Benoit Chassaing

Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome

The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, especially in metabolism and immune development, disturbance of the microbiota–host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and the group of obesity-associated diseases collectively referred to as metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, thereby allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine. Thus, agents that disrupt mucus–bacterial interactions might have the potential to promote diseases associated with gut inflammation. Consequently, it has been hypothesized that emulsifiers, detergent-like molecules that are a ubiquitous component of processed foods and that can increase bacterial translocation across epithelia in vitro, might be promoting the increase in inflammatory bowel disease observed since the mid-twentieth century. Here we report that, in mice, relatively low concentrations of two commonly used emulsifiers, namely carboxymethylcellulose and polysorbate-80, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host–microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.

The Commensal Microbiota and Enteropathogens in the Pathogenesis of Inflammatory Bowel Diseases

Intestinal inflammation arises from abnormal host-microbe interactions. The perturbations of homeostatic coexistence involve host genetic factors, barrier function, innate and adaptive immunity, as well as qualitative and quantitative changes in the composition of the microbiota. Dysbiosis toward selected micro-organisms and decreased complexity of commensal bacteria have been observed in patients with Crohns disease and ulcerative colitis, but it is not clear whether the dysbiosis contributes to development of inflammatory bowel disease or is instead a consequence of the disease. Pathogens with virulence factors that allow them to breach the intestinal barrier and induce chronic inflammation might mediate the pathogenesis of these diseases. To identify new therapeutic approaches for inflammatory bowel disease, it is important to identify host susceptibility factors involved in the control of microbial infection, characterize potential pathogens, and eliminate them or block the expression of their virulence factors.

Dextran sulfate sodium (DSS)-induced colitis in mice.

Inflammatory bowel diseases (IBD), mainly comprising ulcerative colitis and Crohns Disease, are complex and multifactorial diseases with unknown etiology. For the past 20 years, to study human IBD mechanistically, a number of murine models of colitis have been developed. These models are indispensable tools to decipher underlying mechanisms of IBD pathogenesis as well as to evaluate a number of potential therapeutics. Among various chemically induced colitis models, the dextran sulfate sodium (DSS)‐induced colitis model is widely used because of its simplicity and many similarities with human ulcerative colitis. This model has both advantages and disadvantages that must be considered when employed. This protocol describes the DSS‐induced colitis model, focusing on details and factors that could affect DSS‐induced pathology. Curr. Protoc. Immunol. 104:15.25.1‐15.25.14.

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E. coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohns disease-associated E. coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.

Crohn disease–associated adherent-invasive E. coli bacteria target mouse and human Peyer’s patches via long polar fimbriae

Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC)--a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyers patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2(-/-) mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients.

Innate and Adaptive Immunity Interact to Quench Microbiome Flagellar Motility in the Gut

Gut mucosal barrier breakdown and inflammation have been associated with high levels of flagellin, the principal bacterial flagellar protein. Although several gut commensals can produce flagella, flagellin levels are low in the healthy gut, suggesting the existence of control mechanisms. We find that mice lacking the flagellin receptor Toll-like receptor 5 (TLR5) exhibit a profound loss of flagellin-specific immunoglobulins (Igs) despite higher total Ig levels in the gut. Ribotyping of IgA-coated cecal microbiota showed Proteobacteria evading antibody coating in the TLR5(-/-) gut. A diversity of microbiome members overexpressed flagellar genes in the TLR5(-/-) host. Proteobacteria and Firmicutes penetrated small intestinal villi, and flagellated bacteria breached the colonic mucosal barrier. In vitro, flagellin-specific Ig inhibited bacterial motility and downregulated flagellar gene expression. Thus, innate-immunity-directed development of flagellin-specific adaptive immune responses can modulate the microbiomes production of flagella in a three-way interaction that helps to maintain mucosal barrier integrity and homeostasis.

Microbiota-Liver Axis in Hepatic Disease

Accumulating evidence indicates that the gut microbiota, long appreciated to be a key determinant of intestinal inflammation, is also playing a key role in chronic inflammatory disease of the liver. Such studies have yielded a general central hypothesis whereby microbiota products activate the innate immune system to drive proinflammatory gene expression, thus promoting chronic inflammatory disease of the liver. This article reviews the background supporting this hypothesis, outlines how it can potentially explain classic and newly emerging epidemiological chronic inflammatory liver disease, and discusses potential therapeutic means to manipulate the microbiota so as to prevent and/or treat liver disease. (Hepatology 2014;58:328–339)

AIEC pathobiont instigates chronic colitis in susceptible hosts by altering microbiota composition

Background Inflammatory bowel disease (IBD) is driven by a seemingly aberrant immune response to the gut microbiota with disease development dictated by genetics and environmental factors. A model exemplifying this notion is our recent demonstration that colonisation of adherent-invasive Escherichia coli (AIEC) during microbiota acquisition drove chronic colitis in mice lacking the flagellin receptor TLR5 (T5KO). T5KO colitis persisted beyond AIEC clearance and requires TLR4 and the NLRC4 inflammasome. We hypothesised that AIEC instigates chronic inflammation by increasing microbial lipopolysaccharide (LPS) and flagellin levels. Goal Examine if transient AIEC colonisation lastingly alters levels of LPS and flagellin and changes microbiota composition. Methods Germ-free mice (wild type (WT) and T5KO) were inoculated with AIEC strain LF82 and placed in standard housing allowing a complex microbiota that eliminated AIEC in both mice strains. Faeces were assayed for the inflammatory marker, lipocalin-2, bacterial loads, and microbiota composition by pyrosequencing. Faecal LPS and flagellin bioactivity were measured via a cell-based reporter assay. Results Transient AIEC colonisation, in WT mice, did not alter inflammatory markers, bacterial loads, microbiota composition, nor its pro-inflammatory potential. By contrast, transient AIEC colonisation of T5KO mice drove chronic inflammation which correlated with microbiota components having higher levels of bioactive LPS and flagellin. Such AIEC-induced elevation of LPS and flagellin persisted well beyond AIEC clearance, required AIEC be flagellated, and was associated with alteration in microbiota species composition including a loss of species diversity. Conclusions AIEC, and perhaps other pathobionts, may instigate chronic inflammation in susceptible hosts by altering the gut microbiota composition so as to give it an inherently greater ability to activate innate immunity/pro-inflammatory gene expression.

Prevention and cure of rotavirus infection via TLR5/NLRC4–mediated production of IL-22 and IL-18

Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent. Bacterial flagellin protein protects rotavirus-infected mice by stimulating cytokine production. Flagellin gives rotavirus a one-two punch Rotavirus causes gastroenteritis, which can be especially severe in infants and young children. The bacterial protein flagellin activates the innate immune system and protects mice against a variety of infectious and inflammatory agents. Zhang et al. now report that flagellin both prevented rotavirus infection in mice and cured mice chronically infected with rotavirus. It did so by activating two distinct innate immune signaling pathways, which led to cells in the infected mice producing the cytokines interleukin 22 and interleukin 18. Similar to flagellin, treating mice with both of these cytokines prevented or cured rotavirus infection. Science, this issue p. 861

Intestinal Epithelial Cell Toll-like Receptor 5 Regulates the Intestinal Microbiota to Prevent Low-Grade Inflammation and Metabolic Syndrome in Mice

BACKGROUND & AIMS Mice lacking the receptor Toll-like receptor 5 (TLR5-null mice), which recognizes flagellin, have an altered intestinal microbiota composition compared with wild-type mice; they develop low-grade inflammation and metabolic syndrome and are prone to colitis. The relative roles of intestinal epithelial cell (IEC) vs dendritic cell (DC) TLR5 in mediating these phenotypes are not clear; modification of intestinal microbiota composition has been reported to reflect animal husbandry practices rather than loss of TLR5. We generated mice with specific disruption of Tlr5 in IECs or DCs by using a breeding scheme that allows comparison with cohoused siblings as controls. METHODS We generated C57BL/6 mice with LoxP sites flanking Tlr5. These mice were crossed with mice expressing Cre recombinase, regulated by the villin or CD11c promoters, to generate mice that lacked expression of TLR5 by IECs (TLR5(ΔIEC)) or DCs (TLR5(ΔDC)), respectively. Tlr5(fl/fl) siblings were used as controls. On weaning, mice were housed by sex and genotype or by sex only (genotypes cohoused). Mice were examined for basal phenotypes, including microbiota composition; we also analyzed responses to pathobiont challenge, administration of dextran sodium sulfate, and high-fat diets. RESULTS Similar to previous findings from TLR5-null mice, TLR5(ΔIEC) mice had low-grade inflammation (mild splenomegaly, shortened colons, and increased fecal levels of lipocalin 2), metabolic syndrome, and an inability to clear pathobionts and were prone to developing colitis compared with their sibling controls under both housing conditions. Development of this inflammation in the TLR5(ΔIEC) mice was eliminated by administration of antibiotics and associated with alterations in localization of microbiota and levels of fecal lipopolysaccharide and flagellin. The composition of the microbiota clustered more closely according to genotype than housing. Loss of TLR5 from DCs did not associate with development of inflammation-associated phenotypes or alterations in the composition of the microbiota but resulted in complete loss of flagellin-induced production of interleukin-22. CONCLUSIONS In mice, flagellin activation of TLR5 on DCs leads to production of interleukin-22. Expression of TLR5 on IECs regulates the composition and localization of the intestinal microbiota, preventing diseases associated with intestinal inflammation.