Rapid and concomitant gut microbiota and endocannabinoidome response to diet-induced obesity in mice

The intestinal microbiota and the expanded endocannabinoid (eCB) system, or endocannabinoidome (eCBome), have both been implicated in diet-induced obesity and dysmetabolism. These systems were recently suggested to interact during the development of obesity. We aimed at identifying the potential interactions between gut microbiota composition and the eCBome during the establishment of diet-induced obesity and metabolic complications. Male mice were fed a high-fat, high-sucrose (HFHS) diet for 56 days to assess jejunum, ileum, and cecum microbiomes by 16S rRNA gene metataxonomics as well as ileum and plasma eCBome by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). The HFHS diet induced early (3 days) and persistent glucose intolerance followed by weight gain and hyperinsulinemia. Concomitantly, it induced the elevation of the two eCBs, anandamide, in both ileum and plasma, and 2-arachidonoyl-glycerol, in plasma, as well as alterations in several other N-acylethanolamines and 2-acylglycerols. It also promoted segment-specific changes in the relative abundance of several genera in intestinal microbiota, some of which were observed as early as 3 days following HFHS diet. Weight-independent correlations were found between the relative abundances of, among others, Barnesiella, Eubacterium, Adlercreutzia, Parasutterella, Propionibacterium, Enterococcus, and Methylobacterium and the concentrations of anandamide and the anti-inflammatory eCBome mediator N-docosahexaenoyl-ethanolamine. This study highlights for the first time the existence of potential interactions between the eCBome, an endogenous system of multifunctional signaling lipids, and several intestinal genera during early and late HFHS-induced dysmetabolic events, with potential impact on the host capability of adapting to increased intake of fat and sucrose

Human leukocytes differentially express endocannabinoid-glycerol lipases and hydrolyze 2-arachidonoyl-glycerol and its metabolites from the 15-lipoxygenase and cyclooxygenase pathways

2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid with anti-inflammatory properties. Blocking 2-AG hydrolysis to enhance CB2 signaling has proven effective in mouse models of inflammation. However, the expression of 2-AG lipases has never been thoroughly investigated in human leukocytes. Herein, we investigated the expression of seven 2-AG hydrolases by human blood leukocytes and alveolar macrophages (AMs) and found the following protein expression pattern: monoacylglycerol (MAG lipase; eosinophils, AMs, monocytes), carboxylesterase (CES1; monocytes, AMs), palmitoyl-protein thioesterase (PPT1; AMs), α/β-hydrolase domain (ABHD6; mainly AMs), ABHD12 (all), ABHD16A (all), and LYPLA2 (lysophospholipase 2; monocytes, lymphocytes, AMs).We next found that all leukocytes could hydrolyze 2-AG and its metabolites derived from cyclooxygenase-2 (prostaglandin E2-glycerol [PGE2-G]) and the 15-lipoxygenase (15-hydroxy-eicosatetraenoyl-glycerol [15-HETE-G]). Neutrophils and eosinophils were consistently better at hydrolyzing 2-AG and its metabolites than monocytes and lymphocytes. Moreover, the efficacy of leukocytes to hydrolyze 2-AG and its metabolites was 2-AG ≥ 15-HETE-G >> PGE2-G for each leukocyte. Using the inhibitors methylarachidonoyl-fluorophosphonate (MAFP), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), Palmostatin B, 4′-carbamoylbiphenyl-4-yl methyl(3-(pyridin-4-yl)benzyl)carbamate, Nmethyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4′-(aminocarbonyl) [1,1′-biphenyl]-4-yl ester carbamic acid (WWL70), 4′-[[[methyl[[3-(4-pyridinyl)phenyl]methyl]amino]carbonyl]oxy]-[1,1′-biphenyl]-4-carboxylic acid, ethyl ester (WWL113), tetrahydrolipstatin, and ML349, we could not pinpoint a specific hydrolase responsible for the hydrolysis of 2-AG, PGE2-G, and 15-HETE-G by these leukocytes. Furthermore, JZL184, a selective MAG lipase inhibitor, blocked the hydrolysis of 2-AG, PGE2-G, and 15-HETE-G by neutrophils and the hydrolysis of PGE2-G and 15-HETE-G by lymphocytes, two cell types with limited/no MAG lipase. Using an activity-based protein profiling (ABPP) probe to label hydrolases in leukocytes, we found that they expressmanyMAFP-sensitive hydrolases and an unknown JZL184-sensitive hydrolase of ~52 kDa. Altogether, our results indicate that human leukocytes are experts at hydrolyzing 2-AG and its metabolites via multiple lipases and probably via a yet-to-be characterized 52 kDa hydrolase. Blocking 2-AG hydrolysis in humans will likely abrogate the ability of human leukocytes to degrade 2-AG and its metabolites and increase their anti-inflammatory effects in vivo

Evaluating the impact of curfews and other measures on SARS-CoV-2 transmission in French Guiana.

While general lockdowns have proven effective to control SARS-CoV-2 epidemics, they come with enormous costs for society. It is therefore essential to identify control strategies with lower social and economic impact. Here, we report and evaluate the control strategy implemented during a large SARS-CoV-2 epidemic in June-July 2020 in French Guiana that relied on curfews, targeted lockdowns, and other measures. We find that the combination of these interventions coincided with a reduction in the basic reproduction number of SARS-CoV-2 from 1.7 to 1.1, which was sufficient to avoid hospital saturation. We estimate that thanks to the young demographics, the risk of hospitalisation following infection was 0.3 times that of metropolitan France and that about 20% of the population was infected by July. Our model projections are consistent with a recent seroprevalence study. The study showcases how mathematical modelling can be used to support healthcare planning in a context of high uncertainty

Phase II study of olaparib in patients with refractory Ewing sarcoma following failure of standard chemotherapy

Background: Preclinical studies have documented antitumor activity of PARP inhibition both in vitro and in vivo, against Ewing sarcoma cells. This study aimed to translate that observation into a clinical trial to assess the efficacy and tolerability of olaparib, a PARP inhibitor, in patients with advanced Ewing sarcoma (EWS) progressing after prior chemotherapy. Methods: In this nonrandomized phase II trial, adult participants with radiographically measureable metastatic EWS received olaparib tablets, 400 mg orally twice daily, until disease progression or drug intolerance. Tumor measurements were determined by CT or MRI at 6 and 12 weeks after starting olaparib administration, and then every 8 weeks thereafter. Tumor response determinations were made according to RECIST 1.1, and adverse event determinations were made according to CTCAE, version 4.0. A total of 22 participants were planned to be enrolled using a conventional 2-step phase II study design. If no objective responses were observed after 12 participants had been followed for at least 3 months, further accrual would be stopped. Results: 12 participants were enrolled, and all were evaluable. There were no objective responses (PR/CR), 4 SD (duration 10.9, 11.4, 11.9, and 17.9 wks), and 8 PD as best response. Of the SD, 2 had minor responses (−9% and −11.7% by RECIST 1.1). The median time to disease progression was 5.7 weeks. Further enrollment was therefore discontinued. No significant or unexpected toxicities were observed with olaparib, with only a single case each of grade 3 anemia and grade 3 thrombocytopenia observed. Conclusions: This study is the first report of a prospective phase II trial to evaluate the safety and efficacy of a PARP inhibitor in patients with advanced Ewing sarcoma after failure of standard chemotherapy. Olaparib administration was safe and well tolerated when administered to this small heavily pre-treated cohort at the 400 mg BID dose, although the median duration of dosing was for only 5.7 weeks. No significant responses or durable disease control was seen, and the short average interval to disease progression underscores the aggressiveness of this disease. Other studies to combine cytotoxic chemotherapy with PARP inhibition in EWS are actively ongoing. Trial registration ClinicalTrials.gov Identifier: NCT0158354

Assessment of the Effects of Dietary Vitamin D Levels on Olanzapine-Induced Metabolic Side Effects: Focus on the Endocannabinoidome-Gut Microbiome Axis

Vitamin D deficiency is associated with poor mental health and dysmetabolism. Several metabolic abnormalities are associated with psychotic diseases, which can be compounded by atypical antipsychotics that induce weight gain and insulin resistance. These side-effects may be affected by vitamin D levels. The gut microbiota and endocannabinoidome (eCBome) are significant regulators of both metabolism and mental health, but their role in the development of atypical antipsychotic drug metabolic side-effects and their interaction with vitamin D status is unknown. We studied the effects of different combinations of vitamin D levels and atypical antipsychotic drug (olanzapine) exposure on whole-body metabolism and the eCBome-gut microbiota axis in female C57BL/6J mice under a high fat/high sucrose (HFHS) diet in an attempt to identify a link between the latter and the different metabolic outputs induced by the treatments. Olanzapine exerted a protective effect against diet-induced obesity and insulin resistance, largely independent of dietary vitamin D status. These changes were concomitant with olanzapine-mediated decreases in Trpv1 expression and increases in the levels of its agonists, including various N-acylethanolamines and 2-monoacylglycerols, which are consistent with the observed improvement in adiposity and metabolic status. Furthermore, while global gut bacteria community architecture was not altered by olanzapine, we identified changes in the relative abundances of various commensal bacterial families. Taken together, changes of eCBome and gut microbiota families under our experimental conditions might contribute to olanzapine and vitamin D-mediated inhibition of weight gain in mice on a HFHS diet

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.Other UBCNon UBCReviewedFacultyResearche

Leukotriene B₄ Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B₄ <i>In Vitro</i> and <i>In Cellulo</i>

<div><p>LTB₄ is an inflammatory lipid mediator mainly biosynthesized by leukocytes. Since its implication in inflammatory diseases is well recognized, many tools to regulate its biosynthesis have been developed and showed promising results <i>in vitro</i> and <i>in vivo</i>, but mixed results in clinical trials. Recently, the mTOR pathway component p70S6 kinase 1 (p70S6K1) has been linked to LTC₄ synthase and the biosynthesis of cysteinyl-leukotrienes. In this respect, we investigated if p70S6K1 could also play a role in LTB₄ biosynthesis. We thus evaluated the impact of the p70S6K1 inhibitors PF-4708671 and LY2584702 on LTB₄ biosynthesis in human neutrophils. At a concentration of 10 μM, both compounds inhibited S6 phosphorylation, although neither one inhibited the thapsigargin-induced LTB₄ biosynthesis, as assessed by the sum of LTB₄, 20-OH-LTB₄, and 20-COOH-LTB₄. However, PF-4708671, but not LY2584702, inhibited the ω-oxidation of LTB₄ into 20-OH-LTB₄ by intact neutrophils and by recombinant CYP4F3A, leading to increased LTB₄ levels. This was true for both endogenously biosynthesized and exogenously added LTB₄. In contrast to that of 17-octadecynoic acid, the inhibitory effect of PF-4708671 was easily removed by washing the neutrophils, indicating that PF-4708671 was a reversible CYP4F3A inhibitor. At optimal concentration, PF-4708671 increased the half-life of LTB₄ in our neutrophil suspensions by 7.5 fold, compared to 5 fold for 17-octadecynoic acid. Finally, Michaelis-Menten and Lineweaver-Burk plots indicate that PF-4708671 is a mixed inhibitor of CYP4F3A. In conclusion, we show that PF-4708671 inhibits CYP4F3A and prevents the ω-oxidation of LTB₄ <i>in cellulo</i>, which might result in increased LTB₄ levels <i>in vivo</i>.</p></div

Comparison of eight 15-lipoxygenase (LO) inhibitors on the biosynthesis of 15-LO metabolites by human neutrophils and eosinophils.

Neutrophils and eosinophils are important sources of bioactive lipids from the 5- and the 15-lipoxygenase (LO) pathways. Herein, we compared the effectiveness of humans eosinophils and eosinophil-depleted neutrophils to synthesize 15-LO metabolites using a cocktail of different 15-LO substrates as well as their sensitivities to eight documented 15-lipoxygenase inhibitors. The treatment of neutrophils and eosinophils with linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and arachidonyl-ethanolamide, led to the synthesis of 13-HODE, 15-HETrE, 15-HETE, 15-HEPE, 14-HDHA/17-HDHA, and 15-hydroxy-AEA. Neutrophils and eosinophils also metabolized the endocannabinoid 2-arachidonoyl-glycerol into 15-HETE-glycerol, although this required 2-arachidonoyl-glycerol hydrolysis inhibition. Neutrophils and eosinophils differed in regard to dihomo-γ-linolenic acid and linoleic acid utilization with 15-HETrE/13-HODE ratios of 0.014 ± 0.0008 and 0.474 ± 0.114 for neutrophils and eosinophils respectively. 15-LO metabolite synthesis by neutrophils and eosinophils also differed in regard to their relative production of 17-HDHA and 14-HDHA.The synthesis of 15-LO metabolites by neutrophils was concentration-dependent and rapid, reaching a plateau after one minute. While investigating the biosynthetic routes involved, we found that eosinophil-depleted neutrophils express the 15-lipoxygenase-2 but not the 15-LO-1, in contrast to eosinophils which express the 15-LO-1 but not the 15-LO-2. Moreover, 15-LO metabolite synthesis by neutrophils was not inhibited by the 15-LO-1 inhibitors BLX769, BLX3887, and ML351. However, 15-LO product synthesis was partially inhibited by 100 μM NDGA. Altogether, our data indicate that the best 15-LO-1 inhibitors in eosinophils are BLX3887, BLX769, NDGA and ML351 and that the synthesis of 15-LO metabolites by neutrophils does not involve the 15-LO-1 nor the phosphorylation of 5-LO on Ser-663 but is rather the consequence of 15-LO-2 or another unidentified 15-LO

Removal of the inhibitory constraint exerted by CYP4F3A inhibitors on LTB₄ ω-oxidation in neutrophils.

<p>Pre-warmed human neutrophil suspensions (37°C, 5 million cells/ml in HBSS containing 1.6 mM CaCl₂) were incubated with <b>A,B)</b> 30 μM PF-4708671 or vehicle for 5 minutes, <b>C)</b> 30 μM PF-4708671 for 15 minutes, <b>D,E)</b> 30 μM 17-ODYA for 30 minutes, or <b>F)</b> 30 μM 17-ODYA for 15 minutes. Neutrophils were washed (or not) with autologous plasma or HBSS-CaCl₂ as described in methods. <b>A,D)</b> 100 nM thapsigargin or <b>B,C,E,F)</b> 1 μM LTB₄ were then added for 10 and 20 minutes, respectively. Samples then were processed and analyzed for 20-OH-LTB₄ and 20-COOH-LTB₄ as described in methods. Data are the mean (± S.D) of 4 independent experiments, each performed in duplicate.</p