CARD9 in neutrophils protects from colitis and controls mitochondrial metabolism and cell survival.

OBJECTIVES: Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 (Card9), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown. DESIGN: We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of Card9 deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse). RESULTS: Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation. CONCLUSION: These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.

Rewiring the altered tryptophan metabolism as a novel therapeutic strategy in inflammatory bowel diseases.

OBJECTIVE: The extent to which tryptophan (Trp) metabolism alterations explain or influence the outcome of inflammatory bowel diseases (IBDs) is still unclear. However, several Trp metabolism end-products are essential to intestinal homeostasis. Here, we investigated the role of metabolites from the kynurenine pathway. DESIGN: Targeted quantitative metabolomics was performed in two large human IBD cohorts (1069 patients with IBD). Dextran sodium sulphate-induced colitis experiments in mice were used to evaluate effects of identified metabolites. In vitro, ex vivo and in vivo experiments were used to decipher mechanisms involved. Effects on energy metabolism were evaluated by different methods including Single Cell mEtabolism by profiling Translation inHibition. RESULTS: In mice and humans, intestinal inflammation severity negatively correlates with the amount of xanthurenic (XANA) and kynurenic (KYNA) acids. Supplementation with XANA or KYNA decreases colitis severity through effects on intestinal epithelial cells and T cells, involving Aryl hydrocarbon Receptor (AhR) activation and the rewiring of cellular energy metabolism. Furthermore, direct modulation of the endogenous tryptophan metabolism, using the recombinant enzyme aminoadipate aminotransferase (AADAT), responsible for the generation of XANA and KYNA, was protective in rodent colitis models. CONCLUSION: Our study identified a new mechanism linking Trp metabolism to intestinal inflammation and IBD. Bringing back XANA and KYNA has protective effects involving AhR and the rewiring of the energy metabolism in intestinal epithelial cells and CD4+ T cells. This study paves the way for new therapeutic strategies aiming at pharmacologically correcting its alterations in IBD by manipulating the endogenous metabolic pathway with AADAT.

Card9 mediates susceptibility to intestinal pathogens through microbiota modulation and control of bacterial virulence.

OBJECTIVE: In association with innate and adaptive immunity, the microbiota controls the colonisation resistance against intestinal pathogens. Caspase recruitment domain 9 (CARD9), a key innate immunity gene, is required to shape a normal gut microbiota. Card9-/- mice are more susceptible to the enteric mouse pathogen Citrobacter rodentium that mimics human infections with enteropathogenic and enterohaemorrhagic Escherichia coli. Here, we examined how CARD9 controls C. rodentium infection susceptibility through microbiota-dependent and microbiota-independent mechanisms. DESIGN: C. rodentium infection was assessed in conventional and germ-free (GF) wild-type (WT) and Card9-/- mice. To explore the impact of Card9-/-microbiota in infection susceptibility, GF WT mice were colonised with WT (WT→GF) or Card9-/- (Card9-/- →GF) microbiota before C. rodentium infection. Microbiota composition was determined by 16S rDNA gene sequencing. Inflammation severity was determined by histology score and lipocalin level. Microbiota-host immune system interactions were assessed by quantitative PCR analysis. RESULTS: CARD9 controls pathogen virulence in a microbiota-independent manner by supporting a specific humoral response. Higher susceptibility to C. rodentium-induced colitis was observed in Card9-/- →GF mice. The microbiota of Card9-/- mice failed to outcompete the monosaccharide-consuming C. rodentium, worsening the infection severity. A polysaccharide-enriched diet counteracted the ecological advantage of C. rodentium and the defective pathogen-specific antibody response in Card9-/- mice. CONCLUSIONS: CARD9 modulates the susceptibility to intestinal infection by controlling the pathogen virulence in a microbiota-dependent and microbiota-independent manner. Genetic susceptibility to intestinal pathogens can be overridden by diet intervention that restores humoural immunity and a competing microbiota.

Growth factor receptor binding protein 14 inhibition triggers insulin-induced mouse hepatocyte proliferation and is associated with hepatocellular carcinoma.

Metabolic diseases such as obesity and type 2 diabetes are recognized as independent risk factors for hepatocellular carcinoma (HCC). Hyperinsulinemia, a hallmark of these pathologies, is suspected to be involved in HCC development. The molecular adapter growth factor receptor binding protein 14 (Grb14) is an inhibitor of insulin receptor catalytic activity, highly expressed in the liver. To study its involvement in hepatocyte proliferation, we specifically inhibited its liver expression using a short hairpin RNA strategy in mice. Enhanced insulin signaling upon Grb14 inhibition was accompanied by a transient induction of S-phase entrance by quiescent hepatocytes, indicating that Grb14 is a potent repressor of cell division. The proliferation of Grb14-deficient hepatocytes was cell-autonomous as it was also observed in primary cell cultures. Combined Grb14 down-regulation and insulin signaling blockade using pharmacological approaches as well as genetic mouse models demonstrated that Grb14 inhibition-mediated hepatocyte division involved insulin receptor activation and was mediated by the mechanistic target of rapamycin complex 1-S6K pathway and the transcription factor E2F1. In order to determine a potential dysregulation in GRB14 gene expression in human pathophysiology, a collection of 85 human HCCs was investigated. This revealed a highly significant and frequent decrease in GRB14 expression in hepatic tumors when compared to adjacent nontumoral parenchyma, with 60% of the tumors exhibiting a reduced Grb14 mRNA level. CONCLUSION: Our study establishes Grb14 as a physiological repressor of insulin mitogenic action in the liver and further supports that dysregulation of insulin signaling is associated with HCC. (Hepatology 2017;65:1352-1368).

Integration of ChREBP-Mediated Glucose Sensing into Whole Body Metabolism.

Since glucose is the principal energy source for most cells, many organisms have evolved numerous and sophisticated mechanisms to sense glucose and respond to it appropriately. In this context, cloning of the carbohydrate responsive element binding protein has unraveled a critical molecular link between glucose metabolism and transcriptional reprogramming induced by glucose. In this review, we detail major findings that have advanced our knowledge of glucose sensing.

Hypothalamic ventromedial COUP-TFII protects against hypoglycemia-associated autonomic failure.

The nuclear receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) is an important coordinator of glucose homeostasis through its function in different organs such as the endocrine pancreas, adipose tissue, skeletal muscle, and liver. Recently we have demonstrated that COUP-TFII expression in the hypothalamus is restricted to a subpopulation of neurons expressing the steroidogenic factor 1 transcription factor, known to play a crucial role in glucose homeostasis. To understand the functional significance of COUP-TFII expression in the steroidogenic factor 1 neurons, we generated hypothalamic ventromedial nucleus-specific COUP-TFII KO mice using the cyclization recombination/locus of X-overP1 technology. The heterozygous mutant mice display insulin hypersensitivity and a leaner phenotype associated with increased energy expenditure and similar food intake. These mutant mice also present a defective counterregulation to hypoglycemia with altered glucagon secretion. Moreover, the mutant mice are more likely to develop hypoglycemia-associated autonomic failure in response to recurrent hypoglycemic or glucopenic events. Therefore, COUP-TFII expression levels in the ventromedial nucleus are keys in the ability to resist the onset of hypoglycemia-associated autonomic failure.

The role of chicken ovalbumin upstream promoter transcription factor II in the regulation of hepatic fatty acid oxidation and gluconeogenesis in newborn mice.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor involved in the control of numerous functions in various organs (organogenesis, differentiation, metabolic homeostasis, etc.). The aim of the present work was to characterize the regulation and contribution of COUP-TFII in the control of hepatic fatty acid and glucose metabolisms in newborn mice. Our data show that postnatal increase in COUP-TFII mRNA levels is enhanced by glucagon (via cAMP) and PPARα. To characterize COUP-TFII function in the liver of suckling mice, we used a functional (dominant negative form; COUP-TFII-DN) and a genetic (shRNA) approach. Adenoviral COUP-TFII-DN injection induces a profound hypoglycemia due to the inhibition of gluconeogenesis and fatty acid oxidation secondarily to reduced PEPCK, Gl-6-Pase, CPT I, and mHMG-CoA synthase gene expression. Using the crossover plot technique, we show that gluconeogenesis is inhibited at two different levels: 1) pyruvate carboxylation and 2) trioses phosphate synthesis. This could result from a decreased availability in fatty acid oxidation arising cofactors such as acetyl-CoA and reduced equivalents. Similar results are observed using the shRNA approach. Indeed, when fatty acid oxidation is rescued in response to Wy-14643-induced PPARα target genes (CPT I and mHMG-CoA synthase), blood glucose is normalized in COUP-TFII-DN mice. In conclusion, this work demonstrates that postnatal increase in hepatic COUP-TFII gene expression is involved in the regulation of liver fatty acid oxidation, which in turn sustains an active hepatic gluconeogenesis that is essential to maintain an appropriate blood glucose level required for newborn mice survival.

The transcription factor ChREBP Orchestrates liver carcinogenesis by coordinating the PI3K/AKT signaling and cancer metabolism.

Cancer cells integrate multiple biosynthetic demands to drive unrestricted proliferation. How these cellular processes crosstalk to fuel cancer cell growth is still not fully understood. Here, we uncover the mechanisms by which the transcription factor Carbohydrate responsive element binding protein (ChREBP) functions as an oncogene during hepatocellular carcinoma (HCC) development. Mechanistically, ChREBP triggers the expression of the PI3K regulatory subunit p85α, to sustain the activity of the pro-oncogenic PI3K/AKT signaling pathway in HCC. In parallel, increased ChREBP activity reroutes glucose and glutamine metabolic fluxes into fatty acid and nucleic acid synthesis to support PI3K/AKT-mediated HCC growth. Thus, HCC cells have a ChREBP-driven circuitry that ensures balanced coordination between PI3K/AKT signaling and appropriate cell anabolism to support HCC development. Finally, pharmacological inhibition of ChREBP by SBI-993 significantly suppresses in vivo HCC tumor growth. Overall, we show that targeting ChREBP with specific inhibitors provides an attractive therapeutic window for HCC treatment.

Antibiotic treatment using amoxicillin-clavulanic acid impairs gut mycobiota development through modification of the bacterial ecosystem.

BACKGROUND: Effects of antibiotics on gut bacteria have been widely studied, but very little is known about the consequences of such treatments on the fungal microbiota (mycobiota). It is commonly believed that fungal load increases in the gastrointestinal tract following antibiotic treatment, but better characterization is clearly needed of how antibiotics directly or indirectly affect the mycobiota and thus the entire microbiota. DESIGN: We used samples from humans (infant cohort) and mice (conventional and human microbiota-associated mice) to study the consequences of antibiotic treatment (amoxicillin-clavulanic acid) on the intestinal microbiota. Bacterial and fungal communities were subjected to qPCR or 16S and ITS2 amplicon-based sequencing for microbiota analysis. In vitro assays further characterized bacterial-fungal interactions, with mixed cultures between specific bacteria and fungi. RESULTS: Amoxicillin-clavulanic acid treatment triggered a decrease in the total fungal population in mouse feces, while other antibiotics had opposite effects on the fungal load. This decrease is accompanied by a total remodelling of the fungal population with the enrichment in Aspergillus, Cladosporium, and Valsa genera. In the presence of amoxicillin-clavulanic acid, microbiota analysis showed a remodeling of bacterial microbiota with an increase in specific bacteria belonging to the Enterobacteriaceae. Using in vitro assays, we isolated different Enterobacteriaceae species and explored their effect on different fungal strains. We showed that Enterobacter hormaechei was able to reduce the fungal population in vitro and in vivo through yet unknown mechanisms. CONCLUSIONS: Bacteria and fungi have strong interactions within the microbiota; hence, the perturbation initiated by an antibiotic treatment targeting the bacterial community can have complex consequences and can induce opposite alterations of the mycobiota. Interestingly, amoxicillin-clavulanic acid treatment has a deleterious effect on the fungal community, which may have been partially due to the overgrowth of specific bacterial strains with inhibiting or competing effects on fungi. This study provides new insights into the interactions between fungi and bacteria of the intestinal microbiota and might offer new strategies to modulate gut microbiota equilibrium. Video Abstract.

MetaPhlAn 4 profiling of unknown species-level genome bins improves the characterization of diet-associated microbiome changes in mice.

Mouse models are key tools for investigating host-microbiome interactions. However, shotgun metagenomics can only profile a limited fraction of the mouse gut microbiome. Here, we employ a metagenomic profiling method, MetaPhlAn 4, which exploits a large catalog of metagenome-assembled genomes (including 22,718 metagenome-assembled genomes from mice) to improve the profiling of the mouse gut microbiome. We combine 622 samples from eight public datasets and an additional cohort of 97 mouse microbiomes, and we assess the potential of MetaPhlAn 4 to better identify diet-related changes in the host microbiome using a meta-analysis approach. We find multiple, strong, and reproducible diet-related microbial biomarkers, largely increasing those identifiable by other available methods relying only on reference information. The strongest drivers of the diet-induced changes are uncharacterized and previously undetected taxa, confirming the importance of adopting metagenomic methods integrating metagenomic assemblies for comprehensive profiling.

Gut microbiota-derived short-chain fatty acids regulate IL-17 production by mouse and human intestinal γδ T cells.

Gut interleukin-17A (IL-17)-producing γδ T cells are tissue-resident cells that are involved in both host defense and regulation of intestinal inflammation. However, factors that regulate their functions are poorly understood. In this study, we find that the gut microbiota represses IL-17 production by cecal γδ T cells. Treatment with vancomycin, a Gram-positive bacterium-targeting antibiotic, leads to decreased production of short-chain fatty acids (SCFAs) by the gut microbiota. Our data reveal that these microbiota-derived metabolites, particularly propionate, reduce IL-17 and IL-22 production by intestinal γδ T cells. Propionate acts directly on γδ T cells to inhibit their production of IL-17 in a histone deacetylase-dependent manner. Moreover, the production of IL-17 by human IL-17-producing γδ T cells from patients with inflammatory bowel disease (IBD) is regulated by propionate. These data contribute to a better understanding of the mechanisms regulating gut γδ T cell functions and offer therapeutic perspectives of these cells.

Aryl hydrocarbon receptor ligand production by the gut microbiota is decreased in celiac disease leading to intestinal inflammation.

Metabolism of tryptophan by the gut microbiota into derivatives that activate the aryl hydrocarbon receptor (AhR) contributes to intestinal homeostasis. Many chronic inflammatory conditions, including celiac disease involving a loss of tolerance to dietary gluten, are influenced by cues from the gut microbiota. We investigated whether AhR ligand production by the gut microbiota could influence gluten immunopathology in nonobese diabetic (NOD) mice expressing DQ8, a celiac disease susceptibility gene. NOD/DQ8 mice, exposed or not exposed to gluten, were subjected to three interventions directed at enhancing AhR pathway activation. These included a high-tryptophan diet, gavage with Lactobacillus reuteri that produces AhR ligands or treatment with an AhR agonist. We investigated intestinal permeability, gut microbiota composition determined by 16S rRNA gene sequencing, AhR pathway activation in intestinal contents, and small intestinal pathology and inflammatory markers. In NOD/DQ8 mice, a high-tryptophan diet modulated gut microbiota composition and enhanced AhR ligand production. AhR pathway activation by an enriched tryptophan diet, treatment with the AhR ligand producer L. reuteri, or pharmacological stimulation using 6-formylindolo (3,2-b) carbazole (Ficz) decreased immunopathology in NOD/DQ8 mice exposed to gluten. We then determined AhR ligand production by the fecal microbiota and AhR activation in patients with active celiac disease compared to nonceliac control individuals. Patients with active celiac disease demonstrated reduced AhR ligand production and lower intestinal AhR pathway activation. These results highlight gut microbiota-dependent modulation of the AhR pathway in celiac disease and suggest a new therapeutic strategy for treating this disorder.

Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease.

The gut microbiota is a crucial actor in human physiology. Many of these effects are mediated by metabolites that are either produced by the microbes or derived from the transformation of environmental or host molecules. Among the array of metabolites at the interface between these microorganisms and the host is the essential aromatic amino acid tryptophan (Trp). In the gut, the three major Trp metabolism pathways leading to serotonin (5-hydroxytryptamine), kynurenine (Kyn), and indole derivatives are under the direct or indirect control of the microbiota. In this review, we gather the most recent advances concerning the central role of Trp metabolism in microbiota-host crosstalk in health and disease. Deciphering the complex equilibrium between these pathways will facilitate a better understanding of the pathogenesis of human diseases and open therapeutic opportunities.

Enterobacteriaceae are essential for the modulation of colitis severity by fungi.

BACKGROUND: Host-microbe balance maintains intestinal homeostasis and strongly influences inflammatory conditions such as inflammatory bowel diseases (IBD). Here we focused on bacteria-fungi interactions and their implications on intestinal inflammation, a poorly understood area. METHODS: Dextran sodium sulfate (DSS)-induced colitis was assessed in mice treated with vancomycin (targeting gram-positive bacteria) or colistin (targeting Enterobacteriaceae) and supplemented with either Saccharomyces boulardii CNCM I-745 or Candida albicans. Inflammation severity as well as bacterial and fungal microbiota compositions was monitored. RESULTS: While S. boulardii improved DSS-induced colitis and C. albicans worsened it in untreated settings, antibiotic treatment strongly modified DSS susceptibility and effects of fungi on colitis. Vancomycin-treated mice were fully protected from colitis, while colistin-treated mice retained colitis phenotype but were not affected anymore by administration of fungi. Antibacterial treatments not only influenced bacterial populations but also had indirect effects on fungal microbiota. Correlations between bacterial and fungal relative abundance were dramatically decreased in colistin-treated mice compared to vancomycin-treated and control mice, suggesting that colistin-sensitive bacteria are involved in interactions with fungi. Restoration of the Enterobacteriaceae population by administrating colistin-resistant Escherichia coli reestablished both beneficial effects of S. boulardii and pathogenic effects of C. albicans on colitis severity. This effect was at least partly mediated by an improved gut colonization by fungi. CONCLUSIONS: Fungal colonization of the gut is affected by the Enterobacteriaceae population, indirectly modifying effects of mycobiome on the host. This finding provides new insights into the role of inter-kingdom functional interactions in intestinal physiopathology and potentially in IBD.

Bilophila wadsworthia aggravates high fat diet induced metabolic dysfunctions in mice.

Dietary lipids favor the growth of the pathobiont Bilophila wadsworthia, but the relevance of this expansion in metabolic syndrome pathogenesis is poorly understood. Here, we showed that B. wadsworthia synergizes with high fat diet (HFD) to promote higher inflammation, intestinal barrier dysfunction and bile acid dysmetabolism, leading to higher glucose dysmetabolism and hepatic steatosis. Host-microbiota transcriptomics analysis reveal pathways, particularly butanoate metabolism, which may underlie the metabolic effects mediated by B. wadsworthia. Pharmacological suppression of B. wadsworthia-associated inflammation demonstrate the bacterium's intrinsic capacity to induce a negative impact on glycemic control and hepatic function. Administration of the probiotic Lactobacillus rhamnosus CNCM I-3690 limits B. wadsworthia-induced immune and metabolic impairment by limiting its expansion, reducing inflammation and reinforcing intestinal barrier. Our results suggest a new avenue for interventions against western diet-driven inflammatory and metabolic diseases.

Impaired Aryl Hydrocarbon Receptor Ligand Production by the Gut Microbiota Is a Key Factor in Metabolic Syndrome.

The extent to which microbiota alterations define or influence the outcome of metabolic diseases is still unclear, but the byproducts of microbiota metabolism are known to have an important role in mediating the host-microbiota interaction. Here, we identify that in both pre-clinical and clinical settings, metabolic syndrome is associated with the reduced capacity of the microbiota to metabolize tryptophan into derivatives that are able to activate the aryl hydrocarbon receptor. This alteration is not merely an effect of the disease as supplementation with AhR agonist or a Lactobacillus strain, with a high AhR ligand-production capacity, leads to improvement of both dietary- and genetic-induced metabolic impairments, particularly glucose dysmetabolism and liver steatosis, through improvement of intestinal barrier function and secretion of the incretin hormone GLP-1. These results highlight the role of gut microbiota-derived metabolites as a biomarker and as a basis for novel preventative or therapeutic interventions for metabolic disorders.

COUP-TFII controls mouse pancreatic ß-cell mass through GLP-1-ß-catenin signaling pathways.

BACKGROUND: The control of the functional pancreatic ß-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how ß-cell mass is determined. METHODOLOGY/PRINCIPAL FINDINGS: Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal ß-cell proliferation and apoptosis, this suggests decreased ß-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured ß-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the ß-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via ß-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced ß-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat ß-cells providing a feedback loop. CONCLUSIONS/SIGNIFICANCE: Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases ß-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the ß-catenin-dependent pathway and its expression is under the control of TCF7L2.