ABSTRACT: The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis.
Project description:The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis.
Project description:Studying host-microbiota interactions is fundamental to understand mechanisms involved in intestinal inflammation and inflammatory bowel diseases. In this work, we studied these interactions in mice mono-associated with 4 bacteria and 2 yeasts, all representative of intestinal microbiota and/or associated with IBD pathogenesis: Bacteroides thetaiotaomicron, adhesive-invasive Escherichia coli (AIEC), Ruminococcus gnavus, Roseburia intestinalis, Saccharomyces boulardii and Candida albicans. Transcriptomics analyses showed that B. thetaiotaomicron had the highest immunological effect, being able to almost recapitulate the effects of a whole microbiota, and particularly induced Treg pathways. Furthermore, this analysis also pointed out the effects of E. coli AIEC LF82 on IDO activation and of S. boulardii on angiogenesis, as well as major effects of R. gnavus on metabolism. This work therefore reveals information on the role of each micro-organism and proposes several tracks to follow to better understand IBD pathogenesis and identify therapeutic targets 6 mono-associations + 2 controls (germ-free and conventionalized mice), with 5 to 7 mice per group.
Project description:The healthy gut microbiota is composed of three different functional subgroups, symbionts, commensals and pathobionts. Additionally, infection with pathogenic bacteria can occur. Microbe Associated Molecular Patterns (MAMPs) are expressed by most members of both, microbiota and pathogens and interact with Pattern Recognition Receptors (PRRs), resulting in activation of the innate immune system. It is still unclear whether the interaction of PRRs with MAMPs of different origin, leads to a differential activation of innate immunity and whether thereby, distinction can be made between symbionts, commensals, pathobionts and pathogens. Here we addressed the question whether the interaction of flagellin with Toll-like receptor 5 (TLR5) might be a possible mechanism of the innate immune system to distinguish between the different microbial subgroups and initiate the respective immune response. To characterize distinct cellular immune responses to symbiont or pathogen derived flagellin we run transcriptomics, phosphoproteomics and proteomics of TLR5 overexpressing cells. Responses to different flagellins were characterized by differential kinetics and regulation of protein phosphorylation as well as variant protein expression patterns. In vivo experiments revealed that commensal derived flagellin mediated an inert TLR5 signal, thus not affecting Dextran-Sodium-Sulfate (DSS) induced intestinal inflammation. In vitro pathogenic derived flagellin induced a strong pro-inflammatory TLR5 response and did not ameliorate intestinal inflammation. In contrast, symbiont derived flagellin revealed an intermediate TLR5 activation and clearly ameliorated DSS induced intestinal inflammation thus shifting the host immune response in favor of mucosal immune balance. Bone marrow chimeric mice proofed that CD11c+TLR5+ intestinal lamina propria cells mediate the anti-inflammatory symbiotic effect of flagellin. We suggest that the quality of flagellin-induced TL signal might determine the balance between homeostasis and intestinal inflammation and may help the innate immune system to distinguish between symbionts, commensals, pathobionts and pathogenic microbes. We used microarrays to analyze differential expression of genes between treatments with flagellins of E. coli Nissle (EcN) and Salmonella Typhimurium SL1344 (ST).
Project description:The small intestinal epithelial barrier inputs signals from the gut microbiota in order to balance physiological inflammation and tolerance, and to promote homeostasis. Understanding the dynamic relationship between microbes and intestinal epithelial cells has been a challenge given the cellular heterogeneity associated with the epithelium and the inherent difficulty of isolating and identifying individual cell types. Here, we used single-cell RNA sequencing of small intestinal epithelial cells from germ-free and specific pathogen-free mice to study microbe-epithelium crosstalk at the single cell resolution.
Project description:Intestinal microbiota dysbiosis is related to many metabolic diseases in human health. Meanwhile, as an irregular environmental light-dark cycle, short-day (SD) may induce host circadian rhythms disturbances and worsen the risks of gut dysbiosis. Herein, we investigated how LD cycles regulate intestinal metabolism upon the destruction of gut microbes with antibiotic treatments. The transcriptome data indicated that SD have some negative effects on hepatic metabolism, endocrine, digestive, and diseases processes compared with normal light-dark cycle (NLD).The SD induced epithelial and hepatic purine metabolism pathway imbalance in ABX mice, the gut microbes, and their metabolites, all of which could contribute to host metabolism and digestion, endocrine system disorders, and may even cause diseases in the host.
Project description:Choline is a water-soluble nutrient essential for human life. Gut microbial metabolism of choline results in the production of trimethylamine (TMA), which upon absorption by the host is converted in the liver to trimethylamine N-oxide (TMAO). Recent studies revealed that TMAO exacerbates atherosclerosis in mice, and positively correlates with the severity of this disease in human. However, which microbes contribute to TMA production in the human gut; the extent to which host factors, e.g., genotype and diet, affect TMA production and colonization of these microbes; as well as the effects TMA-producing microbes have on bioavailability of dietary choline remain largely unknown. We screened a collection of 78 sequenced human intestinal isolates encompassing the major phyla found in the human gut and identified eight strains capable of producing TMA from choline in vitro. Gnotobiotic mouse studies showed that TMAO accumulates in the serum of animals colonized with TMA-producing species, but not in the serum of animals colonized with intestinal isolates that do not generate TMA from choline in vitro. Remarkably, low levels of colonization of TMA-producing bacteria significantly reduced choline levels available to the host. This effect was more pronounced as the abundance of TMA-producing bacteria increased. Our findings provide a framework for designing strategies aimed at changing the representation or activity of TMA-producing bacteria in the human gut and suggest the TMA producing status of the gut microbiota should be considered when making recommendations about choline intake requirements for humans.
Project description:A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models. 6 samples, 2 biological replicates for each 3 conditions.
Project description:The mammalian gut is inhabited by a large and complex microbial community that lives in a mutualistic relationship with its host. Innate and adaptive mucosal defense mechanisms ensure a homeostatic relationship with this commensal microbiota. Secretory antibodies are generated from the active polymeric Ig receptor (pIgR)-mediated transport of IgA and IgM antibodies to the gut lumen and form the first line of adaptive immune defense of the intestinal mucosa. We probed mucosal homeostasis in pIgR knockout (KO) mice, which lack secretory antibodies. We found that in pIgR KO mice, colonic epithelial cells, the cell type most closely in contact with intestinal microbes, differentially expressed (>2-fold change) more than 200 genes compared with wild type mice, and upregulated the expression of anti-microbial peptides in a commensal-dependent manner. Detailed profiling of microbial communities based on 16S rRNA genes revealed differences in the commensal microbiota between pIgR KO and wild type mice. Furthermore, we found that pIgR KO mice showed increased susceptibility to dextran sulfate sodium (DSS)-induced colitis, and that this was driven by their conventional intestinal microbiota. In conclusion, secretory antibodies or the pIgR itself are required to maintain a stable commensal microbiota. In the absence of these humoral effector components, gut homeostasis is disturbed and the outcome of colitis significantly worsened. 4 groups: wild type mice treated with antibiotic (5 replicates), wild type mice left untreated (5 replicates), pIgR KO mice treated with antibiotic (6 replicates), and pIgR KO mice left untreated (6 replicates).
Project description:Remodeling of the gut microbiota is implicated in various metabolic and inflammatory diseases of the gastrointestinal tract. We hypothesized that the gut microbiota affects the DNA methylation profile of intestinal epithelial cells (IECs) which could, in turn, alter intestinal function. Here, we used mass spectrometry and methylated DNA capture to respectively investigate global and genome-wide DNA methylation of intestinal epithelial cells from germ-free (GF) and conventionally raised mice (CONV-R). In colonic IECs from GF mice, DNA was markedly hypermethylated. This was associated with a dramatic loss of Ten-Eleven-Translocation activity, a lower DNA methyltransferase activity and lower circulating levels of the one carbon metabolites cobalamin and folate. At the gene level, we found an enrichment for differentially methylated regions at proximity of genes regulating cytotoxicity of Natural Killer cells (FDR < 8.9E-6), notably members of the natural killer group 2 member D ligand superfamily Raet. Our results suggest that altered activity of methylation-modifying enzymes in GF mice influences the IEC epigenome at genes involved in the crosstalk between intestinal and immune cells. Epigenetic reprogramming of IECs by the gut microbiota may modulate intestinal function in diseases associated with altered gut microbiota.
Project description:Studying host-microbiota interactions is fundamental to understand mechanisms involved in intestinal inflammation and inflammatory bowel diseases. In this work, we studied these interactions in mice mono-associated with 4 bacteria and 2 yeasts, all representative of intestinal microbiota and/or associated with IBD pathogenesis: Bacteroides thetaiotaomicron, adhesive-invasive Escherichia coli (AIEC), Ruminococcus gnavus, Roseburia intestinalis, Saccharomyces boulardii and Candida albicans. Transcriptomics analyses showed that B. thetaiotaomicron had the highest immunological effect, being able to almost recapitulate the effects of a whole microbiota, and particularly induced Treg pathways. Furthermore, this analysis also pointed out the effects of E. coli AIEC LF82 on IDO activation and of S. boulardii on angiogenesis, as well as major effects of R. gnavus on metabolism. This work therefore reveals information on the role of each micro-organism and proposes several tracks to follow to better understand IBD pathogenesis and identify therapeutic targets