Lucie Etienne-Mesmin

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)

Hepatocyte Toll-Like Receptor 5 Promotes Bacterial Clearance and Protects Mice Against High-Fat Diet–Induced Liver Disease

Background & Aims Innate immune dysfunction can promote chronic inflammatory diseases of the liver. For example, mice lacking the flagellin receptor Toll-like receptor 5 (TLR5) show microbial dysbiosis and predisposition to high-fat diet (HFD)-induced hepatic steatosis. The extent to which hepatocytes play a direct role in detecting bacterial products in general, or flagellin in particular, is poorly understood. In the present study, we investigated the role of hepatocyte TLR5 in recognizing flagellin, policing bacteria, and protecting against liver disease. Methods Mice were engineered to lack TLR5 specifically in hepatocytes (TLR5ΔHep) and analyzed relative to sibling controls (TLR5fl/fl). TLR5 messenger RNA levels, responses to exogenous flagellin, elimination of circulating motile bacteria, and susceptibility of liver injury (concanavalin A, carbon tetrachloride, methionine- and choline-deficient diet, and HFD) were measured. Results TLR5ΔHep expressed similar levels of TLR5 as TLR5fl/fl in all organs examined, except in the liver, which showed a 90% reduction in TLR5 levels, indicating that hepatocytes accounted for the major portion of TLR5 expression in this organ. TLR5ΔHep showed impairment in responding to purified flagellin and clearing flagellated bacteria from the liver. Although TLR5ΔHep mice did not differ markedly from sibling controls in concanavalin A or carbon tetrachloride–induced liver injury models, they showed exacerbated disease in response to a methionine- and choline-deficient diet and HFD. Such predisposition of TLR5ΔHep to diet-induced liver pathology was associated with increased expression of proinflammatory cytokines, which was dependent on the Nod-like-receptor C4 inflammasome and rescued by microbiota ablation. Conclusions Hepatocyte TLR5 plays a critical role in protecting liver against circulating gut bacteria and against diet-induced liver disease.

Tryptophan: A gut microbiota-derived metabolites regulating inflammation.

Inflammatory bowel diseases (IBD), which comprise Crohn’s disease and ulcerative colitis, are chronic intestinal disorders with an increased prevalence and incidence over the last decade in many different regions over the world. The etiology of IBD is still not well defined, but evidence suggest that it results from perturbation of the homeostasis between the intestinal microbiota and the mucosal immune system, with the involvement of both genetic and environmental factors. Genome wide association studies, which involve large-scale genome-wide screening of potential polymorphism, have identified several mutations associated with IBD. Among them, Card9, a gene encoding an adapter molecule involved in innate immune response to fungi (via type C-lectin sensing) through the activation of IL-22 signaling pathway, has been identified as one IBD susceptible genes. Dietary compounds, which represent a source of energy and metabolites for gut bacteria, are also appreciated to be important actors in the etiology of IBD, for example by altering gut microbiota composition and by regulating the generation of short chain fatty acids. A noteworthy study published in the June 2016 issue of Nature Medicine by Lamas and colleagues investigates the interaction between Card9 and the gut microbiota in the generation of the microbiota-derived tryptophan metabolite. This study highlights the role of tryptophan in dampening intestinal inflammation in susceptible hosts.

Dynamic In Vitro Models of the Human Gastrointestinal Tract as Relevant Tools to Assess the Survival of Probiotic Strains and Their Interactions with Gut Microbiota

The beneficial effects of probiotics are conditioned by their survival during passage through the human gastrointestinal tract and their ability to favorably influence gut microbiota. The main objective of this study was to use dynamic in vitro models of the human digestive tract to investigate the effect of fasted or fed state on the survival kinetics of the new probiotic Saccharomyces cerevisiae strain CNCM I-3856 and to assess its influence on intestinal microbiota composition and activity. The probiotic yeast showed a high survival rate in the upper gastrointestinal tract whatever the route of admistration, i.e., within a glass of water or a Western-type meal. S. cerevisiae CNCM I-3856 was more sensitive to colonic conditions, as the strain was not able to colonize within the bioreactor despite a twice daily administration. The main bacterial populations of the gut microbiota, as well as the production of short chain fatty acids were not influenced by the probiotic treatment. However, the effect of the probiotic on the gut microbiota was found to be individual dependent. This study shows that dynamic in vitro models can be advantageously used to provide useful insight into the behavior of probiotic strains in the human digestive environment.

Policing of gut microbiota by the adaptive immune system

The intestinal microbiota is a large and diverse microbial community that inhabits the intestine, containing about 100 trillion bacteria of 500-1000 distinct species that, collectively, provide benefits to the host. The human gut microbiota composition is determined by a myriad of factors, among them genetic and environmental, including diet and medication. The microbiota contributes to nutrient absorption and maturation of the immune system. As reciprocity, the host immune system plays a central role in shaping the composition and localization of the intestinal microbiota. Secretory immunoglobulins A (sIgAs), component of the adaptive immune system, are important player in the protection of epithelium, and are known to have an important impact on the regulation of microbiota composition. A recent study published in Immunity by Fransen and colleagues aimed to mechanistically decipher the interrelationship between sIgA and microbiota diversity/composition. This commentary will discuss these important new findings, as well as how future therapies can ultimately benefit from such discovery.

Toxin-positive Clostridium difficile latently infect mouse colonies and protect against highly pathogenic C. difficile

Objective Clostridium difficile is a toxin-producing bacterium and a leading cause of antibiotic-associated disease. The ability of C. difficile to form spores and infect antibiotic-treated persons at low multiplicity of infection (MOI) underlies its large disease burden. However, C. difficile-induced disease might also result from long-harboured C. difficile that blooms in individuals administered antibiotics. Design Mice purchased from multiple vendors and repeatedly testing negative for this pathogen by quantitative PCR bloomed C. difficile following antibiotic treatment. This endogenous C. difficile strain, herein termed LEM1, which formed spores and produced toxin, was compared with highly pathogenic C. difficile strain VPI10463. Results Whole-genome sequencing revealed that LEM1 and VPI10463 shared 95% of their genes, including all known virulence genes. In contrast to VPI10463, LEM1 did not induce overt disease when administered to antibiotic-treated or germ-free mice, even at high doses. Rather, blooms of LEM1 correlated with survival following VPI10463 inoculation, and exogenous administration of LEM1 before or shortly following VPI10463 inoculation prevented C. difficile-induced death. Accordingly, despite similar growth properties in vitro, LEM1 strongly outcompeted VPI10463 in mice even at 100-fold lower inocula. Conclusions These results highlight the difficulty of determining whether individual cases of C. difficile infection resulted from a bloom of endogenous C. difficile or a new exposure to this pathogen. In addition to impacting the design of studies using mouse models of C. difficile-induced disease, this study identified, isolated and characterised an endogenous murine spore-forming C. difficile strain able to decrease colonisation, associated disease and death induced by a pathogenic C. difficile strain.

Enterohemorrhagic Escherichia coli pathogenesis: role of Long polar fimbriae in Peyer's patches interactions

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer’s patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer’s patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer’s patches and M-cells, and could contribute to intestinal colonization.

30 Role of Long Polar Fimbriae in the Interactions of Enterohemorrhagic Escherichia coli With Peyer's Patches and Inhibition by a Probiotic Yeast

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens associated with diarrhea, hemorrhagic colitis and life-threatening complications such as hemolytic-uremic syndrome. EHEC interact with the Follicule-Associated Epithelium (FAE) of Peyers patches (PPs) of the distal ileum in humans and translocate across the intestinal epithelium via M cells. Molecular mechanisms are still unknown but Long Polar Fimbriae (LPF), which contribute to intestinal colonization, may be involved. Currently no specific treatment is available in EHEC infections and use of antibiotics remains controversial. Probiotic could be an alternative strategy. The objectives of the study were to investigate the role of LPF in EHEC tropism to PPs, and to explore the influence of a probiotic yeast, Saccharomyces cerevisiae, on EHEC interactions with intestinal mucosa. The expression of lpf genes (encoded by two lpf operons) of EHEC O157:H7 strain EDL933 was analyzed using in vitro models of the human upper GI tract and large intestine. To investigate the involvement of LPF in the ability of EDL933 to target PPs, we generated the D lpfA1,DlpfA2, DlpfA1-DlpfA2 isogenic mutants and trans-complemented them with lpf genes. LPF interaction with M-like cells was investigated using an in vitro model of specialized M cells. In vivo interactions of EHEC with murine PPs were analyzed in ileal loop assays. Mice were infected with a mixture of two bacterial strains, and the numbers of PPs-interacting bacteria were determined using a competitive index analysis. To investigate the effect of S. cerevisiae, mice were given the probiotic for 7 days before ileal loops assays were conducted with O157:H7 wild type. Lpf isogenic mutants (i) were not able to interact with ileal biopsies containing PPs compared to the wild type strain in competitive colonization assays and (ii) translocated across M cells at levels significantly lower than those observed for the wild type strain. Trans-complementation of the mutants with the cloned lpf genes restored their ability to interact with PPs and M cells, indicating that expression of lpfA1 or/and lpfA2 genes is required for interactions with PPs. Regarding probiotic strategy, S. cerevisiae exerts a trophic effect on the intestinal mucosa. Bloodshot PPs were macroscopically observed following EHEC infection of murine ileal loops. We showed that pre-treatment with yeast significantly inhibited O157:H7 interactions with PPs and reduced the number of hemorrhagic PPs in murine ileal loops. Yeast cell surface are rich in mannose and the role of such carbohydrates in inhibiting EHEC interactions with PPs will be investigated. We conclude that LPF are involved in the interactions of EHEC with murine PPs and are needed for an active translocation across M cell monolayer. Tropism of EHEC to PPs can be limited by a probiotic S. cerevisiae strain.

Sa1289 Culture-Independent Quantitation of Clostridium difficile in C57BL/6 Mice Aids Mechanistic Study of Disease Pathophysiology and Protective Immunology

was a hematopoietic stem cell transplant recipient patient who was found to have chronic graft-versus-host disease of the terminal ileum. FMT was aborted in this patient despite finding of persistent C. difficile PCR prior to attempted FMT. Two patients were found to have collagenous colitis, which had not been evident on colonoscopies performed within a year of FMT. One patient was found to have a 1.5 cm tubulovillous adenoma. Conclusions: No significant differences in response or AE were seen when comparing UGI tract versus colonoscopy method of FMT delivery as well immunocompromised versus nonimmunocompromised patients. Most patients prefer using a standard donor over a relative. FMT using colonoscopy may help to identify additional pathology that may not be evident when using other modes of delivery. Table 1. Comparison of FMT performed using upper gastrointestinal tract* versus colonoscopy