Set up and validation of a sensitive method to quantify prostaglandins, prostaglandin-glycerol esters and prostaglandin-ethanolamides, as well as their respective precursors.

Arachidonic acid-derived prostaglandins are widely studied for their role in inflammation. However, besides arachidonic acid, other arachidonic moiety-containing lipids can be metabolized by COX-2. Indeed, the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) can follow the same biochemical pathways than arachidonic acid leading to the formation of prostaglandin-glycerol esters (PG-G) and prostaglandin-ethanolamides (or prostamides, PG-EA), respectively. The data reported so far support the interest of these bioactive lipids in inflammatory conditions. However, there is only a handful of methods described for their quantification in biological matrices. Moreover, given the shared biochemical pathways for arachidonic acid, 2-AG and AEA, a method allowing for the quantification of these precursors and the corresponding prostaglandin derivatives appears as largely needed. Thus, we report here the development and validation of a single run UPLC-MS/MS quantification method allowing the quantification of these endocannabinoids-derived mediators together with the classical prostaglandin. Moreover, we applied the method to the quantification of these lipids in vitro (using lipopolysaccharides-activated J774 macrophage cells) and in vivo in several tissues from DSS-induced colitis mice. This femtomole-range method should improve the understanding of the interaction between these lipid mediators and inflammation.

"To brain or not to brain": evaluating the possible direct effects of the satiety factor oleoylethanolamide in the central nervous system.

Introduction: Oleoylethanolamide (OEA), an endogenous N-acylethanolamine acting as a gut-to-brain signal to control food intake and metabolism, has been attracting attention as a target for novel therapies against obesity and eating disorders. Numerous observations suggested that the OEA effects might be peripherally mediated, although they involve central pathways including noradrenergic, histaminergic and oxytocinergic systems of the brainstem and the hypothalamus. Whether these pathways are activated directly by OEA or whether they are downstream of afferent nerves is still highly debated. Some early studies suggested vagal afferent fibers as the main route, but our previous observations have contradicted this idea and led us to consider the blood circulation as an alternative way for OEA's central actions. Methods: To test this hypothesis, we first investigated the impact of subdiaphragmatic vagal deafferentation (SDA) on the OEA-induced activation of selected brain nuclei. Then, we analyzed the pattern of OEA distribution in plasma and brain at different time points after intraperitoneal administration in addition to measuring food intake. Results: Confirming and extending our previous findings that subdiaphragmatic vagal afferents are not necessary for the eating-inhibitory effect of exogenous OEA, our present results demonstrate that vagal sensory fibers are also not necessary for the neurochemical effects of OEA. Rather, within a few minutes after intraperitoneal administration, we found an increased concentration of intact OEA in different brain areas, associated with the inhibition of food intake. Conclusion: Our results support that systemic OEA rapidly reaches the brain via the circulation and inhibits eating by acting directly on selected brain nuclei.

Inulin increases the beneficial effects of rhubarb supplementation on high-fat high-sugar diet-induced metabolic disorders in mice: impact on energy expenditure, brown adipose tissue activity, and microbiota.

Consumption of prebiotics and plant-based compounds have many beneficial health effects through modulation of gut microbiota composition and are considered as promising nutritional strategy for the treatment of metabolic diseases. In the present study, we assessed the separated and combined effects of inulin and rhubarb on diet-induced metabolic disease in mice. We showed that supplementation with both inulin and rhubarb abolished the total body and fat mass gain upon high-fat and high-sucrose diet (HFHS) as well as several obesity-associated metabolic disorders. These effects were associated with increased energy expenditure, lower whitening of the brown adipose tissue, higher mitochondria activity and increased expression of lipolytic markers in white adipose tissue. Despite modifications of intestinal gut microbiota and bile acid compositions by inulin or rhubarb alone, combination of both inulin and rhubarb had minor additional impact on these parameters. However, the combination of inulin and rhubarb increased the expression of several antimicrobial peptides and higher goblet cell numbers, thereby suggesting a reinforcement of the gut barrier. Together, these results suggest that the combination of inulin and rhubarb in mice potentiates beneficial effects of separated rhubarb and inulin on HFHS-related metabolic disease and could be considered as nutritional strategy for the prevention and treatment of obesity and related pathologies.

Lipid nanocapsules for the nose-to-brain delivery of the anti-inflammatory bioactive lipid PGD2-G.

Here, prostaglandin D2-glycerol ester (PGD2-G) was selected to target neuroinflammation. As PGD2-G is reported to have a short plasmatic half-life, we propose to use lipid nanocapsules (LNC) as vehicle to safely transport PGD2-G to the central nervous system (CNS). PGD2-G-loaded LNC (PGD2-G-LNC) reduced pro-inflammatory cytokine expression in activated microglial cells, even so after crossing a primary olfactory cell monolayer. A single nasal administration of PGD2-G-LNC in lipopolysaccharide (LPS)-treated mice reduced pro-inflammatory cytokine expression in the olfactory bulb. Coating LNC's surface with a cell-penetrating peptide, transactivator of transcription (TAT), increased its accumulation in the brain. Although TAT-coated PGD2-G-LNC modestly exerted its anti-inflammatory effect in a mouse model of multiple sclerosis similar to free PGD2-G after nasal administration, TAT-coated LNC surprisingly reduced the expression of pro-inflammatory chemokines in the CNS. These data propose LNC as an interesting drug delivery tool and TAT-coated PGD2-G-LNC remains a good candidate, in need of further work.

Genetic Polymorphisms in SLCO2B1 and ABCC1 Conjointly Modulate Atorvastatin Intracellular Accumulation in HEK293 Recombinant Cell Lines.

BACKGROUND: Although atorvastatin (ATV) is well-tolerated, patients may report muscle complaints. These are difficult to predict owing to high interindividual variability. Such side effects are linked to intramuscular accumulation of ATV. This study aimed to investigate the relative role of transporters expressed in muscle tissue in promoting or limiting drug access to cells. The impact of common single nucleotide polymorphisms (SNPs) in SLCO2B1 coding for OATP2B1 and ABCC1 coding for MRP1 on ATV transport was also evaluated. METHODS: HEK293 cells were stably transfected with plasmids containing cDNA encoding wild-type or variant SLCO2B1 and/or ABCC1 to generate single and double stable transfectant HEK293 recombinant models overexpressing variant or wild-type OATP2B1 (influx) and/or MRP1 (efflux) proteins. Variant plasmids were generated by site-directed mutagenesis. Expression analyses were performed to validate recombinant models. Accumulation and efflux experiments were performed at different concentrations. ATV was quantified by LC-MS/MS, and kinetic parameters were compared between single and double HEK transfectants expressing wild-type and variant proteins. RESULTS: The results confirm the involvement of OATP2B1 and MRP1 in ATV cellular transport because it was demonstrated that intracellular accumulation of ATV was boosted by OATP2B1 overexpression, whereas ATV accumulation was decreased by MRP1 overexpression. In double transfectants, it was observed that increased ATV intracellular accumulation driven by OATP2B1 influx was partially counteracted by MRP1 efflux. The c.935G > A SNP in SLCO2B1 was associated with decreased ATV OATP2B1-mediated influx, whereas the c.2012G > T SNP in ABCC1 seemed to increase MRP1 efflux activity against ATV. CONCLUSIONS: Intracellular ATV accumulation is regulated by OATP2B1 and MRP1 transporters, whose functionality is modulated by natural genetic variants. This is significant because it may play a role in ATV muscle side-effect susceptibility.

Dual-drug loaded nanomedicine hydrogel as a therapeutic platform to target both residual glioblastoma and glioma stem cells.

Glioblastoma (GBM) recurrences are inevitable, and mainly originate from residual tumor cells and the presence of glioma stem cells (GSC) around the resection cavity borders. We previously showed that the local treatment of GBM with nanomedicine-based Lauroyl-gemcitabine lipid nanocapsules (GemC12-LNC) hydrogel delayed tumor onset in various preclinical models and can be used as a scaffold to deliver multiple drugs. However, it does not inhibit tumor relapse in the long-term. In this work, we aim at encapsulating an anti-GSC molecule in the GemC12-LNC hydrogel to eliminate both GBM cells and GSC. We performed a screening on GBM cell lines (GL261 and U-87 MG) and patient-derived GSC (GBM9) to select the anti-GSC molecule that could act synergically with GemC12. Based on our results, salinomycin (Sal) and curcumin (Cur) were selected for further development. Both GemC12-Sal-LNC and GemC12-Cur LNC showed similar size (55 nm), zeta potential (- 2 mV) and viscoelastic properties compared to the GemC12-LNC hydrogel. Encapsulation efficiency was above 95 %. Moreover, the GemC12-Sal-LNC hydrogel was stable for at least 6 months and released both drugs over 30 days in vitro. Both hydrogels inhibited the growth of GL261 and U-87 MG spheroids. Flow cytometry analysis showed that Sal reduced the GSC population in GL261 and U-87 MG cells. Our results show that the co-encapsulation of Sal in the GemC12-LNC hydrogel can reduce both GBM cells and GSC, and therefore might be promising to avoid the onset of GBM recurrences.

Contribution of Membrane Vesicle to Reprogramming of Bacterial Membrane Fluidity in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen capable of resisting environmental insults by applying various strategies, including regulating membrane fluidity and producing membrane vesicles (MVs). This study examined the difference in membrane fluidity between planktonic and biofilm modes of growth in P. aeruginosa and whether the ability to alter membrane rigidity in P. aeruginosa could be transferred via MVs. To this end, planktonic and biofilm P. aeruginosa were compared with respect to the lipid composition of their membranes and their MVs and the expression of genes contributing to alteration of membrane fluidity. Additionally, viscosity maps of the bacterial membrane in planktonic and biofilm lifestyles and under the effect of incubation with bacterial MVs were obtained. Further, the growth rate and biofilm formation capability of P. aeruginosa in the presence of MVs were compared. Results showed that the membrane of the biofilm bacteria is significantly less fluid than the membrane of the planktonic bacteria and is enriched with saturated fatty acids. Moreover, the enzymes involved in altering the structure of existing lipids and favoring membrane rigidification are overexpressed in the biofilm bacteria. MVs of biofilm P. aeruginosa elicit membrane rigidification and delay the bacterial growth in the planktonic lifestyle; conversely, they enhance biofilm development in P. aeruginosa. Overall, the study describes the interplay between the planktonic and biofilm bacteria by shedding light on the role of MVs in altering membrane fluidity. IMPORTANCE Membrane rigidification is a survival strategy in Pseudomonas aeruginosa exposed to stress. Despite various studies dedicated to the mechanism behind this phenomenon, not much attention has been paid to the contribution of the bacterial membrane vesicles (MVs) in this regard. This study revealed that P. aeruginosa rigidifies its membrane in the biofilm mode of growth. Additionally, the capability of decreasing membrane fluidity is transferable to the bacterial population via the bacterial MVs, resulting in reprogramming of bacterial membrane fluidity. Given the importance of membrane rigidification for decreasing the pathogen's susceptibility to antimicrobials, elucidation of the conditions leading to such biophysicochemical modulation of the P. aeruginosa membrane should be considered for the purpose of developing therapeutic approaches against this resistant pathogen.

Tumor Metabolism Is Affected by Obesity in Preclinical Models of Triple-Negative Breast Cancer.

Obesity is characterized by an excessive fat mass accumulation associated with multiple disorders, including impaired glucose homeostasis, altered adipokine levels, and hyperlipidemia. Despite clear associations between tumor progression and obesity, the effects of these disorders on tumor metabolism remain largely unknown. Thus, we studied the metabolic differences between tumors of obese and lean mice in murine models of triple-negative breast cancer (E0771 and PY8819). For this purpose, a real-time hyperpolarized 1-13C-pyruvate-to-lactate conversion was studied before and after glucose administration in fasting mice. This work was completed by U-13C glucose tracing experiments using nuclear magnetic resonance (NMR) spectroscopy, as well as mass spectrometry (MS). Ex vivo analyses included immunostainings of major lipid, glucose, and monocarboxylic acids transporters. On the one hand, we discovered that tumors of obese mice yield higher lactate/pyruvate ratios after glucose administration. On the other hand, we found that the same tumors produce higher levels of lactate and alanine from glucose than tumors from lean mice, while no differences on the expression of key transporters associated with glycolysis (i.e., GLUT1, MCT1, MCT4) have been observed. In conclusion, our data suggests that breast tumor metabolism is regulated by the host's physiological status, such as obesity and diabetes.

Dysosmobacter welbionis is a newly isolated human commensal bacterium preventing diet-induced obesity and metabolic disorders in mice.

OBJECTIVE: To investigate the abundance and the prevalence of Dysosmobacter welbionis J115T, a novel butyrate-producing bacterium isolated from the human gut both in the general population and in subjects with metabolic syndrome. To study the impact of this bacterium on host metabolism using diet-induced obese and diabetic mice. DESIGN: We analysed the presence and abundance of the bacterium in 11 984 subjects using four human cohorts (ie, Human Microbiome Project, American Gut Project, Flemish Gut Flora Project and Microbes4U). Then, we tested the effects of daily oral gavages with live D. welbionis J115T on metabolism and several hallmarks of obesity, diabetes, inflammation and lipid metabolism in obese/diabetic mice. RESULTS: This newly identified bacterium was detected in 62.7%-69.8% of the healthy population. Strikingly, in obese humans with a metabolic syndrome, the abundance of Dysosmobacter genus correlates negatively with body mass index, fasting glucose and glycated haemoglobin. In mice, supplementation with live D. welbionis J115T, but not with the pasteurised bacteria, partially counteracted diet-induced obesity development, fat mass gain, insulin resistance and white adipose tissue hypertrophy and inflammation. In addition, live D. welbionis J115T administration protected the mice from brown adipose tissue inflammation in association with increased mitochondria number and non-shivering thermogenesis. These effects occurred with minor impact on the mouse intestinal microbiota composition. CONCLUSIONS: These results suggest that D. welbionis J115T directly and beneficially influences host metabolism and is a strong candidate for the development of next-generation beneficial bacteria targeting obesity and associated metabolic diseases.

Atorvastatin population pharmacokinetics in a real-life setting: Influence of genetic polymorphisms and association with clinical response.

The purpose of this study was to investigate the potential clinical relevance of estimating the apparent clearance (CL/F) of atorvastatin through population pharmacokinetic (PopPK) modeling with samples collected in a real-life setting in a cohort of ambulatory patients at risk of cardiovascular disease by using an opportunistic sampling strategy easily accessible in clinical routine. A total of 132 pharmacokinetic (PK) samples at a maximum of three visits were collected in the 70 included patients. The effects of demographic, genetic, and clinical covariates were also considered. With the collected data, we developed a two-compartment PopPK model that allowed estimating atorvastatin CL/F relatively precisely and considering the genotype of the patient for SLCO1B1 c.521T>C single-nucleotide polymorphism (SNP). Our results indicate that the estimation of the CL/F of atorvastatin through our PopPK model might help in identifying patients at risk of myalgia. Indeed, we showed that a patient presenting a CL/F lower than 414.67 L h-1 is at risk of suffering from muscle discomfort. We also observed that the CL/F was correlated with the efficacy outcomes, suggesting that a higher CL/F is associated with a better drug efficacy (i.e., a greater decrease in total and LDL-cholesterol levels). In conclusion, our study demonstrates that PopPK modeling can be useful in daily clinics to estimate a patient' atorvastatin clearance. Notifying the clinician with this information can help in identifying patients at risk of myalgia and gives indication about the potential responsiveness to atorvastatin therapy.

N-Acylethanolamine-Hydrolyzing Acid Amidase Inhibition, but Not Fatty Acid Amide Hydrolase Inhibition, Prevents the Development of Experimental Autoimmune Encephalomyelitis in Mice.

N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE.

Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: two sides of the same coin.

BACKGROUND: Leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice are commonly used mice models mimicking the conditions of obesity and type 2 diabetes development. However, although ob/ob and db/db mice are similarly gaining weight and developing massive obesity, db/db mice are more diabetic than ob/ob mice. It remains still unclear why targeting the same pathway-leptin signaling-leads to the development of two different phenotypes. Given that gut microbes dialogue with the host via different metabolites (e.g., short-chain fatty acids) but also contribute to the regulation of bile acids metabolism, we investigated whether inflammatory markers, bacterial components, bile acids, short-chain fatty acids, and gut microbes could contribute to explain the specific phenotype discriminating the onset of an obese and/or a diabetic state in ob/ob and db/db mice. RESULTS: Six-week-old ob/ob and db/db mice were followed for 7 weeks; they had comparable body weight, fat mass, and lean mass gain, confirming their severely obese status. However, as expected, the glucose metabolism and the glucose-induced insulin secretion were significantly different between ob/ob and db/db mice. Strikingly, the fat distribution was different, with db/db mice having more subcutaneous and ob/ob mice having more epididymal fat. In addition, liver steatosis was more pronounced in the ob/ob mice than in db/db mice. We also found very distinct inflammatory profiles between ob/ob and db/db mice, with a more pronounced inflammatory tone in the liver for ob/ob mice as compared to a higher inflammatory tone in the (subcutaneous) adipose tissue for db/db mice. When analyzing the gut microbiota composition, we found that the quantity of 19 microbial taxa was in some way affected by the genotype. Furthermore, we also show that serum LPS concentration, hepatic bile acid content, and cecal short-chain fatty acid profiles were differently affected by the two genotypes. CONCLUSION: Taken together, our results elucidate potential mechanisms implicated in the development of an obese or a diabetic state in two genetic models characterized by an altered leptin signaling. We propose that these differences could be linked to specific inflammatory tones, serum LPS concentration, bile acid metabolism, short-chain fatty acid profile, and gut microbiota composition. Video abstract.

Dietary nanoparticles alter the composition and function of the gut microbiota in mice at dose levels relevant for human exposure.

BACKGROUND: Nanotechnologies provide new opportunities for improving the safety, quality, shelf life, flavor and appearance of foods. The most common nanoparticles (NPs) in human diet are silver metal, mainly present in food packaging and appliances, and silicon and titanium dioxides used as additives. The rapid development and commercialization of consumer products containing these engineered NPs is, however, not well supported by appropriate toxicological studies and risk assessment. Local and systemic toxicity and/or disruption of the gut microbiota (GM) have already been observed after oral administration of NPs in experimental animals, but results are not consistent and doses used were often much higher than the estimated human intakes. In view of the strong evidence linking alterations of the GM to cardiometabolic (CM) diseases, we hypothesized that dietary NPs might disturb this GM-CM axis. MATERIALS AND METHODS: We exposed male C57BL/6JRj mice (n = 13 per dose group) to dietary NPs mixed in food pellets at doses relevant for human exposure: Ag (0, 4, 40 or 400 µg/kg pellet), SiO2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO2 (0, 0.4, 4 or 40 mg/kg pellet). After 24 weeks of exposure, we assessed effects on the GM and CM health (n = 8 per dose group). The reversibility of the effects was examined after 8 additional weeks without NPs exposure (recovery period, n ≤ 5 per dose group). RESULTS: No overt toxicity was recorded. The GM ß-diversity was dose-dependently disrupted by the three NPs, and the bacterial short chain fatty acids (SCFAs) were dose-dependently reduced after the administration of SiO2 and TiO2 NPs. These effects disappeared completely or partly after the recovery period, strengthening the association with dietary NPs. We did not observe atheromatous disease or glucose intolerance after NP exposure. Instead, dose-dependent decreases in the expression of IL-6 in the liver, circulating triglycerides (TG) and urea nitrogen (BUN) were recorded after administration of the NPs. CONCLUSION: We found that long-term oral exposure to dietary NPs at doses relevant for estimated human intakes disrupts the GM composition and function. These modifications did not appear associated with atheromatous or deleterious metabolic outcomes.

Systemic effects and impact on the gut microbiota upon subacute oral exposure to silver acetate in rats.

CONTEXT: The addition of silver (Ag) to food items, and its migration from food packaging and appliances results in a dietary exposure in humans, estimated to 70-90 µg Ag/day. In view of the well-known bactericidal activity of Ag ions, concerns arise about a possible impact of dietary Ag on the gut microbiota (GM), which is a master determinant of human health and diseases. Repeated oral administration of Ag acetate (AgAc) can also cause systemic toxicity in rats with reported NOAELs of 4 mg AgAc/b.w./d for impaired fertility and 0.4 mg AgAc/b.w./d for developmental toxicity. OBJECTIVE: The objective of this study was to investigate whether oral exposure to AgAc can induce GM alterations at doses causing reproductive toxicity in rats. METHODS: Male and female Wistar rats were exposed during 10 weeks to AgAc incorporated into food (0, 0.4, 4 or 40 mg/kg b.w./d), and we analyzed the composition of the GM (α- and ß-diversity). We documented bacterial function by measuring short-chain fatty acid (SCFA) production in cecal content. Ferroxidase activity, a biomarker of systemic Ag toxicity, was measured in serum. RESULTS AND CONCLUSIONS: From 4 mg/kg b.w./d onwards, we recorded systemic toxicity, as indicated by the reduction of serum ferroxidase activity, as well as serum Cu and Se concentrations. This systemic toxic response to AgAc might contribute to explain reprotoxic manifestations. We observed a dose-dependent modification of the GM composition in male rats exposed to AgAc. No impact of AgAc exposure on the production of bacterial SCFA was recorded. The limited GM changes recorded in this study do not appear related to a reprotoxicity outcome.

Effects of R-flurbiprofen and the oxygenated metabolites of endocannabinoids in inflammatory pain mice models.

Pain is one of the cardinal signs accompanying inflammation. The prostaglandins (PGs), synthetized from arachidonic acid by cyclooxygenase (COX)-2, are major bioactive lipids implicated in inflammation and pain. However, COX-2 is also able to metabolize other lipids, including the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), to give glycerol ester (PG-G) and ethanolamide (PG-EA) derivatives of the PGs. Consequently, COX-2 can be considered as a hub not only controlling PG synthesis, but also PG-G and PG-EA synthesis. As they were more recently characterized, these endocannabinoid metabolites are less studied in nociception compared to PGs. Interestingly R-profens, previously considered as inactive enantiomers of nonsteroidal anti-inflammatory drugs (NSAIDs), are substrate-selective COX inhibitors. Indeed, R-flurbiprofen can selectively block PG-G and PG-EA production, without affecting PG synthesis from COX-2. Therefore, we compared the effect of R-flurbiprofen and S-flurbiprofen in models of inflammatory pain triggered by local administration of lipopolysaccharides (LPS) and carrageenan in mice. Remarkably, the effects of flurbiprofen enantiomers on mechanical hyperalgesia seem to depend on (i) the inflammatory stimuli, (ii) the route of administration, and (iii) the timing of administration. We also assessed the effect of administration of the PG-Gs, PG-EAs, and PGs on LPS-induced mechanical hyperalgesia. Our data support the interest of studying the nonhydrolytic endocannabinoid metabolism in the context of inflammatory pain.

25-Hydroxycholesterol metabolism is altered by lung inflammation, and its local administration modulates lung inflammation in mice.

Inflammation is a critical component of many lung diseases including asthma and acute lung injury (ALI). Using high-performance liquid chromatography-mass spectrometry, we quantified the levels of oxysterols in two different murine models of lung diseases. These are lipid mediators derived from cholesterol and known to modulate immunity and inflammation. Interestingly, 25-hydroxycholesterol (25-OHC) was the only oxysterol with altered levels during lung inflammation, and its levels were differently affected according to the model. Therefore, we sought to assess how this oxysterol would affect lung inflammatory responses. In a model of lipopolysaccharide (LPS)-induced acute lung inflammation, 25-OHC levels were increased, and most of the hallmarks of the model (eg, leukocyte recruitment, mRNA expression, and secretion of inflammatory cytokines) were decreased following its intratracheal administration. We also found that, when administered in the lung, 25-OHC is metabolized locally into 25-hydroxycholesterol-3-sulfate and 7α,25-dihydroxycholesterol. Their administration in the lungs did not recapitulate all the effects of 25-OHC. Conversely, in a model of allergic asthma induced by intranasal administration of house dust mites (HDM), 25-OHC levels were decreased, and when intranasally administered, this oxysterol worsened the hallmarks of the model (eg, leukocyte recruitment, tissue remodeling [epithelium thickening and peribranchial fibrosis], and cytokine expression) and induced changes in leukotriene levels. Ex vivo, we found that 25-OHC decreases LPS-induced primary alveolar macrophage activation while having no effect on neutrophil activation. Its sulfated metabolite, 25-hydroxycholesterol-3-sulfate, decreased neutrophil, but not macrophage activation. Taken together, our data support a differential role of 25-OHC in ALI and allergic inflammation models.

Diesel exhaust particles alter the profile and function of the gut microbiota upon subchronic oral administration in mice.

BACKGROUND: Ambient air pollution by particulate matters, including diesel exhaust particles (DEP), is a major cause of cardiovascular and metabolic mortality worldwide. The mechanisms by which DEP cause these adverse outcomes are not completely understood. Because the gut microbiota controls cardiovascular and metabolic health, we hypothesized that the fraction of inhaled DEP which reach the gut after mucociliary clearance and swallowing might induce gut dysbiosis and, in turn, contribute to aggravate or induce cardiovascular and metabolic diseases. RESULTS: Female ApoE-/- mice fed a Western diet, and wild-type (C57Bl/6) mice fed standard diet were gavaged with DEP (SRM2975) doses corresponding to mucociliary clearance from inhalation exposure (200 or 1000 ng/day, 3 times a week for 3 months; and 40, 200 or 1000 ng/day, 3 times a week for 6 months, respectively). No mortality, overt systemic or digestive toxicity was observed. A dose-dependent alteration of the gut microbiota was recorded in both strains. In ApoE-/-, ß-diversity was modified by DEP, but no significant modification of the relative abundance of the phyla, families or genera was identified. In C57BL/6 mice, DEP reduced α-diversity (Shannon and Simpson indices), and modified ß-diversity, including a reduction of the Proteobacteria and Patescibacteria phyla, and an increase of the Campylobacterota phylum. In both mouse models, perturbation of the gut microbiota composition was associated with a dose-dependent reduction of bacterial short chain fatty acids (butyrate and propionate) in cecal content. However, DEP ingestion did not aggravate (ApoE-/-), or induce (C57BL/6 mice) atherosclerotic plaques, and no metabolic alteration (glucose tolerance, resistance to insulin, or lipidemia) was recorded. CONCLUSIONS: We show here that oral exposure to DEP, at doses relevant for human health, changes the composition and function of the gut microbiota. These modifications were, however, not translated into ultimate atherosclerotic or metabolic outcomes.

Identification of new enterosynes using prebiotics: roles of bioactive lipids and mu-opioid receptor signalling in humans and mice.

OBJECTIVE: The enteric nervous system (ENS) plays a key role in controlling the gut-brain axis under normal and pathological conditions, such as type 2 diabetes. The discovery of intestinal actors, such as enterosynes, able to modulate the ENS-induced duodenal contraction is considered an innovative approach. Among all the intestinal factors, the understanding of the role of gut microbes in controlling glycaemia is still developed. We studied whether the modulation of gut microbiota by prebiotics could permit the identification of novel enterosynes. DESIGN: We measured the effects of prebiotics on the production of bioactive lipids in the intestine and tested the identified lipid on ENS-induced contraction and glucose metabolism. Then, we studied the signalling pathways involved and compared the results obtained in mice to human. RESULTS: We found that modulating the gut microbiota with prebiotics modifies the actions of enteric neurons, thereby controlling duodenal contraction and subsequently attenuating hyperglycaemia in diabetic mice. We discovered that the signalling pathway involved in these effects depends on the synthesis of a bioactive lipid 12-hydroxyeicosatetraenoic acid (12-HETE) and the presence of mu-opioid receptors (MOR) on enteric neurons. Using pharmacological approaches, we demonstrated the key role of the MOR receptors and proliferator-activated receptor γ for the effects of 12-HETE. These findings are supported by human data showing a decreased expression of the proenkephalin and MOR messanger RNAs in the duodenum of patients with diabetic. CONCLUSIONS: Using a prebiotic approach, we identified enkephalin and 12-HETE as new enterosynes with potential real beneficial and safety impact in diabetic human.

From correlation to causality: the case of Subdoligranulum.

Gut microbes are considered as major factors contributing to human health. Nowadays, the vast majority of the data available in the literature are mostly exhibiting negative or positive correlations between specific bacteria and metabolic parameters. From these observations, putative detrimental or beneficial effects are then inferred. Akkermansia muciniphila is one of the unique examples for which the correlations with health benefits have been causally validated in vivo in rodents and humans. In this study, based on available metagenomic data in overweight/obese population and clinical variables that we obtained from two cohorts of individuals (n = 108) we identified several metagenomic species (MGS) strongly associated with A. muciniphila with one standing out: Subdoligranulum. By analyzing both qPCR and shotgun metagenomic data, we discovered that the abundance of Subdoligranulum was correlated positively with microbial richness and HDL-cholesterol levels and negatively correlated with fat mass, adipocyte diameter, insulin resistance, levels of leptin, insulin, CRP, and IL6 in humans. Therefore, to further explore whether these strong correlations could be translated into causation, we investigated the effects of the unique cultivated strain of Subdoligranulum (Subdoligranulum variabile DSM 15176 T) in obese and diabetic mice as a proof-of-concept. Strikingly, there were no significant difference in any of the hallmarks of obesity and diabetes measured (e.g., body weight gain, fat mass gain, glucose tolerance, liver weight, plasma lipids) at the end of the 8 weeks of treatment. Therefore, the absence of effect following the supplementation with S. variabile indicates that increasing the intestinal abundance of this bacterium is not translated into beneficial effects in mice. In conclusion, we demonstrated that despite the fact that numerous strong correlations exist between a given bacteria and health, proof-of-concept experiments are required to be further validated or not in vivo. Hence, an urgent need for causality studies is warranted to move from human observations to preclinical validations.

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis.

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.