The metabolic benefits of substituting sucrose for maple syrup are associated with a shift in carbohydrate digestion and gut microbiota composition in high-fat high-sucrose diet-fed mice.

Overconsumption of added sugars is now largely recognized as a major culprit in the global situation of obesity and metabolic disorders. Previous animal studies reported that maple syrup (MS) is less deleterious than refined sugars on glucose metabolism and hepatic health, but the mechanisms remain poorly studied. Beyond its content in sucrose, MS is a natural sweetener containing several bioactive compounds, such as polyphenols and inulin, which are potential gut microbiota modifiers. We aimed to investigate the impact of MS on metabolic health and gut microbiota in male C57Bl/6J mice fed a high-fat high-sucrose (HFHS + S) diet or an isocaloric HFHS diet in which a fraction (10% of the total caloric intake) of the sucrose was substituted by MS (HFHS + MS). Insulin and glucose tolerance tests were performed at 5 and 7 wk into the diet, respectively. The fecal microbiota was analyzed by whole-genome shotgun sequencing. Liver lipids and inflammation were determined, and hepatic gene expression was assessed by transcriptomic analysis. Maple syrup was less deleterious on insulin resistance and decreased liver steatosis compared with mice consuming sucrose. This could be explained by the decreased intestinal α-glucosidase activity, which is involved in carbohydrate digestion and absorption. Metagenomic shotgun sequencing analysis revealed that MS intake increased the abundance of Faecalibaculum rodentium, Romboutsia ilealis, and Lactobacillus johnsonii, which all possess gene clusters involved in carbohydrate metabolism, such as sucrose utilization and butyric acid production. Liver transcriptomic analyses revealed that the cytochrome P450 (Cyp450) epoxygenase pathway was differently modulated between HFHS + S- and HFHS + MS-fed mice. These results show that substituting sucrose for MS alleviated dysmetabolism in diet-induced obese mice, which were associated with decreased carbohydrate digestion and shifting gut microbiota.NEW & NOTEWORTHY The natural sweetener maple syrup has sparked much interest as an alternative to refined sugars. This study aimed to investigate whether the metabolic benefits of substituting sucrose with an equivalent dose of maple syrup could be linked to changes in gut microbiota composition and digestion of carbohydrates in obese mice. We demonstrated that maple syrup is less detrimental than sucrose on metabolic health and possesses a prebiotic-like activity through novel gut microbiota and liver mechanisms.

Hypoabsorptive surgeries cause limb-dependent changes in the gut endocannabinoidome and microbiome in association with beneficial metabolic effects.

OBJECTIVE: To determine whether the metabolic benefits of hypoabsorptive surgeries are associated with changes in the gut endocannabinoidome (eCBome) and microbiome. METHODS: Biliopancreatic diversion with duodenal switch (BPD-DS) and single anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) were performed in diet-induced obese (DIO) male Wistar rats. Control groups fed a high-fat diet (HF) included sham-operated (SHAM HF) and SHAM HF-pair-weighed to BPD-DS (SHAM HF-PW). Body weight, fat mass gain, fecal energy loss, HOMA-IR, and gut-secreted hormone levels were measured. The levels of eCBome lipid mediators and prostaglandins were quantified in different intestinal segments by LC-MS/MS, while expression levels of genes encoding eCBome metabolic enzymes and receptors were determined by RT-qPCR. Metataxonomic (16S rRNA) analysis was performed on residual distal jejunum, proximal jejunum, and ileum contents. RESULTS: BPD-DS and SADI-S reduced fat gain and HOMA-IR, while increasing glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) levels in HF-fed rats. Both surgeries induced potent limb-dependent alterations in eCBome mediators and in gut microbial ecology. In response to BPD-DS and SADI-S, changes in gut microbiota were significantly correlated with those of eCBome mediators. Principal component analyses revealed connections between PYY, N-oleoylethanolamine (OEA), N-linoleoylethanolamine (LEA), Clostridium, and Enterobacteriaceae_g_2 in the proximal and distal jejunum and in the ileum. CONCLUSIONS: BPD-DS and SADI-S caused limb-dependent changes in the gut eCBome and microbiome. The present results indicate that these variables could significantly influence the beneficial metabolic outcome of hypoabsorptive bariatric surgeries.

Altered endocannabinoidome bioactive lipid levels accompany reduced DNBS-induced colonic inflammation in germ-free mice.

BACKGROUND: Gut microbiota are involved in the onset and development of chronic intestinal inflammation. The recently described endocannabinoidome (eCBome), a diverse and complex system of bioactive lipid mediators, has been reported to play a role in various physio-pathological processes such as inflammation, immune responses and energy metabolism. The eCBome and the gut microbiome (miBIome) are closely linked and form the eCBome - miBIome axis, which may be of special relevance to colitis. METHODS: Colitis was induced in conventionally raised (CR), antibiotic-treated (ABX) and germ-free (GF) mice with dinitrobenzene sulfonic acid (DNBS). Inflammation was assessed by Disease Activity Index (DAI) score, body weight change, colon weight-length ratio, myeloperoxidase (MPO) activity and cytokine gene expression. Colonic eCBome lipid mediator concentrations were measured by HPLC-MS /MS. RESULTS: GF mice showed increased levels of anti-inflammatory eCBome lipids (LEA, OEA, DHEA and 13- HODE-EA) in the healthy state and higher MPO activity. DNBS elicited reduced inflammation in GF mice, having lower colon weight/length ratios and lower expression levels of Il1b, Il6, Tnfa and neutrophil markers compared to one or both of the other DNBS-treated groups. Il10 expression was also lower and the levels of several N-acyl ethanolamines and 13-HODE-EA levels were higher in DNBS-treated GF mice than in CR and ABX mice. The levels of these eCBome lipids negatively correlated with measures of colitis and inflammation. CONCLUSIONS: These results suggest that the depletion of the gut microbiota and subsequent differential development of the gut immune system in GF mice is followed by a compensatory effect on eCBome lipid mediators, which may explain, in part, the observed lower susceptibility of GF mice to develop DNBS-induced colitis.

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.

Chemokines and eicosanoids fuel the hyperinflammation within the lungs of patients with severe COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to a variety of clinical outcomes, ranging from the absence of symptoms to severe acute respiratory disease and ultimately death. A feature of patients with severe coronavirus disease 2019 (COVID-19) is the abundance of inflammatory cytokines in the blood. Elevated levels of cytokines are predictive of infection severity and clinical outcome. In contrast, studies aimed at defining the driving forces behind the inflammation in lungs of subjects with severe COVID-19 remain scarce. OBJECTIVE: Our aim was to analyze and compare the plasma and bronchoalveolar lavage (BAL) fluids of patients with severe COVID-19 (n = 45) for the presence of cytokines and lipid mediators of inflammation (LMIs). METHODS: Cytokines were measured by using Luminex multiplex assay, and LMIs were measured by using liquid chromatography-tandem mass spectrometry. RESULTS: We revealed high concentrations of numerous cytokines, chemokines, and LMIs in the BAL fluid of patients with severe COVID-19. Of the 13 most abundant mediators in BAL fluid, 11 were chemokines, with CXCL1 and CXCL8 being 200 times more abundant than IL-6 and TNF-α. Eicosanoid levels were also elevated in the lungs of subjects with severe COVID-19. Consistent with the presence chemotactic molecules, BAL fluid samples were enriched for neutrophils, lymphocytes, and eosinophils. Inflammatory cytokines and LMIs in plasma showed limited correlations with those present in BAL fluid, arguing that circulating inflammatory molecules may not be a reliable proxy of the inflammation occurring in the lungs of patients with severe COVID-19. CONCLUSIONS: Our findings indicate that hyperinflammation of the lungs of patients with severe COVID-19 is fueled by excessive production of chemokines and eicosanoids. Therapeutic strategies to dampen inflammation in patients with COVID-19 should be tailored accordingly.

Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust biosynthesis of 2-arachidonoyl-glycerol and its congeners in human myeloid leukocytes.

The endocannabinoid (eCB) 2-arachidonoyl-gycerol (2-AG) modulates immune responses by activating cannabinoid receptors or through its multiple metabolites, notably eicosanoids. Thus, 2-AG hydrolysis inhibition might represent an interesting anti-inflammatory strategy that would simultaneously increase the levels of 2-AG and decrease those of eicosanoids. Accordingly, 2-AG hydrolysis inhibition increased 2-AG half-life in neutrophils. Under such setting, neutrophils, eosinophils, and monocytes synthesized large amounts of 2-AG and other monoacylglycerols (MAGs) in response to arachidonic acid (AA) and other unsaturated fatty acids (UFAs). Arachidonic acid and UFAs were ~1000-fold more potent than G protein-coupled receptor (GPCR) agonists. Triascin C and thimerosal, which, respectively, inhibit fatty acyl-CoA synthases and acyl-CoA transferases, prevented the UFA-induced MAG biosynthesis, implying glycerolipid remodeling. 2-AG and other MAG biosynthesis was preceded by that of the corresponding lysophosphatidic acid (LPA). However, we could not directly implicate LPA dephosphorylation in MAG biosynthesis. While GPCR agonists poorly induced 2-AG biosynthesis, they inhibited that induced by AA by 25%-50%, suggesting that 2-AG biosynthesis is decreased when leukocytes are surrounded by a pro-inflammatory entourage. Our data strongly indicate that human leukocytes use AA and UFAs to biosynthesize biologically significant concentrations of 2-AG and other MAGs and that hijacking the immune system with 2-AG hydrolysis inhibitors might diminish inflammation in humans.

Intuitive eating is associated with elevated levels of circulating omega-3-polyunsaturated fatty acid-derived endocannabinoidome mediators.

The regulation of food intake and eating behaviours involves interactions between different systems. The endocannabinoidome, comprising several fatty acid-derived mediators, plays a central role in the regulation of food intake. Alterations of this system have been suggested to intervene in the aetiology of eating disorders. This study aimed to examine the associations between non-pathological eating behaviours and circulating endocannabinoidome mediators in a heterogeneous human population. Plasma 2-monoacyl-glycerol and N-acyl-ethanolamine congeners were measured by LC-MS/MS in a sample of 190 men and women. Eating behaviours were assessed using the Three-Factor Eating Questionnaire (TFEQ) and the Intuitive Eating Scale-2 (IES-2). Following adjustment for body mass index and age, plasma levels of omega-3 polyunsaturated fatty acid-derived 2-monoacyl-glycerols, 2-eicosapentaenoyl-glycerol (2-EPG) and 2-docosapentaenoyl-glycerol (2-DPG), were associated with higher intuitive eating scores (0.15 ≤ rho ≤ 0.20; p < 0.05). These associations were independent of the dietary intake of the fatty acid precursors of these 2-monoacyl-glycerols. However, almost no association was found between plasma levels of N-acyl-ethanolamine congeners and the TFEQ or the IES-2 scores. The results of the present study suggest the association of 2-monoacyl-glycerols, especially 2-EPG and 2-DPG, in the regulation of intuitive eating and the potential implication therein of bioactive lipids.

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis.

BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

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.

Three of a Kind: Control of the Expression of Liver-Expressed Antimicrobial Peptide 2 (LEAP2) by the Endocannabinoidome and the Gut Microbiome.

The endocannabinoidome (expanded endocannabinoid system, eCBome)-gut microbiome (mBIome) axis plays a fundamental role in the control of energy intake and processing. The liver-expressed antimicrobial peptide 2 (LEAP2) is a recently identified molecule acting as an antagonist of the ghrelin receptor and hence a potential effector of energy metabolism, also at the level of the gastrointestinal system. Here we investigated the role of the eCBome-gut mBIome axis in the control of the expression of LEAP2 in the liver and, particularly, the intestine. We confirm that the small intestine is a strong contributor to the circulating levels of LEAP2 in mice, and show that: (1) intestinal Leap2 expression is profoundly altered in the liver and small intestine of 13 week-old germ-free (GF) male mice, which also exhibit strong alterations in eCBome signaling; fecal microbiota transfer (FMT) from conventionally raised to GF mice completely restored normal Leap2 expression after 7 days from this procedure; in 13 week-old female GF mice no significant change was observed; (2) Leap2 expression in organoids prepared from the mouse duodenum is elevated by the endocannabinoid noladin ether, whereas in human Caco-2/15 epithelial intestinal cells it is elevated by PPARγ activation by rosiglitazone; (3) Leap2 expression is elevated in the ileum of mice with either high-fat diet-or genetic leptin signaling deficiency-(i.e., ob/ob and db/db mice) induced obesity. Based on these results, we propose that LEAP2 originating from the small intestine may represent a player in eCBome- and/or gut mBIome-mediated effects on food intake and energy metabolism.

Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.

The gut microbiota is a unique ecosystem of microorganisms interacting with the host through several biochemical mechanisms. The endocannabinoidome (eCBome), a complex signaling system including the endocannabinoid system, approximately 50 receptors and metabolic enzymes, and more than 20 lipid mediators with important physiopathologic functions, modulates gastrointestinal tract function and may mediate host cell-microbe communications there. Germ-free (GF) mice, which lack an intestinal microbiome and so differ drastically from conventionally raised (CR) mice, offer a unique opportunity to explore the eCBome in a microbe-free model and in the presence of a reintroduced functional gut microbiome through fecal microbiota transplant (FMT). We aimed to gain direct evidence for a link between the microbiome and eCBome systems by investigating eCBome alterations in the gut in GF mice before and after FMT. Basal eCBome gene expression and lipid profiles were measured in various segments of the intestine of GF and CR mice at juvenile and adult ages using targeted quantitative PCR transcriptomics and LC-MS/MS lipidomics. GF mice exhibited age-dependent modifications in intestinal eCBome gene expression and lipid mediator levels. FMT from CR donor mice to age-matched GF male mice reversed several of these alterations, particularly in the ileum and jejunum, after only 1 week, demonstrating that the gut microbiome directly impacts the host eCBome and providing a cause-effect relationship between the presence or absence of intestinal microbes and eCBome signaling. These results open the way to new studies investigating the mechanisms through which intestinal microorganisms exploit eCBome signaling to exert some of their physiopathologic functions.

Lipopolysaccharide impacts murine CD103+ DC differentiation, altering the lung DC population balance.

Conventional DCs are a heterogeneous population that bridge the innate and adaptive immune systems. The lung DC population comprises CD103+ XCR1+ DC1s and CD11b+ DC2s; their various combined functions cover the whole spectrum of immune responses needed to maintain homeostasis. Here, we report that in vivo exposure to LPS leads to profound alterations in the proportions of CD103+ XCR1+ DCs in the lung. Using ex vivo LPS and TNF stimulations of murine lung and spleen-isolated DCs, we show that this is partly due to a direct downregulation of the GM-CSF-induced DC CD103 expression. Furthermore, we demonstrate that LPS-induced systemic inflammation alters the transcriptional signature of DC precursors toward a lower capacity to differentiate into XCR1+ DCs. Also, we report that TNF prevents the capacity of pre-DCs to express CD103 upon maturation. Overall, our results indicate that exposure to LPS directly impacts the capacity of pre-DCs to differentiate into XCR1+ DCs, in addition to lowering their capacity to express CD103. This leads to decreased proportions of CD103+ XCR1+ DCs in the lung, favoring CD11b+ DCs, which likely plays a role in the break in homeostasis following LPS exposure, and in determining the nature of the immune response to LPS.

The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by eicosanoid biosynthetic enzymes. For example, N-arachidonoyl-ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study's aim was to define the effects of these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo.

Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12.

Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.

Activation of TAK1 by Chemotactic and Growth Factors, and Its Impact on Human Neutrophil Signaling and Functional Responses.

The MAP3 kinase, TAK1, is known to act upstream of IKK and MAPK cascades in several cell types, and is typically activated in response to cytokines (e.g., TNF, IL-1) and TLR ligands. In this article, we report that in human neutrophils, TAK1 can also be activated by different classes of inflammatory stimuli, namely, chemoattractants and growth factors. After stimulation with such agents, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the inducible phosphorylation of ERK occurring in response to these stimuli but had little or no effect on that of p38 MAPK or PI3K. Inhibition of TAK1 also impaired MEKK3 (but not MEKK1) activation by fMLF. Moreover, both TAK1 and the MEK/ERK module were found to influence inflammatory cytokine expression and release in fMLF- and GM-CSF-activated neutrophils, whereas the PI3K pathway influenced this response independently of TAK1. Besides cytokine production, other responses were found to be under TAK1 control in neutrophils stimulated with chemoattractants and/or GM-CSF, namely, delayed apoptosis and leukotriene biosynthesis. Our data further emphasize the central role of TAK1 in controlling signaling cascades and functional responses in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.

The CB2 receptor and its role as a regulator of inflammation.

The CB2 receptor is the peripheral receptor for cannabinoids. It is mainly expressed in immune tissues, highlighting the possibility that the endocannabinoid system has an immunomodulatory role. In this respect, the CB2 receptor was shown to modulate immune cell functions, both in cellulo and in animal models of inflammatory diseases. In this regard, numerous studies have reported that mice lacking the CB2 receptor have an exacerbated inflammatory phenotype. This suggests that therapeutic strategies aiming at modulating CB2 signaling could be promising for the treatment of various inflammatory conditions. Herein, we review the pharmacology of the CB2 receptor, its expression pattern, and the signaling pathways induced by its activation. We next examine the regulation of immune cell functions by the CB2 receptor and the evidence obtained from primary human cells, immortalized cell lines, and animal models of inflammation. Finally, we discuss the possible therapies targeting the CB2 receptor and the questions that remain to be addressed to determine whether this receptor could be a potential target to treat inflammatory disease.

Plasma eicosanoid profiles determined by high-performance liquid chromatography coupled with tandem mass spectrometry in stimulated peripheral blood from healthy individuals and sickle cell anemia patients in treatment.

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Correlation between CCL26 production by human bronchial epithelial cells and airway eosinophils: Involvement in patients with severe eosinophilic asthma.

BACKGROUND: High pulmonary eosinophil counts are associated with asthma symptoms and severity. Bronchial epithelial cells (BECs) produce CC chemokines, notably CCL26 (eotaxin-3), which recruits and activates eosinophils from asthmatic patients. This suggests that CCL26 production by BECs might be involved in persistent eosinophilia in patients with severe asthma despite treatment with high corticosteroid doses. OBJECTIVE: We sought to determine whether CCL26 levels correlate with eosinophilia and asthma severity. METHODS: Human CC chemokine expression was assessed by means of quantitative PCR or a quantitative PCR array in vehicle- or IL-13-treated BECs. CCL26 was quantitated by means of ELISA. Immunohistochemistry analyses of CCL26 and major basic protein were done on bronchial biopsy specimens. RESULTS: IL-13 selectively induced CCL26 expression by BECs. This increase was time-dependent and more prominent in BECs from patients with severe eosinophilic asthma. CCL26 levels measured in supernatants of IL-13-stimulated BECs also increased with asthma severity as follows: patients with severe eosinophilic asthma > patients with mild asthma ≈ healthy subjects. Immunohistochemistry analyses of bronchial biopsy specimens confirmed increased levels of CCL26 in the epithelium of patients with mild and those with severe eosinophilic asthma. Tissue eosinophil counts did not correlate with CCL26 staining. However, sputum CCL26 levels significantly correlated with sputum eosinophil counts (P < .0001), suggesting that CCL26 participates in the movement of eosinophils from the tissues to the airway lumen. CONCLUSIONS: These results show a relation between CCL26 production by IL-13-stimulated BECs, sputum eosinophil counts, and asthma severity. They also suggest a role for CCL26 in the sustained inflammation observed in patients with severe eosinophilic asthma and reveal CCL26 as a potential target for treating patients with eosinophilic asthma that are refractory to classic therapies.

Pulmonary CD103 expression regulates airway inflammation in asthma.

Although CD103(+) cells recently emerged as key regulatory cells in the gut, the role of CD103 ubiquitous expression in the lung and development of allergic airway disease has never been studied. To answer this important question, we evaluated the response of Cd103(-/-) mice in two separate well-described mouse models of asthma (ovalbumin and house dust mite extract). Pulmonary inflammation was assessed by analysis of bronchoalveolar lavage content, histology, and cytokine response. CD103 expression was analyzed on lung dendritic cells and T cell subsets by flow cytometry. Cd103(-/-) mice exposed to antigens developed exacerbated lung inflammation, characterized by increased eosinophilic infiltration, severe tissue inflammation, and altered cytokine response. In wild-type mice exposed to house dust mite, CD103(+) dendritic cells are increased in the lung and an important subset of CD4(+) T cells, CD8(+) T cells, and T regulatory cells express CD103. Importantly, Cd103(-/-) mice presented a deficiency in the resolution phase of inflammation, which supports an important role for this molecule in the control of inflammation severity. These results suggest an important role for CD103 in the control of airway inflammation in asthma.

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma.

BACKGROUND: In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus. METHODS: BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes. RESULTS: AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4(+) T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs. CONCLUSION: Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.