Arachidonic Acid-derived Lipid Mediators Research Articles

Effect of FADS1 SNPs rs174546, rs174547 and rs174550 on blood fatty acid profiles and plasma free oxylipins.

Previous studies have indicated that activity of fatty acid desaturase 1 (FADS1), is involved in cardiometabolic risk. Recent experimental data have shown that FADS1 knockdown can promote lipid accumulation and lipid droplet formation in liver cells. In this study, we aimed to characterize whether different FADS1 genotypes affect liver fat content, essential fatty acid content and free oxylipin mediators in the blood. We analyzed the impact of FADS1 single-nucleotide polymorphisms (SNPs) rs174546, rs174547, and rs174550 on blood fatty acids and free oxylipins in a cohort of 85 patients from an academic metabolic medicine outpatient center. Patients were grouped based on their genotype into the homozygous major (derived) allele group, the heterozygous allele group, and the homozygous minor (ancestral) allele group. Omega-3 polyunsaturated fatty acids (n-3 PUFA) and omega-6 polyunsaturated fatty acids (n-6 PUFA) in the blood cell and plasma samples were analyzed by gas chromatography. Free Oxylipins in plasma samples were analyzed using HPLC-MS/MS. Liver fat content and fibrosis were evaluated using Fibroscan technology. Patients with the homozygous ancestral (minor) FADS1 genotype exhibited significantly lower blood levels of the n-6 PUFA arachidonic acid (AA), but no significant differences in the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). There were no significant differences in liver fat content or arachidonic acid-derived lipid mediators, such as thromboxane B2 (TXB2), although there was a trend toward lower levels in the homozygous ancestral genotype group. Our findings suggest that FADS1 genotypes influence the blood levels of n-6 PUFAs, while not significantly affecting the n-3 PUFAs EPA and DHA. The lack of significant differences in liver fat content and arachidonic acid-derived lipid mediators suggests that the genotype-related variations in fatty acid levels may not directly translate to differences in liver fat or inflammatory lipid mediators in this cohort. However, the trend towards lower levels of certain lipid mediators in the homozygous ancestral genotype group warrants further investigation to elucidate the underlying mechanisms of different FADS1 genotypes and potential implications for cardiometabolic risk.

Association of Arachidonic Acid-Derived Lipid Mediators With Disease Severity in Patients With Relapsing and Progressive Multiple Sclerosis.

Excessive activation of certain lipid mediator (LM) pathways plays a role in the complex pathogenesis of multiple sclerosis (MS). However, the relationship between bioactive LMs and different aspects of CNS-related pathophysiologic processes remains largely unknown. Therefore, in this study, we assessed the association of bioactive LMs belonging to the ω-3/ω-6 lipid classes with clinical and biochemical (serum neurofilament light [sNfL] and serum glial fibrillary acidic protein [sGFAP]) parameters and MRI-based brain volumes in patients with MS (PwMS) and healthy controls (HCs). A targeted high-performance liquid chromatography-tandem mass spectrometry approach was used on plasma samples of PwMS and HCs of the Project Y cohort, a cross-sectional population-based cohort that contains PwMS all born in 1966 in the Netherlands and age-matched HCs. LMs were compared between PwMS and HCs and were correlated with levels of sNfL, sGFAP, disability (Expanded Disability Status Scale [EDSS]), and brain volumes. Finally, significant correlates were included in a backward multivariate regression model to identify which LMs best related to disability. The study sample consisted of 170 patients with relapsing remitting MS (RRMS), 115 patients with progressive MS (PMS), and 125 HCs. LM profiles of patients with PMS significantly differed from those of patients with RRMS and HCs, particularly patients with PMS showed elevated levels of several arachidonic acid (AA) derivatives. In particular, 15-hydroxyeicosatetraenoic acid (HETE) (r = 0.24, p < 0.001) correlated (average r = 0.2, p < 0.05) with clinical and biochemical parameters such as EDSS and sNfL. In addition, higher 15-HETE levels were related to lower total brain (r = -0.24, p = 0.04) and deep gray matter volumes (r = -0.27, p = 0.02) in patients with PMS and higher lesion volume (r = 0.15, p = 0.03) in all PwMS. In PwMS of the same birth year, we show that ω-3 and ω-6 LMs are associated with disability, biochemical parameters (sNfL, GFAP), and MRI measures. Furthermore, our findings indicate that, particularly, in patients with PMS, elevated levels of specific products of the AA pathway, such as 15-HETE, associate with neurodegenerative processes. Our findings highlight the potential relevance of ω-6 LMs in the pathogenesis of MS.

Prostaglandin F2α regulates mitochondrial dynamics and mitophagy in the bovine corpus luteum.

Prostaglandins are arachidonic acid-derived lipid mediators involved in numerous physiological and pathological processes. PGF2α analogues are therapeutically used for regulating mammalian reproductive cycles and blood pressure, inducing term labor, and treating ocular disorders. PGF2α exerts effects via activation of calcium and PKC signaling, however, little is known about the cellular events imposed by PGF2α signaling. Here, we explored the early effects of PGF2α on mitochondrial dynamics and mitophagy in the bovine corpus luteum employing relevant and well characterized in vivo and in vitro approaches. We identified PKC/ERK and AMPK as critical protein kinases essential for activation of mitochondrial fission proteins, DRP1 and MFF. Furthermore, we report that PGF2α elicits increased intracellular reactive oxygen species and promotes receptor-mediated activation of PINK-Parkin mitophagy. These findings place the mitochondrium as a novel target in response to luteolytic mediator, PGF2α. Understanding intracellular processes occurring during early luteolysis may serve as a target for improving fertility.

Comprehensive elucidation of developmental functions of arachidonic acid-derived lipid mediators in zebrafish.

Prostaglandins (PGs) and leukotrienes (LTs) are major physiologically active lipid, which are biosynthesized from arachidonic acid (AA). AA is released from membrane phospholipids by phospholipase A2 (PLA2), and thus PGs and LTs are derived from membrane phospholipids. Zebrafish is a vertebrate model organism that has been widely used in the studies on development and drug discovery for the reasons as follows: (i) embryo development occurs outside the maternal body, (ii) embryo is transparent, (iii) high conservation between human and zebrafish genes, etc. Nevertheless, the systematic identification and characterization of zebrafish PG and LT receptors have not been performed. Therefore, we identified twelve zebrafish PG receptors and characterized their pharmacological properties. Furthermore, to identify the physiological role of zebrafish PG receptors, we generated each receptor-deficient zebrafish, and analyzed developmental processes. Here, we report that PGE2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Using pharmacological and genetic approaches, we demonstrated that the COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of critical factors for the lymphatic specification. We recently focused the zebrafish cysteinyl LT (cysLT) receptors, cloned four zebrafish cysLT receptors, and analyzed the expression profile of these receptors in developmental stages. As results, we revealed that the four zebrafish cysLT receptors show unique tissue distribution patterns. Each receptor may hence be involved in unique physiological functions. This work provides further insights into the diverse functions of arachidonate metabolites in zebrafish.

Excessive dietary linoleic acid promotes plasma accumulation of pronociceptive fatty acyl lipid mediators

Various fatty acyl lipid mediators are derived from dietary polyunsaturated fatty acids (PUFAs) and modulate nociception. The modern diet is rich in linoleic acid, which is associated with nociceptive hypersensitivities and may present a risk factor for developing pain conditions. Although recommendations about fatty acid intake exist for some diseases (e.g. cardiovascular disease), the role of dietary fatty acids in promoting pain disorders is not completely understood. To determine how dietary linoleic acid content influences the accumulation of pro- and anti-nociceptive fatty acyl lipid mediators, we created novel rodent diets using custom triglyceride blends rich in either linoleic acid or oleic acid. We quantified the fatty acyl lipidome in plasma of male and female rats fed these custom diets from the time of weaning through nine weeks of age. Dietary fatty acid composition determined circulating plasma fatty acyl lipidome content. Exposure to a diet rich in linoleic acid was associated with accumulation of linoleic and arachidonic acid-derived pro-nociceptive lipid mediators and reduction of anti-nociceptive lipid mediators derived from the omega-3 PUFAs. Our findings provide mechanistic insights into exaggerated nociceptive hypersensitivity associated with excessive dietary linoleic acid intake and highlight potential biomarkers for pain risk stratification.

Correction: Myeloid-Specific Deficiency of Long-Chain Acyl CoA Synthetase 4 Reduces Inflammation by Remodeling Phospholipids and Reducing Production of Arachidonic Acid-Derived Proinflammatory Lipid Mediators.

Andrew R. Reeves, Brian E. Sansbury, Meixia Pan, Xianlin Han, Matthew Spite, and Andrew S. Greenberg. 2021. Myeloid-Specific Deficiency of Long-Chain Acyl CoA Synthetase 4 Reduces Inflammation by Remodeling Phospholipids and Reducing Production of Arachidonic Acid–Derived Proinflammatory Lipid

Dietary intake of n-3 polyunsaturated fatty acids alters the lipid mediator profile of the kidney but does not attenuate renal insufficiency

The efficacy of n-3 polyunsaturated fatty acids (PUFAs) in improving outcomes in a renal ischemia-reperfusion injury (IRI) model has previously been reported. However, the underlying mechanisms remain poorly understood and few reports demonstrate how dietary n-3 PUFAs influence the composition of membrane phospholipids in the kidney. Additionally, it has not been elucidated whether perilla oil (PO), which is mainly composed of the n-3 alpha-linolenic acid, mitigates renal IRI. In this study, we investigated the effect of dietary n-3 PUFAs (PO), compared with an n-6 PUFA-rich soybean oil (SO) diet, on IRI-induced renal insufficiency in a rat model. Levels of membrane phospholipids containing n-3 PUFAs were higher in the kidney of PO-rich diet-fed rats than the SO-rich diet-fed rats. Levels of blood urea nitrogen and serum creatinine were significantly higher in the ischemia-reperfusion group than the sham group under both dietary conditions. However, no significant differences were observed in blood urea nitrogen, serum creatinine, or histological damage between PO-rich diet-fed rats and SO-rich diet-fed rats. In the kidney of PO-rich diet-fed rats, levels of arachidonic acid and arachidonic acid-derived pro-inflammatory lipid mediators were lower than SO-rich diet-fed rats. Eicosapentaenoic acid and eicosapentaenoic acid-derived lipid mediators were significantly higher in the kidney of PO-rich than SO-rich diet-fed rats. These results suggest that dietary n-3 PUFAs alter the fatty acid composition of membrane phospholipids and lipid mediators in the kidney; however, this does not attenuate renal insufficiency or histological damage in a renal IRI model.

Unraveling the Role of 12- and 20- HETE in Cardiac Pathophysiology: G-Protein-Coupled Receptors, Pharmacological Inhibitors, and Transgenic Approaches.

Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450-derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

Association of Arachidonic Acid-derived Lipid Mediators with Subsequent Onset of Acute Myocardial Infarction in Patients with Coronary Artery Disease

Polyunsaturated fatty acids (PUFAs) have been suggested for cardiovascular health. This study was conducted to investigate the prognostic impacts of the PUFA metabolites, oxylipins, on clinical outcomes in coronary artery disease (CAD). A total of 2,239 patients with stable CAD were prospectively enrolled and followed up regularly. Among them, twenty-five consecutive patients with new onset of acute myocardial infarction (AMI) within 2-year follow-up were studied. Another 50 gender- and age-matched patients without clinical cardiovascular events for more than 2 years were studied for control. Baseline levels of specific arachidonic acid metabolites were significantly higher in patients with subsequent AMI than in the controls. In Kaplan-Meier analysis, the incidence of future AMI was more frequently seen in patients with higher baseline levels of 8-hydroxyeicosatetraenoic acid (HETE), 9-HETE, 11-HETE, 12-HETE, 15-HETE, 19-HETE, 20-HETE, 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, 11,12-EET, or 14-15-EET when compared to their counterparts (all the P < 0.01). Further, serum levels of these specific HETEs, except for 11,12-EET, were positively correlated to the levels of some inflammatory and cardiac biomarker such as tumor necrosis factor-α and N-terminal pro B-type natriuretic peptide. Accordingly, serum specific oxylipins levels are increased and associated with the consequent onset of AMI, suggesting their potential role for secondary prevention in clinically stable CAD.

The effects of n-6 polyunsaturated fatty acid deprivation on the inflammatory gene response to lipopolysaccharide in the mouse hippocampus

BackgroundNeuroinflammation is thought to contribute to psychiatric and neurological disorders such as major depression and Alzheimer’s disease (AD). N-6 polyunsaturated fatty acids (PUFA) and molecules derived from them, including linoleic acid- and arachidonic acid-derived lipid mediators, are known to have pro-inflammatory properties in the periphery; however, this has yet to be tested in the brain. Lowering the consumption of n-6 PUFA is associated with a decreased risk of depression and AD in human observational studies. The purpose of this study was to investigate the inflammation-modulating effects of lowering dietary n-6 PUFA in the mouse hippocampus.MethodsC57BL/6 male mice were fed either an n-6 PUFA deprived (2% of total fatty acids) or an n-6 PUFA adequate (23% of total fatty acids) diet from weaning to 12 weeks of age. Animals then underwent intracerebroventricular surgery, in which lipopolysaccharide (LPS) was injected into the left lateral ventricle of the brain. Hippocampi were collected at baseline and following LPS administration (1, 3, 7, and 14 days). A microarray (n = 3 per group) was used to identify candidate genes and results were validated by real-time PCR in a separate cohort of animals (n = 5–8 per group).ResultsMice administered with LPS had significantly increased Gene Ontology categories associated with inflammation and immune responses. These effects were independent of changes in gene expression in any diet group. Results were validated for the effect of LPS treatment on astrocyte, cytokine, and chemokine markers, as well as some results of the diets on Ifrd2 and Mfsd2a expression.ConclusionsLPS administration increases pro-inflammatory and lipid-metabolizing gene expression in the mouse hippocampus. An n-6 PUFA deprived diet modulated inflammatory gene expression by both increasing and decreasing inflammatory gene expression, without impairing the resolution of neuroinflammation following LPS administration.

Roles of Toll-like receptor 2/4, monoacylglycerol lipase, and cyclooxygenase in social defeat stress-induced prostaglandin E2 synthesis in the brain and their behavioral relevance

Inflammation in the brain and periphery has been associated with stress-related pathology of mental illness. We have shown that prostaglandin (PG) E2, an arachidonic acid-derived lipid mediator, and innate immune receptors Toll-like receptor (TLR) 2/4 are crucial for repeated stress-induced behavioral changes in rodents. However, how the stress induces PGE2 synthesis in the brain and whether TLR2/4 are involved in the PGE2 synthesis remain unknown. Using mice lacking TLR2 and TLR4 in combination, here we show that social defeat stress (SDS) induced the PGE2 synthesis in subcortical, but not cortical, tissues in a TLR2/4-dependent manner. It is known that PGE2 in the brain is mainly derived by monoacylglycerol lipase (MAGL)-mediated conversion of endocannabinoid 2-arachidonoylglycerol to free-arachidonic acid, a substrate for cyclooxygenase (COX) for PGE2 synthesis. We found that TLR2/4 deletion reduced the mRNA expression of MAGL and COX1 in subcortical tissues after repeated SDS. Perturbation of MAGL and COX1 as well as COX2 abolished SDS-induced PGE2 synthesis in subcortical tissues. Furthermore, systemic administration of JZL184, an MAGL inhibitor, abolished repeated SDS-induced social avoidance. These results suggest that SDS induces PGE2 synthesis in subcortical regions of the brain via the MAGL-COX pathway in a TLR2/4-dependent manner, thereby leading to social avoidance.

Arachidonic Acid Derived Lipid Mediators Influence Kaposi's Sarcoma-Associated Herpesvirus Infection and Pathogenesis.

Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, particularly latent infection is often associated with inflammation. The arachidonic acid pathway, the home of several inflammation and resolution associated lipid mediators, is widely altered upon viral infections. Several in vitro studies show that these lipid mediators help in the progression of viral pathogenesis. This review summarizes the findings related to human herpesvirus KSHV infection and arachidonic acid pathway metabolites. KSHV infection has been shown to promote inflammation by upregulating cyclooxygenase-2 (COX-2), 5 lipoxygenase (5LO), and their respective metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) to promote latency and an inflammatory microenvironment. Interestingly, the anti-inflammatory lipid mediator lipoxin is downregulated during KSHV infection to facilitate infected cell survival. These studies aid in understanding the role of arachidonic acid pathway metabolites in the progression of viral infection, the host inflammatory response, and pathogenesis. With limited therapeutic options to treat KSHV infection, use of inhibitors to these inflammatory metabolites and their synthetic pathways or supplementing anti-inflammatory lipid mediators could be an effective alternative therapeutic.

Endocannabinoid Signalling in Atherosclerosis and Related Metabolic Complications.

Endocannabinoids are a group of arachidonic acid-derived lipid mediators binding to cannabinoid receptors CB1 and CB2. An overactivity of the endocannabinoid system plays a pathophysiological role in the development of visceral obesity and insulin resistance. Moreover, elevated circulating endocannabinoid levels are also prevalent in atherosclerosis. The pathophysiological increase of endocannabinoid levels is due to an altered expression of endocannabinoid synthesizing and degrading enzymes induced by inflammatory mediators such as cytokines or lipids. Emerging experimental evidence suggests that enhanced endocannabinoid signalling affects atherosclerosis via multiple effects, including a modulation of vascular inflammation, leukocyte recruitment, macrophage cholesterol metabolism and consequently atherosclerotic plaque stability. In addition, recent findings in various metabolic disease models highlight the relevance of peripheral CB1 cannabinoid receptors in adipose tissue, liver and pancreas, which crucially regulate lipid and glucose metabolism as well as macrophage properties in these organs. This suggests that targeting the endocannabinoid system in the vasculature and peripheral organs might have a therapeutic potential for atherosclerosis by inhibiting vascular inflammation and improving metabolic risk factors. This review will provide a brief update on the effects of endocannabinoid signalling in atherosclerosis and related metabolic complications.

Prostaglandin E2 suppresses hCAP18/LL-37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection.

Prostaglandin (PG)E2 is an arachidonic acid-derived lipid mediator that plays an important role in inflammation and immunity. In this study, we demonstrate that PGE2 suppresses basal and 1,25-dihydroxy vitamin D3 (VD3)-induced expression of hCAP18/LL-37 via E prostanoid (EP)2 and EP4 receptors. In humans, VD3 up-regulates vitamin D receptor (VDR) expression and promotes transcription of the cathelicidin hCAP18/LL-37 gene, whereas PGE2 counteracts this effect. We find that PGE2 induces the cAMP/PKA-signaling pathway and enhances the expression of the inhibitory transcription factor cAMP-responsive modulator/inducible cAMP early repressor, which prevents VDR expression and induction of hCAP18/LL-37 in human macrophages. The negative regulation by PGE2 was evident in M1- and M2-polarized human macrophages, although PGE2 displayed more profound inhibitory effects in M2 cells. PGE2 impaired VD3-induced expression of cathelicidin and concomitant activation of autophagy during Mycobacterium tuberculosis (Mtb) infection and facilitated intracellular Mtb growth in human macrophages. An EP4 agonist also significantly promoted Mtb survival in human macrophages. Our results indicate that PGE2 inhibits hCAP18/LL-37 expression, especially VD3-induced cathelicidin and autophagy, which may reduce host defense against Mtb. Accordingly, antagonists of EP4 may constitute a novel adjunctive therapy in Mtb infection.-Wan, M., Tang, X., Rekha, R. S., Muvva, S. S. V. J. R., Brighenti, S., Agerberth, B., Haeggström, J. Z. Prostaglandin E2 suppresses hCAP18/LL-37 expression in human macrophages via EP2/EP4: implications for treatment of Mycobacterium tuberculosis infection.

Lipid mediators of inflammation in neurological injury: shifting the balance toward resolution.

Lipid mediators of inflammation in neurological injury: shifting the balance toward resolution.

Lipid mediator lipoxin A4 inhibits tumor growth by targeting IL-10-producing regulatory B (Breg) cells

Lipoxin A4 (LXA4), an arachidonic acid-derived anti-inflammatory lipid mediator, shows anti-tumor potential by regulating tumor immune microenvironments. However, the underlying molecular and cellular basis of this function remains unclear. IL-10-producing B (Breg) cells display tumor-promoting effects by negatively regulating anti-tumor immunity. Here we show that LXA4 inhibits tumor growth by suppressing the generation of Breg cells in tumor-bearing mice. The administration of LXA4 inhibited the induction of Breg cells. Breg cell deficiency, in turn, resulted in LXA4 losing its anti-tumor properties. Intriguingly, regulatory T (Treg) cells also had a role in this process. Targeting Breg cells by LXA4 decreased the number of Treg cells in draining lymph nodes and tumor tissues as well as enhanced cytotoxic T cell activities. In addition, we further demonstrated that LXA4 inhibited Breg cells through its dephosphorylating STAT3 and ERK. These findings unveil a new anti-tumor mechanism underlying LXA4 targeting Breg cells with potential clinical applications.

PPAR-γ activation by Tityus serrulatus venom regulates lipid body formation and lipid mediator production

Tityus serrulatus venom (TsV) consists of numerous peptides with different physiological and pharmacological activities. Studies have shown that scorpion venom increases pro-inflammatory cytokine production, contributing to immunological imbalance, multiple organ dysfunction, and patient death. We have previously demonstrated that TsV is a venom-associated molecular pattern (VAMP) recognized by TLRs inducing intense inflammatory reaction through the production of pro-inflammatory cytokines and arachidonic acid-derived lipid mediators prostaglandin (PG)E2 and leukotriene (LT)B4. Lipid bodies (LBs) are potential sites for eicosanoid production by inflammatory cells. Moreover, recent studies have shown that the peroxisome proliferator-activated receptor gamma (PPAR-γ) is implicated in LB formation and acts as an important modulator of lipid metabolism during inflammation. In this study, we used murine macrophages to evaluate whether the LB formation induced by TsV after TLR recognition correlates with lipid mediator generation by macrophages and if it occurs through PPAR-γ activation. We demonstrate that TsV acts through TLR2 and TLR4 stimulation and PPAR-γ activation to induce LB formation and generation of PGE2 and LTB4. Our data also show that PPAR-γ negatively regulates the pro-inflammatory NF-κB transcription factor. Based on these results, we suggest that during envenomation, LBs constitute functional organelles for lipid mediator production through signaling pathways that depend on cell surface and nuclear receptors. These findings point to the inflammatory mechanisms that might also be triggered during human envenomation by TsV.

SAT0024 Increased production of leukotrienes by peripheral blood mononuclear cells is associated with more severe disease and worse prognosis in patients with systemic sclerosis

BackgroundLeukotrienes (LTs) are a group of pro-inflammatory and pro-fibrotic, arachidonic acid-derived lipid mediators. We have shown that LTs are increased in the lungs of patients with interstitial lung disease (ILD)...

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling*

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).