Investigating the alterations of endocannabinoidome signaling in the human small intestine in the context of obesity and type 2 diabetes.

Background: Human studies have linked obesity-related diseases, such as type-2 diabetes (T2D), to the modulation of endocannabinoid signaling. Cannabinoid CB1 and CB2 receptor activation by the endocannabinoids (eCBs) 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA), both derived from arachidonic acid, play a role in homeostatic regulation. Other long chain fatty acid-derived endocannabinoid-like molecules have extended the metabolic role of this signaling system through other receptors. In this study, we aimed to assess in depth the interactions between the circulating and intestinal tone of this extended eCB system, or endocannabinoidome (eCBome), and their involvement in the pathogenesis of diabetes. Methods: Plasma and ileum samples were collected from subjects with obesity and harboring diverse degrees of insulin resistance or T2D, who underwent bariatric surgery. The levels of eCBome mediators and their congeners were then assessed by liquid chromatography coupled to tandem mass spectrometry, while gene expression was screened with qPCR arrays. Findings: Intestinal and circulating levels of eCBome mediators were higher in subjects with T2D. We found an inverse correlation between the intestinal and circulating levels of monoacylglycerols (MAGs). Additionally, we identified genes known to be implicated in both lipid metabolism and intestinal function that are altered by the context of obesity and glucose homeostasis. Interpretation: Although the impact of glucose metabolism on the eCBome remains poorly understood in subjects with advanced obesity state, our results suggest a strong causative link between altered glucose homeostasis and eCBome signaling in the intestine and the circulation.

Pharmacological evidences that the inhibitory effects of prostaglandin E2 are mediated by the EP2 and EP4 receptors in human neutrophils.

Prostaglandin (PG) E2 is a recognized inhibitor of granulocyte functions. However, most of the data supporting this was obtained when available pharmacological tools mainly targeted the EP2 receptor. Herein, we revisited the inhibitory effect of PGE2 on reactive oxygen species production, leukotriene biosynthesis and migration in human neutrophils. Our data confirm the inhibitory effect of PGE2 on these functions and unravel that the effect of PGE2 on human neutrophils is obtained by the combined action of EP2 and EP4 agonism. Accordingly, we also demonstrate that the inhibitory effect of PGE2 is fully prevented only by the combination of EP2 and EP4 receptor antagonists, underscoring the importance of targeting both receptors in the effect of PGE2. Conversely, we also show that the inhibition of reactive oxygen species production by human eosinophils only involves the EP4 receptor, despite the fact that they also express the EP2 receptor.

Influence of diet on acute endocannabinoidome mediator levels post exercise in active women, a crossover randomized study.

The extended endocannabinoid system, also termed endocannabinoidome, participates in multiple metabolic functions in health and disease. Physical activity can both have an acute and chronic impact on endocannabinoid mediators, as does diet. In this crossover randomized controlled study, we investigated the influence of diet on the peripheral response to acute maximal aerobic exercise in a sample of active adult women (n = 7) with no underlying metabolic conditions. We compared the impact of 7-day standardized Mediterranean diet (MedDiet) and control diet inspired by Canadian macronutrient intake (CanDiet) on endocannabinoidome and short-chain fatty acid metabolites post maximal aerobic exercise. Overall, plasmatic endocannabinoids, their congeners and some polyunsaturated fatty acids increased significantly post maximal aerobic exercise upon cessation of exercise and recovered their initial values within 1 h after exercise. Most N-acylethanolamines and polyunsaturated fatty acids increased directly after exercise when the participants had consumed the MedDiet, but not when they had consumed the CanDiet. This impact was different for monoacylglycerol endocannabinoid congeners, which in most cases reacted similarly to acute exercise while on the MedDiet or the CanDiet. Fecal microbiota was only minimally affected by the diet in this cohort. This study demonstrates that endocannabinoidome mediators respond to acute maximal aerobic exercise in a way that is dependent on the diet consumed in the week prior to exercise.

Expression and Functions of the CB2 Receptor in Human Leukocytes.

The cannabinoid CB2 receptor was cloned from the promyeloid cell line HL-60 and is notably expressed in most, if not all leukocyte types. This relatively restricted localization, combined to the absence of psychotropic effects following its activation, make it an attractive drug target for inflammatory and autoimmune diseases. Therefore, there has been an increasing interest in the past decades to identify precisely which immune cells express the CB2 receptor and what are the consequences of such activation. Herein, we provide new data on the expression of both CB1 and CB2 receptors by human blood leukocytes and discuss the impact of CB2 receptor activation in human leukocytes. While the expression of the CB2 mRNA can be detected in eosinophils, neutrophils, monocytes, B and T lymphocytes, this receptor is most abundant in human eosinophils and B lymphocytes. We also review the evidence obtained from primary human leukocytes and immortalized cell lines regarding the regulation of their functions by the CB2 receptor, which underscore the urgent need to deepen our understanding of the CB2 receptor as an immunoregulator in humans.

Biosynthesis of the Novel Endogenous 15-Lipoxygenase Metabolites N-13-Hydroxy-octodecadienoyl-ethanolamine and 13-Hydroxy-octodecadienoyl-glycerol by Human Neutrophils and Eosinophils.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine are lipids regulating many physiological processes, notably inflammation. Endocannabinoid hydrolysis inhibitors are now being investigated as potential anti-inflammatory agents. In addition to 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine, the endocannabinoidome also includes other monoacylglycerols and N-acyl-ethanolamines such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). By increasing monoacylglycerols and/or N-acyl-ethanolamine levels, endocannabinoid hydrolysis inhibitors will likely increase the levels of their metabolites. Herein, we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, given that both possess a 1Z,4Z-pentadiene motif, near their omega end. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. N-13-hydroxy-octodecadienoyl-ethanolamine (13-HODE-EA) and 13-hydroxy-octodecadienoyl-glycerol (13-HODE-G), the 15-lipoxygenase metabolites of LEA and 1-LG, were synthesized using Novozym 435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after five minutes. Substrate preference of eosinophils was LA > LEA > 1-LG in presence of 13-HODE-G hydrolysis inhibition with methyl-arachidonoyl-fluorophosphonate. Human neutrophils also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 15-30 s. Substrate preference was LA ≫ 1-LG > LEA. Importantly, 13-HODE-G was found in humans and mouse tissue samples. In conclusion, our data show that human eosinophils and neutrophils metabolize 1-LG and LEA into the novel endogenous 15-lipoxygenase metabolites 13-HODE-G and 13-HODE-EA. The full biological importance of 13-HODE-G and 13-HODE-EA remains to be explored.

Role of the Endocannabinoid System in the Adipose Tissue with Focus on Energy Metabolism.

The endocannabinoid system is involved in a wide range of processes including the control of energy acquisition and expenditure. Endocannabinoids and their receptors are present in the central nervous system but also in peripheral tissues, notably the adipose tissues. The endocannabinoid system interacts with two main hormones regulating appetite, namely leptin and ghrelin. The inhibitory effect of the cannabinoid receptor 1 (CB1) antagonist rimonabant on fat mass suggested that the endocannabinoid system can also have a peripheral action in addition to its effect on appetite reduction. Thus, several investigations have focused on the peripheral role of the endocannabinoid system in the regulation of metabolism. The white adipose tissue stores energy as triglycerides while the brown adipose tissue helps to dissipate energy as heat. The endocannabinoid system regulates several functions of the adipose tissues to favor energy accumulation. In this review we will describe the presence of the endocannabinoid system in the adipose tissue. We will survey the role of the endocannabinoid system in the regulation of white and brown adipose tissue metabolism and how the eCB system participates in obesity and metabolic diseases.

High levels of eicosanoids and docosanoids in the lungs of intubated COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 is responsible for coronavirus disease 2019 (COVID-19). While COVID-19 is often benign, a subset of patients develops severe multilobar pneumonia that can progress to an acute respiratory distress syndrome. There is no cure for severe COVID-19 and few treatments significantly improved clinical outcome. Dexamethasone and possibly aspirin, which directly/indirectly target the biosynthesis/effects of numerous lipid mediators are among those options. Our objective was to define if severe COVID-19 patients were characterized by increased bioactive lipids modulating lung inflammation. A targeted lipidomic analysis of bronchoalveolar lavages (BALs) by tandem mass spectrometry was done on 25 healthy controls and 33 COVID-19 patients requiring mechanical ventilation. BALs from severe COVID-19 patients were characterized by increased fatty acids and inflammatory lipid mediators. There was a predominance of thromboxane and prostaglandins. Leukotrienes were also increased, notably LTB4 , LTE4 , and eoxin E4 . Monohydroxylated 15-lipoxygenase metabolites derived from linoleate, arachidonate, eicosapentaenoate, and docosahexaenoate were also increased. Finally yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-series resolvins, were also increased, underscoring that the lipid mediator storm occurring in severe COVID-19 involves pro- and anti-inflammatory lipids. Our data unmask the lipid mediator storm occurring in the lungs of patients afflicted with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and enhancing the effects of anti-inflammatory and/or pro-resolving lipid mediators.

Synthesis and molecular targets of N-13-hydroxy-octadienoyl-ethanolamine, a novel endogenous bioactive 15-lipoxygenase-derived metabolite of N-linoleoyl-ethanolamine found in the skin and saliva.

N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.

Chemokines in human obesity.

Obesity and type 2 diabetes have been shown to be associated with chronic inflammation. Despite extensive evidence for inflammatory mediators in the obese patients and multiple clinical trials, the outcome has been disappointing. In murine models recruitment of immune cells during inflammation has been shown to contribute to the chronic inflammation. Clearcut evidence for the differential expression of chemokines that mediate this recruitment is not available. In this short review we discuss the observations on CCL2 and CCL5 in human obesity.

Inflammation in human adipose tissues-Shades of gray, rather than white and brown.

Chronic inflammation in adipose tissues has been associated with obesity and metabolic syndrome over the years. Various studies using animal models have contributed to our knowledge on the pro- and anti- inflammatory mediators that regulate obesity. Analyses of cytokine profiles in humans have not revealed a clear scenario. Likewise, treatments targeting inflammation to control obesity and insulin resistance has not yielded promising results. In this review we summarize the data available in human obesity and discuss the possible reasons that could explain the difficulties in treating obesity and insulin resistance by targeting pro-inflammatory cytokines.

Inflammatory Cytokine Profiles in Visceral and Subcutaneous Adipose Tissues of Obese Patients Undergoing Bariatric Surgery Reveal Lack of Correlation With Obesity or Diabetes.

Population studies have linked insulin resistance to systemic low-grade chronic inflammation and have reported elevated levels of inflammatory cytokines such as TNFα, IL-1ß and IL-6, individually or in certain combinations, in adipose tissues or in the serum. We undertook this comprehensive study to simultaneously evaluate the expression of several pro-inflammatory and anti-inflammatory cytokines in serum and in the visceral and subcutaneous adipose tissues from obese patients undergoing bariatric surgery. We observed that several inflammatory cytokines implicated in obesity-associated inflammation showed no significant difference in protein or gene expression between obese patients with or without diabetes and control groups. IL1B gene expression was significantly elevated in the visceral adipose tissues of obese patients, but did not correlate with their diabetes status. Despite the significant increase in IL1B expression in the obese group, a significant proportion of obese patients did not express TNFA, IL1B or IL6 in visceral adipose tissues. Certain inflammatory cytokines showed correlation with the chemokine CCL2 and VEGF-A in visceral adipose tissues. Our findings suggest that the inflammatory cytokine profile in metabolic syndrome is more complex than what is currently perceived and that chronic inflammation in obese patients likely results from incremental contribution from different cytokines and possibly other inflammatory mediators from within and outside the adipose tissues. It is possible that this obesity associated chronic inflammation is not predicted by a single mediator, but rather includes a large spectrum of possible profiles.

Correction: Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues.

[This corrects the article DOI: 10.1371/journal.pone.0162995.].

Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues.

OBJECTIVE: IL-15 is an inflammatory cytokine secreted by many cell types. IL-15 is also produced during physical exercise by skeletal muscle and has been reported to reduce weight gain in mice. Contrarily, our findings on IL-15 knockout (KO) mice indicate that IL-15 promotes obesity. The aim of this study is to investigate the mechanisms underlying the pro-obesity role of IL-15 in adipose tissues. METHODS: Control and IL-15 KO mice were maintained on high fat diet (HFD) or normal control diet. After 16 weeks, body weight, adipose tissue and skeletal mass, serum lipid levels and gene/protein expression in the adipose tissues were evaluated. The effect of IL-15 on thermogenesis and oxygen consumption was also studied in primary cultures of adipocytes differentiated from mouse preadipocyte and human stem cells. RESULTS: Our results show that IL-15 deficiency prevents diet-induced weight gain and accumulation of lipids in visceral and subcutaneous white and brown adipose tissues. Gene expression analysis also revealed elevated expression of genes associated with adaptive thermogenesis in the brown and subcutaneous adipose tissues of IL-15 KO mice. Accordingly, oxygen consumption was increased in the brown adipocytes from IL-15 KO mice. In addition, IL-15 KO mice showed decreased expression of pro-inflammatory mediators in their adipose tissues. CONCLUSIONS: Absence of IL-15 results in decreased accumulation of fat in the white adipose tissues and increased lipid utilization via adaptive thermogenesis. IL-15 also promotes inflammation in adipose tissues that could sustain chronic inflammation leading to obesity-associated metabolic syndrome.

Interleukin-15-mediated inflammation promotes non-alcoholic fatty liver disease.

Interleukin-15 (IL-15) is essential for the homeostasis of lymphoid cells particularly memory CD8(+) T cells and NK cells. These cells are abundant in the liver, and are implicated in obesity-associated pathogenic processes. Here we characterized obesity-associated metabolic and cellular changes in the liver of mice lacking IL-15 or IL-15Rα. High fat diet-induced accumulation of lipids was diminished in the livers of mice deficient for IL-15 or IL-15Rα. Expression of enzymes involved in the transport of lipids in the liver showed modest differences. More strikingly, the liver tissues of IL15-KO and IL15Rα-KO mice showed decreased expression of chemokines CCl2, CCL5 and CXCL10 and reduced infiltration of mononuclear cells. In vitro, IL-15 stimulation induced chemokine gene expression in wildtype hepatocytes, but not in IL15Rα-deficient hepatocytes. Our results show that IL-15 is implicated in the high fat diet-induced lipid accumulation and inflammation in the liver, leading to fatty liver disease.

Homeostasis of IL-15 dependent lymphocyte subsets in the liver.

IL-15 is a member of the gamma chain family of cytokines (γc - CD132). The IL-15 receptor (IL-15R) complex consists of 3 subunits: the ligand-binding IL-15Rα chain (CD215), the ß chain (CD122; also used by IL-2), and the common γ chain. The biological activities of IL-15 are mostly mediated by the IL-15:IL-15Rα complex, produced by the same cell and 'trans-presented' to responder cells expressing the IL-15Rßγc. The peculiar and almost unique requirement for IL-15 to be trans-presented by IL-15Rα suggests that the biological effects of IL-15 signaling are tightly regulated even at the level of availability of IL-15. Tissue-specific deletion of IL-15Rα has shown macrophage-and dendritic cell-derived IL-15Rα mediate the homeostasis of different CD8(+) T cell subsets. Here we show that hepatocyte and macrophage- specific expression of IL-15Rα is required to maintain the homeostasis of NK and NKT cells in the liver. Thus, homeostasis of IL-15-dependent lymphocyte subsets is also regulated by trans-presentation of IL-15 by non-hematopoietic cells in the tissue environment.