Biosynthesis Of Lipid Mediators Research Articles

Aspergillus fumigatus-derived gliotoxin impacts innate immune cell activation through modulating lipid mediator production in macrophages.

Gliotoxin (GT), a secondary metabolite and virulence factor of the fungal pathogen Aspergillus fumigatus, suppresses innate immunity and supports the suppression of host immune responses. Recently, we revealed that GT blocks the formation of the chemotactic lipid mediator leukotriene (LT)B4 in activated human neutrophils and monocytes, and in rodents in vivo, by directly inhibiting LTA4 hydrolase. Here, we elucidated the impact of GT on LTB4 biosynthesis and the entire lipid mediator networks in human M1- and M2-like monocyte-derived macrophages (MDMs) and in human tissue-resident alveolar macrophages. In activated M1-MDMs with high capacities to generate LTs, the formation of LTB4 was effectively suppressed by GT, connected to attenuated macrophage phagocytic activity as well as human neutrophil movement and migration. In resting macrophages, especially in M1-MDMs, GT elicited strong formation of prostaglandins, while bacterial exotoxins from Staphylococcus aureus evoked a broad spectrum of lipid mediator biosynthesis in both MDM phenotypes. We conclude that GT impairs functions of activated innate immune cells through selective suppression of LTB4 biosynthesis, while GT may also prime the immune system by provoking prostaglandin formation in macrophages.

Hyphal-associated protein expression is crucial for Candida albicans-induced eicosanoid biosynthesis in immune cells.

Candida albicans causes opportunistic infections ranging from mucosal mycoses to life-threatening systemic infections in immunocompromised patients. During C. albicans infection, leukotrienes and prostaglandins are formed from arachidonic acid by 5-lipoxygenase (5-LOX) and cyclooxygenases, respectively to amplify inflammatory conditions, but also to initiate macrophage infiltration to achieve tissue homeostasis. Since less is known about the cellular mechanisms triggering such lipid mediator biosynthesis, we investigated the eicosanoid formation in monocyte-derived M1 and M2 macrophages, neutrophils and HEK293 cells transfected with 5-LOX and 5-LOX-activating protein (FLAP) in response to C. albicans yeast or hyphae. Leukotriene biosynthesis was exclusively induced by hyphae in neutrophils and macrophages, whereas prostaglandin E2 was also formed in response to yeast cells by M1 macrophages. Eicosanoid biosynthesis was significantly higher in M1 compared to M2 macrophages. In HEK_5-LOX/FLAP cells only hyphae activated the essential 5-LOX translocation to the nuclear membrane. Using yeast-locked C. albicans mutants, we demonstrated that hyphal-associated protein expression is critical in eicosanoid formation. For neutrophils and HEK_5-LOX/FLAP cells, hyphal wall protein 1 was identified as the essential surface protein that stimulates leukotriene biosynthesis. In summary, our data suggest that hyphal-associated proteins of C. albicans are central triggers of eicosanoid biosynthesis in human phagocytes.

Fatty acid desaturation and lipoxygenase pathways support trained immunity

Infections and vaccines can induce enhanced long-term responses in innate immune cells, establishing an innate immunological memory termed trained immunity. Here, we show that monocytes with a trained immunity phenotype, due to exposure to the Bacillus Calmette-Guérin (BCG) vaccine, are characterized by an increased biosynthesis of different lipid mediators (LM) derived from long-chain polyunsaturated fatty acids (PUFA). Pharmacological and genetic approaches show that long-chain PUFA synthesis and lipoxygenase-derived LM are essential for the BCG-induced trained immunity responses of human monocytes. Furthermore, products of 12-lipoxygenase activity increase in monocytes of healthy individuals after BCG vaccination. Grasping the underscoring lipid metabolic pathways contributes to our understanding of trained immunity and may help to identify therapeutic tools and targets for the modulation of innate immune responses.

Epigenetic regulation of 15-lipoxygenase-1 expression in human chondrocytes by promoter methylation.

15-Lipoxygenase-1 (15-LOX-1) catalyzes the biosynthesis of many anti-inflammatory and immunomodulatory lipid mediators and was reported to have protective properties in several inflammatory conditions, including osteoarthritis (OA). This study was designed to evaluate the expression of 15-LOX-1 in cartilage from normal donors and patients with OA, and to determine whether it is regulated by DNA methylation. Cartilage samples were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee joint replacement surgery. The expression of 15-LOX-1 was evaluated using real-time polymerase chain reaction (PCR). The role of DNA methylation in 15-LOX-1 expression was assessed using the DNA methyltransferase inhibitor 5-Aza-2'-desoxycytidine (5-Aza-dC). The effect of CpG methylation on 15-LOX-1 promoter activity was evaluated using a CpG-free luciferase vector. The DNA methylation status of the 15-LOX-1 promoter was determined by pyrosequencing. Expression of 15-LOX-1 was upregulated in OA compared to normal cartilage. Treatment with 5-Aza-dC increased 15-LOX-1 mRNA levels in chondrocytes, and in vitro methylation decreased 15-LOX-1 promoter activity. There was no difference in the methylation status of the 15-LOX-1 gene promoter between normal and OA cartilage. The expression level of 15-LOX-1 was elevated in OA cartilage, which may be part of a repair process. The upregulation of 15-LOX-1 in OA cartilage was not associated with the methylation status of its promoter, suggesting that other mechanisms are involved in its upregulation.

Cannabidiol acts as molecular switch in innate immune cells to promote the biosynthesis of inflammation-resolving lipid mediators

Cannabinoids are phytochemicals from cannabis with anti-inflammatory actions in immune cells. Lipid mediators (LM), produced from polyunsaturated fatty acids (PUFA), are potent regulators of the immune response and impact all stages of inflammation. How cannabinoids influence LM biosynthetic networks is unknown. Here, we reveal cannabidiol (CBD) as a potent LM class-switching agent that stimulates the production of specialized pro-resolving mediators (SPMs) but suppresses pro-inflammatory eicosanoid biosynthesis. Detailed metabololipidomics analysis in human monocyte-derived macrophages showed that CBD (i)upregulates exotoxin-stimulated generation of SPMs, (ii) suppresses 5-lipoxygenase (LOX)-mediated leukotriene production, and (iii) strongly induces SPM and 12/15-LOX product formation in resting cells by stimulation of phospholipase A2-dependent PUFA release and through Ca2+-independent, allosteric 15-LOX-1 activation. Finally, in zymosan-induced murine peritonitis, CBD increased SPM and 12/15-LOX products and suppressed pro-inflammatory eicosanoid levels invivo. Switching eicosanoid to SPM production is a plausible mode of action of CBD and a promising inflammation-resolving strategy.

Discovering selective antiferroptotic inhibitors of the 15LOX/PEBP1 complex noninterfering with biosynthesis of lipid mediators

Programmed ferroptotic death eliminates cells in all major organs and tissues with imbalanced redox metabolism due to overwhelming iron-catalyzed lipid peroxidation under insufficient control by thiols (Glutathione (GSH)). Ferroptosis has been associated with the pathogenesis of major chronic degenerative diseases and acute injuries of the brain, cardiovascular system, liver, kidneys, and other organs, and its manipulation offers a promising new strategy for anticancer therapy. This explains the high interest in designing new small-molecule-specific inhibitors against ferroptosis. Given the role of 15-lipoxygenase (15LOX) association with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) in initiating ferroptosis-specific peroxidation of polyunsaturated PE, we propose a strategy of discovering antiferroptotic agents as inhibitors of the 15LOX/PEBP1 catalytic complex rather than 15LOX alone. Here we designed, synthesized, and tested a customized library of 26 compounds using biochemical, molecular, and cell biology models along with redox lipidomic and computational analyses. We selected two lead compounds, FerroLOXIN-1 and 2, which effectively suppressed ferroptosis invitro and invivo without affecting the biosynthesis of pro-/anti-inflammatory lipid mediators invivo. The effectiveness of these lead compounds is not due to radical scavenging or iron-chelation but results from their specific mechanisms of interaction with the 15LOX-2/PEBP1 complex, which either alters the binding pose of the substrate [eicosatetraenoyl-PE (ETE-PE)] in a nonproductive way or blocks the predominant oxygen channel thus preventing the catalysis of ETE-PE peroxidation. Our successful strategy may be adapted to the design of additional chemical libraries to reveal new ferroptosis-targeting therapeutic modalities.

Cell-Type-Specific Gene Regulatory Networks of Pro-Inflammatory and Pro-Resolving Lipid Mediator Biosynthesis in the Immune System.

Lipid mediators are important regulators in inflammatory responses, and their biosynthetic pathways are targeted by commonly used anti-inflammatory drugs. Switching from pro-inflammatory lipid mediators (PIMs) to specialized pro-resolving (SPMs) is a critical step toward acute inflammation resolution and preventing chronic inflammation. Although the biosynthetic pathways and enzymes for PIMs and SPMs have now been largely identified, the actual transcriptional profiles underlying the immune cell type-specific transcriptional profiles of these mediators are still unknown. Using the Atlas of Inflammation Resolution, we created a large network of gene regulatory interactions linked to the biosynthesis of SPMs and PIMs. By mapping single-cell sequencing data, we identified cell type-specific gene regulatory networks of the lipid mediator biosynthesis. Using machine learning approaches combined with network features, we identified cell clusters of similar transcriptional regulation and demonstrated how specific immune cell activation affects PIM and SPM profiles. We found substantial differences in regulatory networks in related cells, accounting for network-based preprocessing in functional single-cell analyses. Our results not only provide further insight into the gene regulation of lipid mediators in the immune response but also shed light on the contribution of selected cell types in their biosynthesis.

Exercise-induced specialized proresolving mediators stimulate AMPK phosphorylation to promote mitochondrial respiration in macrophages

ObjectivePhysical activity has been shown to reduce the risk of CVD mortality in large-cohort longitudinal studies; however, the mechanisms underpinning the beneficial effects of exercise remain incompletely understood. Emerging data suggest that the risk reducing effect of exercise extends beyond changes in traditional CVD risk factors alone and involves alterations in immunity and reductions in inflammatory mediator production. Our study aimed to determine whether exercise-enhanced production of proresolving lipid mediators contribute to alterations in macrophage intermediary metabolism, which may contribute to the anti-inflammatory effects of exercise. MethodsChanges in lipid mediators and macrophage metabolism were assessed in C57Bl/6 mice following 4 weeks of voluntary exercise training. To investigate whether exercise-stimulated upregulation of specialized proresolving lipid mediators (SPMs) was sufficient to enhance mitochondrial respiration, both macrophages from control mice and human donors were incubated in vitro with SPMs and mitochondrial respiratory parameters were measured using extracellular flux analysis. Compound-C, an ATP-competitive inhibitor of AMPK kinase activity, was used to investigate the role of AMPK activity in SPM-induced mitochondrial metabolism. To assess the in vivo contribution of 5-lipoxygenase in AMPK activation and exercise-induced mitochondrial metabolism in macrophages, Alox5−/− mice were also subjected to exercise training. ResultsFour weeks of exercise training enhanced proresolving lipid mediator production, while also stimulating the catabolism of inflammatory lipid mediators (e.g., leukotrienes and prostaglandins). This shift in lipid mediator balance following exercise was associated with increased macrophage mitochondrial metabolism. We also find that treating human and murine macrophages in vitro with proresolving lipid mediators enhances mitochondrial respiratory parameters. The proresolving lipid mediators RvD1, RvE1, and MaR1, but not RvD2, stimulated mitochondrial respiration through an AMPK-dependent signaling mechanism. Additionally, in a subset of macrophages, exercise-induced mitochondrial activity in vivo was dependent upon 5-lipoxygenase activity. ConclusionCollectively, these results suggest that exercise stimulates proresolving lipid mediator biosynthesis and mitochondrial metabolism in macrophages via AMPK, which might contribute to the anti-inflammatory and CVD risk reducing effect of exercise.

Clinical significance of polyunsaturated fatty acids in the prevention of cardiovascular diseases.

Cardiovascular diseases are one of the most important problems of modern medicine. They are associated with a large number of health care visits, hospitalizations and mortality. Prevention of atherosclerosis is one of the most effective strategies and should start as early as possible. Correction of lipid metabolism disorders is associated with definite clinical successes, both in primary prevention and in the prevention of complications of many cardiovascular diseases. A growing body of evidence suggests a multifaceted role for polyunsaturated fatty acids. They demonstrate a variety of functions in inflammation, both participating directly in a number of cellular processes and acting as a precursor for subsequent biosynthesis of lipid mediators. Extensive clinical data also support the importance of polyunsaturated fatty acids, but all questions have not been answered to date, indicating the need for further research.

Phytochemical Characterization of Cannabis sativa L. Chemotype V Reveals Three New Dihydrophenanthrenoids That Favorably Reprogram Lipid Mediator Biosynthesis in Macrophages

The growing general interest surrounding Cannabis sativa L. has led to a renewal in breeding and resulted in an impressive variability of chemotypical characteristics that required the division of cannabis into different recognized chemotypes. The chemotype V has been overlooked in terms of phytochemical composition due to the almost total absence of cannabinoids, on which biomedical attention is focused. Systematic approaches addressing diverse chemotypes are, however, needed to discriminate and define phytochemical aspects beyond cannabinoids. Such thoroughly characterized chemotypes guarantee blinding in controlled studies by mimicking the sensory properties of hemp and may help to unravel the “entourage effect”. Capitalizing on the ability of cannabis to synthesize a large number of non-cannabinoid phenolic compounds, we here investigated, for the first time, the composition of the Ermo chemotype V and identified new compounds: two dihydrophenanthrenes and the methoxy-dihydrodenbinobin. All three compounds suppress pro-inflammatory leukotriene biosynthesis in activated macrophage subtypes by targeting 5-lipoxygenase, but substantially differ in their capacity to elevate the levels of specialized pro-resolving lipid mediators and their precursors in M2 macrophages. We conclude that the discovered compounds likely contribute to the anti-inflammatory properties of Cannabis sativa L. chemotype V and might promote inflammation resolution by promoting a lipid mediator class switch.

Linoleate-Rich Safflower Oil Diet Increases Linoleate-Derived Bioactive Lipid Mediators in Plasma, and Brown and White Adipose Depots of Healthy Mice.

Polyunsaturated fats are energy substrates and precursors to the biosynthesis of lipid mediators of cellular processes. Adipose tissue not only provides energy storage, but influences whole-body energy metabolism through endocrine functions. How diet influences adipose–lipid mediator balance may have broad impacts on energy metabolism. To determine how dietary lipid sources modulate brown and white adipose tissue and plasma lipid mediators, mice were fed low-fat (15% kcal fat) isocaloric diets, containing either palm oil (POLF) or linoleate-rich safflower oil (SOLF). Baseline and post body weight, adiposity, and 2-week and post fasting blood glucose were measured and lipid mediators were profiled in plasma, and inguinal white and interscapular brown adipose tissues. We identified over 30 species of altered lipid mediators between diets and found that these changes were unique to each tissue. We identified changes to lipid mediators with known functional roles in the regulation of adipose tissue expansion and function, and found that there was a relationship between the average fold difference in lipid mediators between brown adipose tissue and plasma in mice consuming the SOLF diet. Our findings emphasize that even with a low-fat diet, dietary fat quality has a profound effect on lipid mediator profiles in adipose tissues and plasma.

Modulation of Inflammation-Related Lipid Mediator Pathways by Celastrol During Human Macrophage Polarization.

Background and PurposeCelastrol (CS) is a major active ingredient of the Chinese/Asian herb Tripterygium wilfordii that is frequently used as phytomedicine to treat inflammation and autoimmune diseases. We showed before that short-term exposure to CS (1 µM) favorably impacts the biosynthesis of inflammation-related lipid mediators (LM) in human polarized macrophages by modulating the activities of different lipoxygenases (LOXs). However, whether CS regulates the expression of LOXs and other related LM-biosynthetic enzymes during macrophage polarization is unknown. Here, we investigated how CS affects LM-biosynthetic enzyme expression on the protein level and studied concomitant LM signature profiles during polarization of human monocyte-derived macrophages (MDM) towards M1- and M2-like phenotypes.Methods and ResultsWe used LM metabololipidomics to study the long-term effects of CS on LM profile signatures after manipulation of human monocyte-derived macrophages (MDM) during polarization. Exposure of MDM to low concentrations of CS (ie, 0.2 µM) during polarization to an inflammatory M1 phenotype potently suppressed the formation of pro-inflammatory cyclooxygenase (COX)- and 5-LOX-derived LM, especially prostaglandin (PG)E2. Notably, gene and enzyme expression of COX-2 and microsomal PGE2 synthase (mPGES)-1 as well as M1 markers were strongly decreased by CS during M1-MDM polarization, along with impaired activation of nuclear factor-κB and p38 mitogen-activated protein kinase. During IL-4-induced M2 polarization, CS decreased the capacity of the resulting M2-MDM to generate pro-inflammatory COX and 5-LOX products as well but it also reduced the formation of 12/15-LOX products and specialized pro-resolving mediators, without affecting the levels of liberated fatty acid substrates.ConclusionDepending on the timing and concentration, CS not only favorably affects LOX activities in macrophages but also the expression of LM-biosynthetic enzymes during macrophage polarization connected to changes of inflammation-related LM which might be of relevance for potential application of CS to treat inflammatory disorders.

Fatty Acid Fingerprints and Hyaluronic Acid in Extracellular Vesicles from Proliferating Human Fibroblast-like Synoviocytes.

Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1–3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.

Inflammation and resolution signaling in cardiac repair and heart failure.

SummaryUnresolved inflammation is a key mediator of advanced heart failure. Especially, damage, pathogen, and lifestyle-associated molecular patterns are the major factors in initiating baseline inflammatory diseases, particularly in cardiac pathology. After a significant cardiac injury like a heart attack, splenic and circulating leukocytes begin a highly optimized sequence of immune cell recruitment (neutrophils and monocytes) to coordinate effective tissue repair. An injured cardiac tissue repair and homeostasis are dependent on clearance of cellular debris where the recruited leukocytes transition from a pro-inflammatory to a reparative program through resolution process. After a cardiac injury, macrophages play a decisive role in cardiac repair through the biosynthesis of endogenous lipid mediators that ensure a timely tissue repair while avoiding chronic inflammation and impaired cardiac repair. However, dysregulation of resolution of inflammation processes due to cardiometabolic defects (obesity, hypertension, and diabetes), aging, or co-medication(s) lead to impaired cardiac repair. Hence, the presented review demonstrates the fundamental role of leukocytes, in particular macrophages orchestrate the inflammation and resolution biology, focusing on the biosynthesis of specialized lipid mediators in cardiac repair and heart failure. This work was supported by research funds from National Institutes of Health (AT006704, HL132989, and HL144788) to G.V.H. The authors acknowledges the use of Servier Medical Art image bank and Biorender that is used to create schematic Figures 1–3.

Reduced Plasma Extracellular Vesicle CD5L Content in Patients With Acute-On-Chronic Liver Failure: Interplay With Specialized Pro-Resolving Lipid Mediators.

Acute-on chronic liver failure (ACLF) is a syndrome that develops in patients with acutely decompensated cirrhosis (AD). It is characterized by a systemic hyperinflammatory state, leading to multiple organ failure. Our objective was to analyze macrophage anti-inflammatory protein CD5L in plasma extracellular vesicles (EVs) and assess its as yet unknown relationship with lipid mediators in ACLF. With this aim, EVs were purified by size exclusion chromatography from the plasma of healthy subjects (HS) (n=6) and patients with compensated cirrhosis (CC) (n=6), AD (n=11) and ACLF (n=11), which were defined as positive for CD9, CD5L and CD63 and their size, number, morphology and lipid mediator content were characterized by NTA, EM, and LC-MS/MS, respectively. Additionally, plasma CD5L was quantified by ELISA in 10 HS, 20 CC and 149 AD patients (69 ACLF). Moreover, macrophage CD5L expression and the biosynthesis of specialized lipid mediators (SPMs) were characterized in vitro in primary cells. Our results indicate that circulating EVs were significantly suppressed in cirrhosis, regardless of severity, and showed considerable alterations in CD5L and lipid mediator content as the disease progressed. In AD, levels of EV CD5L correlated best with those of the SPM RvE1. Analysis of total plasma supported these data and showed that, in ACLF, low CD5L levels were associated with circulatory (p<0.001), brain (p<0.008) and respiratory (p<0.05) failure (Mann-Whitney test). Functional studies in macrophages indicated a positive feedback loop between CD5L and RvE1 biosynthesis. In summary, we have determined a significant alteration of circulating EV contents in ACLF, with a loss of anti-inflammatory and pro-resolving molecules involved in the control of acute inflammation in this condition.

Resolvins, Protectins, and Maresins: DHA-Derived Specialized Pro-Resolving Mediators, Biosynthetic Pathways, Synthetic Approaches, and Their Role in Inflammation.

Marine organisms are an important source of natural products with unique and diverse chemical structures that may hold the key for the development of novel drugs. Docosahexaenoic acid (DHA) is an omega-3 fatty acid marine natural product playing a crucial regulatory role in the resolution of inflammation and acting as a precursor for the biosynthesis of the anti-inflammatory specialized pro-resolving mediators (SPMs) resolvins, protectins, and maresins. These metabolites exert many beneficial actions including neuroprotection, anti-hypertension, or anti-tumorigenesis. As dysregulation of SPMs is associated with diseases of prolonged inflammation, the disclosure of their bioactivities may be correlated with anti-inflammatory and pro-resolving capabilities, offering new targets for drug design. The availability of these SPMs from natural resources is very low, but the evaluation of their pharmacological properties requires their access in larger amounts, as achieved by synthetic routes. In this report, the first review of the total organic syntheses carried out for resolvins, protectins, and maresins is presented. Recently, it was proposed that DHA-derived pro-resolving mediators play a key role in the treatment of COVID-19. In this work we also review the current evidence on the structures, biosynthesis, and functional and new-found roles of these novel lipid mediators of disease resolution.

Human and Mouse Eosinophils Differ in Their Ability to Biosynthesize Eicosanoids, Docosanoids, the Endocannabinoid 2-Arachidonoyl-glycerol and Its Congeners.

High eosinophil (EOS) counts are a key feature of eosinophilic asthma. EOS notably affect asthmatic response by generating several lipid mediators. Mice have been utilized in hopes of defining new pharmacological targets to treat asthma. However, many pinpointed targets in mice did not translate into clinics, underscoring that key differences exist between the two species. In this study, we compared the ability of human (h) and mouse (m) EOS to biosynthesize key bioactive lipids derived from arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). hEOS were isolated from the blood of healthy subjects and mild asthmatics, while mEOSs were differentiated from the bone marrow. EOSs were treated with fatty acids and lipid mediator biosynthesis assessed by LC-MS/MS. We found that hEOS biosynthesized leukotriene (LT) C4 and LTB4 in a 5:1 ratio while mEOS almost exclusively biosynthesized LTB4. The biosynthesis of the 15-lipoxygenase (LO) metabolites 15-HETE and 12-HETE also differed, with a 15-HETE:12-HETE ratio of 6.3 for hEOS and 0.727 for mEOS. EOS biosynthesized some specialized pro-resolving mediators, and the levels from mEOS were 9-times higher than those of hEOS. In contrast, hEOS produced important amounts of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) and its congeners from EPA and DHA, a biosynthetic pathway that was up to ~100-fold less prominent in mEOS. Our data show that hEOS and mEOS biosynthesize the same lipid mediators but in different amounts. Compared to asthmatics, mouse models likely have an amplified involvement of LTB4 and specialized pro-resolving mediators and a diminished impact of the endocannabinoid 2-arachidonoyl-glycerol and its congeners.

Paralog- and ortholog-specificity of inhibitors of human and mouse lipoxygenase-isoforms.

Lipoxygenases (ALOX-isoforms) are lipid peroxidizing enzymes, which have been implicated in cell differentiation and maturation but also in the biosynthesis of lipid mediators playing important roles in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. In mammals these enzymes are widely distributed and the human genome involves six functional genes encoding for six distinct human ALOX paralogs. In mice, there is an orthologous enzyme for each human ALOX paralog but the catalytic properties of human and mouse ALOX orthologs show remarkable differences. ALOX inhibitors are frequently employed for deciphering the biological role of these enzymes in mouse models of human diseases but owing to the functional differences between mouse and human ALOX orthologs the uncritical use of such inhibitors is sometimes misleading. In this study we evaluated the paralog- and ortholog-specificity of 13 frequently employed ALOX-inhibitors against four recombinant human and mouse ALOX paralogs (ALOX15, ALOX15B, ALOX12, ALOX5) under different experimental conditions. Our results indicated that except for zileuton, which exhibits a remarkable paralog-specificity for mouse and human ALOX5, no other inhibitor was strictly paralog specific but some compounds exhibit an interesting ortholog-specificity. Because of the variable isoform specificities of the currently available ALOX inhibitors care must be taken when the biological effects of these compounds observed in complex in vitro and in vivo systems are interpreted.

Inflammatory Effects of Bothrops Phospholipases A2: Mechanisms Involved in Biosynthesis of Lipid Mediators and Lipid Accumulation.

Phospholipases A2s (PLA2s) constitute one of the major protein groups present in the venoms of viperid and crotalid snakes. Snake venom PLA2s (svPLA2s) exhibit a remarkable functional diversity, as they have been described to induce a myriad of toxic effects. Local inflammation is an important characteristic of snakebite envenomation inflicted by viperid and crotalid species and diverse svPLA2s have been studied for their proinflammatory properties. Moreover, based on their molecular, structural, and functional properties, the viperid svPLA2s are classified into the group IIA secreted PLA2s, which encompasses mammalian inflammatory sPLA2s. Thus, research on svPLA2s has attained paramount importance for better understanding the role of this class of enzymes in snake envenomation and the participation of GIIA sPLA2s in pathophysiological conditions and for the development of new therapeutic agents. In this review, we highlight studies that have identified the inflammatory activities of svPLA2s, in particular, those from Bothrops genus snakes, which are major medically important snakes in Latin America, and we describe recent advances in our collective understanding of the mechanisms underlying their inflammatory effects. We also discuss studies that dissect the action of these venom enzymes in inflammatory cells focusing on molecular mechanisms and signaling pathways involved in the biosynthesis of lipid mediators and lipid accumulation in immunocompetent cells.

Natural chalcones elicit formation of specialized pro-resolving mediators and related 15-lipoxygenase products in human macrophages

Specialized pro-resolving mediators (SPMs) comprise lipid mediators (LMs) produced from polyunsaturated fatty acids (PUFAs) via stereoselective oxygenation particularly involving 12/15-lipoxygenases (LOXs). In contrast to pro-inflammatory LMs such as leukotrienes formed by 5-LOX and prostaglandins formed by cyclooxygenases, the SPMs have anti-inflammatory and inflammation-resolving properties. Although glucocorticoids and non-steroidal anti-inflammatory drugs (NSAIDs) that block prostaglandin production are still prime therapeutics for inflammation-related diseases despite severe side effects, novel concepts focus on SPMs as immunoresolvents for anti-inflammatory pharmacotherapy. Here, we studied the natural chalcone MF-14 and the corresponding dihydrochalcone MF-15 from Melodorum fruticosum, for modulating the biosynthesis of LM including leukotrienes, prostaglandins, SPM and their 12/15-LOX-derived precursors in human monocyte-derived macrophage (MDM) M1- and M2-like phenotypes. In MDM challenged with Staphylococcus aureus-derived exotoxins both compounds (10 µM) significantly suppressed 5-LOX product formation but increased the biosynthesis of 12/15-LOX products, especially in M2-MDM. Intriguingly, in resting M2-MDM, MF-14 and MF-15 strikingly evoked generation of 12/15-LOX products and of SPMs from liberated PUFAs, along with translocation of 15-LOX-1 to membranous compartments. Enhanced 12/15-LOX product formation by the chalcones was evident also when exogenous PUFAs were supplied, excluding increased substrate supply as sole underlying mechanism. Rather, MF-14 and MF-15 stimulate the activity of 15-LOX-1, supported by experiments with HEK293 cells transfected with either 5-LOX, 15-LOX-1 or 15-LOX-2. Together, the natural chalcone MF-14 and the dihydrochalcone MF-15 favorably modulate LM biosynthesis in human macrophages by suppressing pro-inflammatory leukotrienes but stimulating formation of SPMs by differential interference with 5-LOX and 15-LOX-1.