjuvants.

Lipoxin A4 modulates adaptive immunity by decreasing memory B‐cell responses via an ALX/FPR2‐dependent mechanism

Specialized proresolving mediators are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. Lipoxins and other specialized proresolving mediators have been identified in important immunological tissues including bone marrow, spleen, and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A4 (LXA4) and its receptor ALX/FPR2 on human and mouse B cells. LXA4 decreased IgM and IgG production on activated human B cells through ALX/FPR2‐dependent signaling, which downregulated NF‐κB p65 nuclear translocation. LXA4 also inhibited human memory B‐cell antibody production and proliferation, but not naïve B‐cell function. Lastly, LXA4 decreased antigen‐specific antibody production in an OVA immunization mouse model. To our knowledge, this is the first description of the actions of lipoxins on human B cells, demonstrating a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B‐cell antibody production can be beneficial to threat inflammatory and autoimmune disorders.

Implication of the anti-inflammatory bioactive lipid prostaglandin D2-glycerol ester in the control of macrophage activation and inflammation by ABHD6

Proinflammatory macrophages are key mediators in several pathologies; thus, controlling their activation is necessary. The endocannabinoid system is implicated in various inflammatory processes. Here we show that in macrophages, the newly characterized enzyme α/β-hydrolase domain 6 (ABHD6) controls 2-arachidonoylglycerol (2-AG) levels and thus its pharmacological effects. Furthermore, we characterize a unique pathway mediating the effects of 2-AG through its oxygenation by cyclooxygenase-2 to give rise to the anti-inflammatory prostaglandin D2-glycerol ester (PGD2-G). Pharmacological blockade of cyclooxygenase-2 or of prostaglandin D synthase prevented the effects of increasing 2-AG levels by ABHD6 inhibition in vitro, as well as the 2-AG-induced increase in PGD2-G levels. Together, our data demonstrate the physiological relevance of the interaction between the endocannabinoid and prostanoid systems. Moreover, we show that ABHD6 inhibition in vivo allows for fine-tuning of 2-AG levels in mice, therefore reducing lipopolysaccharide-induced inflammation, without the characteristic central side effects of strong increases in 2-AG levels obtained following monoacylglycerol lipase inhibition. In addition, administration of PGD2-G reduces lipopolysaccharide-induced inflammation in mice, thus confirming the biological relevance of this 2-AG metabolite. This points to ABHD6 as an interesting therapeutic target that should be relevant in treating inflammation-related conditions, and proposes PGD2-G as a bioactive lipid with potential anti-inflammatory properties in vivo.

Lipoxin A4 decreases human memory B cell antibody production via an ALX/FPR2-dependent mechanism: A link between resolution signals and adaptive immunity (P5151)

Abstract Specialized proresolving mediators (SPMs) are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. SPMs are classified into lipoxins, resolvins, protectins and maresins. Lipoxins and other SPM have been identified in immunological tissues including bone marrow, spleen and blood. Lipoxins regulate functions of the innate immune system such as monocyte recruitment and inhibition of neutrophil infiltration to the site of inflammation. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we discovered that B cells express the lipoxin A4 (LXA4) receptor, ALX/FPR2, and upregulate its expression following mitogen activation. Furthermore, LXA4 decreased IgM and IgG production on activated B cells through an ALX/FPR2-dependent mechanism. This caused a reduction in NF-κB p65 nuclear translocation. We found that memory B cells express higher levels of ALX/FPR2 compared to naïve B cells. Interestingly, LXA4 inhibited human memory B cell antibody production and proliferation, but not naïve B cell function. LXA4 also decreased antigen-specific antibody production in vivo. To our knowledge, this is the first description of the effects of lipoxins on B cells, which provides a link between resolution signals and adaptive immunity. The ability of lipoxins to decrease memory B cell antibody production could be particularly beneficial to threat chronic inflammatory diseases and autoimmune disorders.

Update on the endocannabinoid-mediated regulation of gelatinase release in arterial wall physiology and atherosclerotic pathophysiology

Endocannabinoids are endogenous bioactive lipids ubiquitously distributed in several tissues (e.g., brain, adipose tissue, liver, heart and arterial vessels), which play a crucial role in atherosclerosis. Endocannabinoids have been shown to promote cell homeostasis and modulate inflammatory bioactivities mainly via the binding to transmembrane receptors (called cannabinoid type 1 and cannabinoid type 2 receptors, respectively). Although other cannabinoid receptors have been recently identified and shown to play a crucial role in cardiovascular pathophysiology, so far, the pharmacological targeting of both cannabinoid type 1 and cannabinoid type 2 receptors has been described as a promising therapeutic target in atherogenesis and associated inflammatory processes. In particular, endocannabinoids have been shown to modulate the release and activation of matrix degrading enzymes (i.e., matrix metalloproteinases [MMPs]) increasing intraplaque vulnerability. In this article the authors describe the pivotal regulatory activity of the endocannabinoid system on gelatinase (MMP-2 and -9) bioactivity in the arterial wall physiology and pathophysiology.

N-Acylated phospholipid metabolism and seedling growth

N-Acylphosphatidylethanolamines (NAPEs) are precursors of endogenous bioactive lipids, N-acylethanolamines (NAEs). NAPEs, which occur as a minor membrane lipid, are hydrolyzed in a single enzymatic step catalyzed by a type of phospholipase D (PLD) to generate fatty acid ethanolamides. Although, the occurrence of NAPE is widespread in the plant kingdom, the physiological roles remain under appreciated due to the lack of sensitive tools to quantify the pathway metabolites. In Kilaru et al. (2012, Planta, DOI 10.1007/s00425-012-1669-z), comprehensive mass spectrometry (MS)-based methods were developed to gain a clearer understanding of the complex network of metabolites that participate in NAE metabolic pathway. This targeted lipidomics approach allowed insights to be drawn into the implications of altered NAE levels on NAPE content and composition, and the overall regulation of PLD-mediated hydrolysis in Arabidopsis. Based on these results, we point out here the important need for the identification of the precise isoform(s) of PLD in plants that is (are) involved in the regulated hydrolysis of NAPE and formation of NAE lipid mediators in vivo.

Interplay of cannabinoid 2 (CB2) receptors with nitric oxide synthases, oxidative and nitrative stress, and cell death during remote neurodegeneration

Remote neuronal degeneration and death/injury, which often occur in regions remote but functionally connected to the primary lesion site, may play a pivotal role in extending neuronal damage/dysfunction following traumatic brain injury, stroke, or peripheral nerve injury, as well as in chronic neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Even though the precise mechanisms of remote neuronal injury are poorly understood and no efficacious treatment options are available, it involves glial activation, inflammation, oxidative/nitrative stress, and apoptotic cell death. The newly discovered endocannabinoid signaling system consisting of endocannabinoids (endogenous bioactive lipid mediators), their synthetic and metabolizing enzymes, and their primary G protein-coupled cannabinoid 1 and 2 (CB(1) and CB(2)) receptors has been implicated in the regulation of numerous physiological and pathological processes/functions, including those associated with neurodegeneration. Using a well-characterized rodent model of remote neuronal degeneration, Oddi et al. (J Mol Med 2012, in press, DOI 10.1007/s00109-012-0884-1 ) have demonstrated that targeting CB(2) cannabinoid receptors may represent a promising novel approach to attenuate this pathological process. This editorial discusses the clinical significance of these interesting observations and the mechanisms of the possible interplay of CB(2) receptors with nitric oxide synthases, oxidative and nitrative stress, and cell death during remote neurodegeneration.

The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus laevis

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids which mediate a variety of biological cell responses such as cell proliferation, migration, differentiation and apoptosis. Their actions are mediated by binding to the G-protein-coupled endothelial differentiation gene (Edg) receptor subfamily, referred to as S1P1-5 and LPA1-5, and regulate a variety of signalling pathways involved in numerous physiological processes and pathological conditions. Their importance during embryogenesis has been demonstrated by the generation of knock-out mice and specific roles have been assigned to these receptors. However, potential functional redundancy and the lethality of some mutants have complicated functional analysis in these models. Here we report the cloning of the S1P and LPA receptors in Xenopus laevis and tropicalis. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. We have conducted a comparative expression analysis of these receptors during development and in the adult frog, by both RT-PCR and whole mount in situ hybridisation. In particular, we show that S1P1, 2 and 5 display distinct embryonic specific expression patterns, suggesting potentially different developmental roles for these receptors, and therefore for their ligands, during amphibian embryogenesis.

Effect of synthetic and natural phospholipids on N-acylphosphatidylethanolamine-hydrolyzing phospholipase D activity

N-Acylethanolamines (NAEs) constitute a family of endogenous bioactive lipids that includes arachidonoylethanolamide (anandamide), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). These lipids are formed from their respective N-acylated ethanolamine phospholipid (NAPE) precursor by the action of a phospholipase D enzyme (NAPE-PLD). Anandamide, OEA, and PEA are all bioactive lipids that may influence, amongst others: neuroinflammation, food intake, and oocyte implantation. Here we have synthesized a number of NAPE analogues with variation in the phosphoester structure. The NAPE analogues as well as selected phospholipids and beta-lactamase substrates were tested as potential modifiers of cloned human NAPE-PLD in an enzyme assay involving a 14C-labeled diether-NAPE substrate. One hit was identified, namely 1,2-dihexanoyl-glycero- N-(3-(tetradecanoylamino)propyl)phosphoramidate (AHP-71B) which showed inhibitory activity and may serve as template for further structure–activity developments. Furthermore, it was found that NAPE-PLD was activated by phosphatidylethanolamine and inhibited by the beta-lactamase substrate nitrocefin.

The emerging role of the endocannabinoid system in cardiovascular disease

Endocannabinoids are endogenous bioactive lipid mediators present both in the brain and various peripheral tissues, which exert their biological effects via interaction with specific G-protein-coupled cannabinoid receptors, the CB(1) and CB(2). Pathological overactivation of the endocannabinoid system (ECS) in various forms of shock and heart failure may contribute to the underlying pathology and cardiodepressive state by the activation of the cardiovascular CB(1) receptors. Furthermore, tonic activation of CB(1) receptors by endocannabinoids has also been implicated in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes, such as plasma lipid alterations, abdominal obesity, hepatic steatosis, inflammation, and insulin and leptin resistance. In contrast, activation of CB(2) receptors in immune cells exerts various immunomodulatory effects, and the CB(2) receptors in endothelial and inflammatory cells appear to limit the endothelial inflammatory response, chemotaxis, and inflammatory cell adhesion and activation in atherosclerosis and reperfusion injury. Here, we will overview the cardiovascular actions of endocannabinoids and the growing body of evidence implicating the dysregulation of the ECS in a variety of cardiovascular diseases. We will also discuss the therapeutic potential of the modulation of the ECS by selective agonists/antagonists in various cardiovascular disorders associated with inflammation and tissue injury, ranging from myocardial infarction and heart failure to atherosclerosis and cardiometabolic disorders.

Endogenous bioactive lipids and the regulation of conventional outflow facility

Perturbation of paracrine signaling within the human conventional outflow pathway influences tissue homeostasis and outflow function. For example, exogenous introduction of the bioactive lipids, sphingosine-1-phosphate, anandamide or prostaglandin F2α, to conventional outflow tissues alters the rate of drainage of aqueous humor through the trabecular meshwork and into Schlemm’s canal. This review summarizes recent data that characterize endogenous bioactive lipids, their receptors and associated signaling partners in the conventional outflow tract. We also discuss the potential of targeting such signaling pathways as a strategy for the development of therapeutics to treat ocular hypertension and glaucoma.

Molecular characterization of a novel lysosomal enzyme degrading the anti-inflammatory lipid mediator N-acylethanolamine

N-Acylethanolamines (NAEs) represent a class of endogenous bioactive lipids generated from glycerophospholipids, and they include N-palmitoylethanolamine (an anti-inflammatory substance) and N-arachidonoylethanolamine (anandamide, an endogenous ligand of cannabinoid receptor). It is generally accepted that NAEs are hydrolyzed to free fatty acids and ethanolamine by the catalysis of fatty acid amide hydrolase (FAAH) acting principally at alkaline and neutral pH. Several years ago, our laboratory found another enzyme catalyzing the same reaction only at acidic pH. However, the molecular cloning and detailed analysis of this enzyme, termed “N-acylethanolamine-hydrolyzing acid amidase (NAAA)”, have not yet been performed. In the present study, we purified the enzyme from rat lung, and cloned its cDNA from human, mouse, and rat. The primary structures revealed that NAAA had no homology to FAAH, but revealed 33-35% amino acid identity to lysosomal acid ceramidase, which hydrolyzes ceramide to free fatty acids and sphingosine at acidic pH. Recombinant human NAAA hydrolyzed various NAEs, and exhibited the highest activity toward N-palmitoylethanolamine. In addition, a low ceramide-hydrolyzing activity was detected with NAAA. We also observed a lysosome-like distribution of NAAA-green fluorescence protein fusion protein, as expressed in HEK293 cells. The organ distribution of mRNA and enzyme activity in rats revealed its wide distribution with the highest level in lung. Interestingly, macrophages expressed NAAA abundantly, suggesting a role of this enzyme in inflammation. By using an NAAA-selective inhibitor which we developed, we showed that NAAA and FAAH cooperatively degraded NAEs in macrophages. Taken together, our results indicated that NAAA is a novel lysosomal enzyme having structural and functional similarity to acid ceramidase, and functions as a second NAE hydrolase in mammalian tissues.

Determination of endogenous tissue inflammation profiles by LC/MS/MS: COX- and LOX-derived bioactive lipids

Cyclooxygenase and lipoxygenase arachidonate products, including prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), are known to modulate inflammation within tissues and can serve as important etiologic factors in carcinogenesis. Eicosanoid content in tissues is typically determined either as a single molecular species through antibody-based assays or by high-performance liquid chromatography after addition of an exogenous substrate such as arachidonic acid. Unfortunately, the methods currently in use are either time-consuming or complicated. Here we report a method for simultaneously identifying eicosanoids appearing as endogenous bioactive lipids in in vivo settings using LC/MS/MS. The analyses indicate marked differences in endogenous eicosanoid content between malignant tissue types suggesting a need for selective therapeutic approaches. As a demonstration of the utility of the method, we present data to show that the technique can be used to distinguish eicosapentaenoic acid-derived formation of PGE 3 from PGE 2 in murine prostate tissue. The method has also been applied to an examination of endogenous eicosanoid metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer in hamsters demonstrating the inflammatory nature of this type of cancer with elevated levels of both PGE 2 and LTB 4. In addition, the concentration of the eicosanoid 13-hydroxyoctadecadienoic acid was 67.6% lower in DMBA treated specimens than in control specimens. Thus, our method provides a powerful tool for measuring modulation of eicosanoid metabolites in various preclinical and clinical tissues and may be useful in studies of the endogenous changes in eicosanoid metabolism at various stages of cancer development.

High-Throughput Screening for the Discovery of Inhibitors of Fatty Acid Amide Hydrolase Using a Microsome-Based Fluorescent Assay

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.

Lysophospholipids in development: Miles apart and edging in.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids that participate in the regulation of mammalian cell proliferation, apoptosis, migration, and angiogenesis. These processes are each critical for successful embryogenesis, raising the possibility that lysophospholipid signaling may contribute to normal animal development. In fact, recent studies in developmental model systems have established that S1P and LPA are necessary for diverse developmental programs including those required for morphogenesis of vertebrate reproductive, cardiovascular and central and peripheral nervous systems (PNS), as well as the establishment of maternal-fetal circulation and the immune system. Genetic, morphological, and biochemical characterization of developmental model systems offer powerful approaches to elucidating the molecular mechanisms of lysophospholipid signaling and its contributions to animal development and postnatal physiology. In this review, the routes of S1P and LPA metabolism and our current understanding of lysophospholipid-mediated signal transduction in mammalian cells will be summarized. The evidence implicating lysophospholipid signaling in the development of specific vertebrate systems will then be reviewed, with an emphasis on signals mediated through G protein-coupled receptors of the Edg family. Lastly, recent insights derived from the study of simple metazoan models and implications regarding lysophospholipid signaling in organisms in which Edg receptors are not conserved will be explored.