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.

Saccharomyces boulardii CNCM I-745 supplementation during and after antibiotic treatment positively influences the bacterial gut microbiota.

Introduction: Antibiotic effects on gut bacteria have been widely studied, but very little is known about the consequences of such treatments on the mycobiota, the fungal part of the microbiota and how the length of administration influences both microbiota. Here, we examined the effect of antibiotics (ATB) on the composition of bacterial and fungal microbiota and how the administration of Saccharomyces boulardii CNCM I-745 influences both microbiota. Methods: In order to get closer to the human microbiota, the mice used in this study were subjected to fecal microbiota transfer (FMT) using human feces and subsequently called human microbiotaassociated (HMA) mice. These mice were then treated with amoxicillinclavulanate antibiotics and supplemented with S. boulardii during and after ATB treatment to understand the effect of the yeast probiotic on both bacterial and fungal microbiota. Bacterial and fungal microbiota analyses were done using 16S and ITS2 rRNA amplicon-based sequencing. Results: We showed that the administration of S. boulardii during ATB treatment had very limited effect on the fungal populations on the long term, once the yeast probiotic has been cleared from the gut. Concerning bacterial microbiota, S. boulardii administration allowed a better recovery of bacterial populations after the end of the ATB treatment period. Additionally, 16S and ITS2 rRNA sequence analysis revealed that 7 additional days of S. boulardii administration (17 days in total) enhanced the return of the initial bacterial equilibrium. Discussion: In this study, we provide a comprehensive analysis of how probiotic yeast administration can influence the fungal and bacterial microbiota in a model of broad-spectrum antibiotherapy.

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.

Cyberlindnera jadinii and Kluyveromyces lactis, two fungi used in food processes, have potential probiotic effects on gut inflammation.

IMPORTANCE: The food industry has always used many strains of microorganisms including fungi in their production processes. These strains have been widely characterized for their biotechnological value, but we still know very little about their interaction capacities with the host at a time when the intestinal microbiota is at the center of many pathologies. In this study, we characterized five yeast strains from food production which allowed us to identify two new strains with high probiotic potential and beneficial effects in a model of intestinal inflammation.

Bacterial-fungal metabolic interactions within the microbiota and their potential relevance in human health and disease: a short review.

The composition of the microbiota is the focus of many recent publications describing the effects of the microbiota on host health. In recent years, research has progressed further, investigating not only the diversity of genes and functions but also metabolites produced by microorganisms composing the microbiota of various niches and how these metabolites affect and shape the microbial community. While an abundance of data has been published on bacterial interactions, much less data are available on the interactions of bacteria with another component of the microbiota: the fungal community. Although present in smaller numbers, fungi are essential to the balance of this complex microbial ecosystem. Both bacterial and fungal communities produce metabolites that influence their own population but also that of the other. However, to date, interkingdom interactions occurring through metabolites produced by bacteria and fungi have rarely been described. In this review, we describe the major metabolites produced by both kingdoms and discuss how they influence each other, by what mechanisms and with what consequences for the host.

Effects of Five Filamentous Fungi Used in Food Processes on In Vitro and In Vivo Gut Inflammation.

Food processes use different microorganisms, from bacteria to fungi. Yeast strains have been extensively studied, especially Saccharomyces cerevisiae. However, to date, very little is known about the potential beneficial effects of molds on gut health as part of gut microbiota. We undertook a comprehensive characterization of five mold strains, Penicillium camemberti, P. nalgiovense, P. roqueforti, Fusarium domesticum, and Geotrichum candidum used in food processes, on their ability to trigger or protect intestinal inflammation using in vitro human cell models and in vivo susceptibility to sodium dextran sulfate-induced colitis. Comparison of spore adhesion to epithelial cells showed a very wide disparity in results, with F. domesticum and P. roqueforti being the two extremes, with almost no adhesion and 20% adhesion, respectively. Interaction with human immune cells showed mild pro-inflammatory properties of all Penicillium strains and no effect of the others. However, the potential anti-inflammatory abilities detected for G. candidum in vitro were not confirmed in vivo after oral gavage to mice before and during induced colitis. According to the different series of experiments carried out in this study, the impact of the spores of these molds used in food production is limited, with no specific beneficial or harmful effect on the gut.

Deletion of both Dectin-1 and Dectin-2 affects the bacterial but not fungal gut microbiota and susceptibility to colitis in mice.

BACKGROUND: Innate immunity genes have been reported to affect susceptibility to inflammatory bowel diseases (IBDs) and colitis in mice. Dectin-1, a receptor for fungal cell wall ß-glucans, has been clearly implicated in gut microbiota modulation and modification of the susceptibility to gut inflammation. Here, we explored the role of Dectin-1 and Dectin-2 (another receptor for fungal cell wall molecules) deficiency in intestinal inflammation. DESIGN: Susceptibility to dextran sodium sulfate (DSS)-induced colitis was assessed in wild-type, Dectin-1 knockout (KO), Dectin-2KO, and double Dectin-1KO and Dectin-2KO (D-1/2KO) mice. Inflammation severity, as well as bacterial and fungal microbiota compositions, was monitored. RESULTS: While deletion of Dectin-1 or Dectin-2 did not have a strong effect on DSS-induced colitis, double deletion of Dectin-1 and Dectin-2 significantly protected the mice from colitis. The protection was largely mediated by the gut microbiota, as demonstrated by fecal transfer experiments. Treatment of D-1/2KO mice with opportunistic fungal pathogens or antifungal agents did not affect the protection against gut inflammation, suggesting that the fungal microbiota had no role in the protective phenotype. Amplicon-based microbiota analysis of the fecal bacterial and fungal microbiota of D-1/2KO mice confirmed the absence of changes in the mycobiota but strong modification of the bacterial microbiota. We showed that bacteria from the Lachnospiraceae family were at least partly involved in this protection and that treatment with Blautia hansenii was enough to recapitulate the protection. CONCLUSIONS: Deletion of both the Dectin-1 and Dectin-2 receptors triggered a global shift in the microbial gut environment, affecting, surprisingly, mainly the bacterial population and driving protective effects in colitis. Members of the Lachnospiraceae family seem to play a central role in this protection. These findings provide new insights into the role of the Dectin receptors, which have been described to date as affecting only the fungal population, in intestinal physiopathology and in IBD. Video Abstract.

BMP Antagonists Secreted by Mesenchymal Stromal Cells Improve Colonic Organoid Formation: Application for the Treatment of Radiation-induced Injury.

Radiation therapy is crucial in the therapeutic arsenal to cure cancers; however, non-neoplastic tissues around an abdominopelvic tumor can be damaged by ionizing radiation. In particular, the radio-induced death of highly proliferative stem/progenitor cells of the colonic mucosa could induce severe ulcers. The importance of sequelae for patients with gastrointestinal complications after radiotherapy and the absence of satisfactory management has opened the field to the testing of innovative treatments. The aim of this study was to use adult epithelial cells from the colon, to reduce colonic injuries in an animal model reproducing radiation damage observed in patients. We demonstrated that transplanted in vitro-amplified epithelial cells from colonic organoids (ECO) of C57/Bl6 mice expressing green fluorescent protein implant, proliferate, and differentiate in irradiated mucosa and reduce ulcer size. To improve the therapeutic benefit of ECO-based treatment with clinical translatability, we performed co-injection of ECO with mesenchymal stromal cells (MSCs), cells involved in niche function and widely used in clinical trials. We observed in vivo an improvement of the therapeutic benefit and in vitro analysis highlighted that co-culture of MSCs with ECO increases the number, proliferation, and size of colonic organoids. We also demonstrated, using gene expression analysis and siRNA inhibition, the involvement of bone morphogenetic protein antagonists in MSC-induced organoid formation. This study provides evidence of the potential of ECO to limit late radiation effects on the colon and opens perspectives on combined strategies to improve their amplification abilities and therapeutic effects.

Prophylactic Faecalibacterium prausnitzii treatment prevents the acute breakdown of colonic epithelial barrier in a preclinical model of pelvic radiation disease.

Every year, millions of people around the world benefit from radiation therapy to treat cancers localized in the pelvic area. Damage to healthy tissue in the radiation field can cause undesirable toxic effects leading to gastrointestinal complications called pelvic radiation disease. A change in the composition and/or function of the microbiota could contribute to radiation-induced gastrointestinal toxicity. In this study, we tested the prophylactic effect of a new generation of probiotic like Faecalibacterium prausnitzii (F. prausnitzii) on acute radiation-induced colonic lesions. Experiments were carried out in a preclinical model of pelvic radiation disease. Rats were locally irradiated at 29 Gray in the colon resulting in colonic epithelial barrier rupture. Three days before the irradiation and up to 3 d after the irradiation, the F. prausnitzii A2-165 strain was administered daily (intragastrically) to test its putative protective effects. Results showed that prophylactic F. prausnitzii treatment limits radiation-induced para-cellular hyperpermeability, as well as the infiltration of neutrophils (MPO+ cells) in the colonic mucosa. Moreover, F. prausnitzii treatment reduced the severity of the morphological change of crypts, but also preserved the pool of Sox-9+ stem/progenitor cells, the proliferating epithelial PCNA+ crypt cells and the Dclk1+/IL-25+ differentiated epithelial tuft cells. The benefit of F. prausnitzii was associated with increased production of IL-18 by colonic crypt epithelial cells. Thus, F. prausnitzii treatment protected the epithelial colonic barrier from colorectal irradiation. New-generation probiotics may be promising prophylactic treatments to reduce acute side effects in patients treated with radiation therapy and may improve their quality of life.