An adherent-invasive Escherichia coli-colonized mouse model to evaluate microbiota-targeting strategies in Crohn's disease.

Adherent-invasive Escherichia coli (AIEC) were investigated for their involvement in the induction/chronicity of intestinal inflammation in Crohn's disease (CD). AIEC gut establishment is favoured by overexpression of the glycoprotein CEACAM6 in the ileal epithelium. We generated a transgenic mouse model, named 'Vill-hCC6', in which the human CEACAM6 gene was under the control of the villin promoter, conditioning expression in the small intestine. We demonstrated that CEACAM6 is strongly expressed in the small intestine mucosa and is correlated with numerous glycosylations displayed at the brush border of enterocytes. Ex vivo, the AIEC-enterocyte interaction was enhanced by CEACAM6 expression and necessitated the presence of the bacterial adhesive factor FimH. Finally, AIEC bacteria preferentially persisted in a FimH-dependent manner in the ileal mucosa of Vill-hCC6 mice compared to wild-type mice. This preclinical model opens new perspectives in the mechanistic study of the AIEC pathobiont and represents a valuable tool to evaluate the efficacy of new strategies to eliminate AIEC implanted in the ileal mucosa, such as phages, inhibitory and/or anti-virulence molecules, or CRISPR-based strategies targeting virulence or fitness factors of AIEC bacteria.

Epigenetic master regulators HDAC1 and HDAC5 control pathobiont Enterobacteria colonization in ileal mucosa of Crohn's disease patients.

ABBREVIATIONS: AIEC Adherent-Invasive Escherichia coli; BSA Bovine serum albumin; CD Crohn's disease; CEABAC10 Carcinoembryonic antigen bacterial artificial chromosome 10; CEACAM Carcinoembryonic antigen-related cell adhesion molecule; FBS Fetal bovine serum; IBD Inflammatory Bowel Disease; HAT Histone acetyltransferase; HDAC Histone deacetylase; kDa KiloDalton; SAHA Suberoylanilide Hydroxamic Acid; Scr Scramble.

Propionate catabolism by CD-associated adherent-invasive E. coli counteracts its anti-inflammatory effect.

Crohn's disease (CD) is a chronic and disabling inflammatory disorder of the gut that is profoundly influenced by intestinal microbiota composition, host genetics and environmental factors. Several groups worldwide have described an imbalance of the gut microbiome composition, called dysbiosis, in CD patients, with an increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes. A high prevalence of adherent-invasive Escherichia coli (AIEC) pathobionts has been identified in the intestinal mucosa of CD patients. A significant loss in the bacteria that produce short-chain fatty acids (SCFAs) with anti-inflammatory properties, such as propionate, is also a consequence of dysbiosis in CD patients. Here, the AIEC reference strain LF82 was able to degrade propionate in the gut, which was sufficient to counteract the anti-inflammatory effect of propionate both in in vitro models and in mice with DSS-induced colitis. The consumption of propionate by AIEC pathobionts leads to an increase in TNF-α production by macrophages upon infection through the bacterial methyl-citrate pathway. To induce the protective effects of SCFAs on the inflamed gut, we used a G-protein-coupled receptor 43 agonist (GPR43 agonist) that is not metabolizable by intestinal bacteria. Interestingly, this agonist showed anti-inflammatory properties and decreased the severity of colitis in AIEC-infected mice, as assessed by an improvement in the disease activity index (DAI) and a decrease in AIEC pathobiont encroachment. Taken together, these results highlight the effectiveness of GPR43 agonist treatment in the control of gut inflammation and improved our understanding of the ability of AIEC to modulate propionate availability to create an infectious niche to its advantage.

Methyl-donor supplementation prevents intestinal colonization by Adherent-Invasive E. coli in a mouse model of Crohn's disease.

Deficiencies in methyl-donor molecules (folate, B12 vitamin), DNA methylation alteration and high prevalence of Adherent-Invasive Escherichia coli (AIEC) are frequently observed in Crohn's disease (CD) patients. AIEC bacteria adhere to the enterocytes through abnormally expressed carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) glycoprotein on host cells. This work aims at studying the relationship between methyl-donor molecules and AIEC-induced intestinal inflammatory response. CEABAC10 mice, a mouse model of CD, were fed a control or Methyl-donor Supplemented diet (MS diet). CEACAM6 promoter was hypermethylated in intestinal epithelial cells from mice fed an MS diet, which was associated with a significant decrease in CEACAM6 expression. Transcriptomic analysis revealed increased expression of anti-microbial peptides, increase in HSP70 gene family expression and a decreased expression of inflammatory marker Calprotectin upon MS diet, associated to a lower ability of AIEC bacteria to colonize gut mucosa. We observed in a cohort of CD patients that serum folate concentration was inversely correlated to Crohn's disease endoscopic index of severity and to fecal inflammatory markers. This study demonstrates that methyl-donor supplementation through the diet induces a specific intestinal micro-environment limiting pathobiont colonization of the gut. Clinicians may wish to consider methyl-donor supplementation for methyl-donor deficient CD patients.

Adherent-Invasive E. coli: Update on the Lifestyle of a Troublemaker in Crohn's Disease.

Besides genetic polymorphisms and environmental factors, the intestinal microbiota is an important factor in the etiology of Crohn's disease (CD). Among microbiota alterations, a particular pathotype of Escherichia coli involved in the pathogenesis of CD abnormally colonizes the intestinal mucosa of patients: the adherent-invasive Escherichia coli (AIEC) pathobiont bacteria, which have the abilities to adhere to and to invade intestinal epithelial cells (IECs), as well as to survive and replicate within macrophages. AIEC have been the subject of many studies in recent years to unveil some genes linked to AIEC virulence and to understand the impact of AIEC infection on the gut and consequently their involvement in CD. In this review, we describe the lifestyle of AIEC bacteria within the intestine, from the interaction with intestinal epithelial and immune cells with an emphasis on environmental and genetic factors favoring their implantation, to their lifestyle in the intestinal lumen. Finally, we discuss AIEC-targeting strategies such as the use of FimH antagonists, bacteriophages, or antibiotics, which could constitute therapeutic options to prevent and limit AIEC colonization in CD patients.

Smarcad1 mediates microbiota-induced inflammation in mouse and coordinates gene expression in the intestinal epithelium.

BACKGROUND: How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. RESULTS: Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. CONCLUSIONS: Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases.

Adaptation of adherent-invasive E. coli to gut environment: Impact on flagellum expression and bacterial colonization ability.

The pathogenesis of Crohn's disease (CD) is multifactorial and involves genetic susceptibility, environmental triggers and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are flagellated bacteria more prevalent in CD patients than in healthy subjects and promote chronic intestinal inflammation. We aim at deciphering the role of flagella and flagellin modulation by intestinal conditions. AIEC flagellum expression is required for optimal adhesion to and invasion of intestinal epithelial cells. Interestingly, differential flagellin regulation was observed between commensal E. coli (HS) and AIEC (LF82) strains: flagellum expression by AIEC bacteria, in contrast to that of commensal E. coli, is enhanced under intestinal conditions (the presence of bile acids and mucins). Flagella are involved in the ability of the AIEC LF82 strain to cross a mucus layer in vitro and in vivo, conferring a selective advantage in penetrating the mucus layer and reaching the epithelial surface. In a CEABAC10 mouse model, a non-motile mutant (LF82-ΔfliC) exhibits reduced colonization that is restored by a dextran sodium sulfate treatment that alters mucus layer integrity. Moreover, a mutant that continuously secretes flagellin (LF82-ΔflgM) triggers a stronger inflammatory response than the wild-type strain, and the mutant's ability to colonize the CEABAC10 mouse model is decreased. Overexpression of flagellin in bacteria in contact with epithelial cells can be detrimental to their virulence by inducing acute inflammation that enhances AIEC clearance. AIEC pathobionts must finely modulate flagellum expression during the infection process, taking advantage of their specific virulence gene regulation to improve their adaptability and flexibility within the gut environment.

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.

BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.

Microbiota derived short chain fatty acids promote histone crotonylation in the colon through histone deacetylases.

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.

Western diet induces a shift in microbiota composition enhancing susceptibility to Adherent-Invasive E. coli infection and intestinal inflammation.

Recent advances have shown that the abnormal inflammatory response observed in CD involves an interplay among intestinal microbiota, host genetics and environmental factors. The escalating consumption of fat and sugar in Western countries parallels an increased incidence of CD during the latter 20(th) century. The impact of a HF/HS diet in mice was evaluated for the gut micro-inflammation, intestinal microbiota composition, function and selection of an E. coli population. The HF/HS diet created a specific inflammatory environment in the gut, correlated with intestinal mucosa dysbiosis characterized by an overgrowth of pro-inflammatory Proteobacteria such as E. coli, a decrease in protective bacteria, and a significantly decreased of SCFA concentrations. The expression of GPR43, a SCFA receptor was reduced in mice treated with a HF/HS diet and reduced in CD patients compared with controls. Interestingly, mice treated with an agonist of GPR43 were protected against DSS-induced colitis. Finally, the transplantation of feces from HF/HS treated mice to GF mice increased susceptibility to AIEC infection. Together, our results demonstrate that a Western diet could aggravate the inflammatory process and that the activation of the GPR43 receptor pathway could be used as a new strategy to treat CD patients.

Saccharomyces cerevisiae CNCM I-3856 prevents colitis induced by AIEC bacteria in the transgenic mouse model mimicking Crohn's disease.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with Crohn's disease (CD), are able to adhere to and invade intestinal epithelial cells. Overexpression of the glycoprotein CEACAM6 on host cells favors AIEC attachment and inflammation. We investigated the ability of Saccharomyces cerevisiae CNCM I-3856 to inhibit AIEC adhesion and to reduce colitis. METHODS: Adhesion experiments were performed on T84 cells and on enterocytes from patients with CD with AIEC LF82 in the presence of S. cerevisiae. Colonization and symptoms of colitis were assessed in LF82-infected transgenic CEABAC10 mice treated with live S. cerevisiae or S. cerevisiae derivatives. Proinflammatory cytokines were quantified by enzyme linked immunosorbent assay. Intestinal permeability was assessed by measuring the 4 kDa dextran-FITC flux in the serum. RESULTS: S. cerevisiae strongly inhibited LF82 adhesion to T84 cells and to the brush border of CD enterocytes. Yeasts decreased LF82 colonization and colitis in CEABAC10 mice and restored barrier function through prevention of the LF82-induced expression of pore-forming tight junction claudin-2 at the plasma membrane of intestinal epithelial cells. These effects were accompanied by a decrease in proinflammatory cytokines IL-6, IL-1ß, and KC release by the gut mucosa. Yeast derivatives exerted similar effects on LF82 colonization and colitis demonstrating that yeast viability was not essential to exert beneficial effects. CONCLUSIONS: S. cerevisiae yeasts reduce colitis induced by AIEC bacteria in CEACAM6-expressing mice. Such a probiotic strategy could be envisaged in a subgroup of patients with CD abnormally expressing CEACAM6 at the ileal mucosa and therefore susceptible to being colonized by AIEC bacteria.

Diet-induced hypoxia responsive element demethylation increases CEACAM6 expression, favouring Crohn's disease-associated Escherichia coli colonisation.

OBJECTIVE: Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC strains adhere to enterocytes via interaction between type 1 pili and CEACAM6 receptors abnormally expressed on CD ileal mucosa, leading to gut inflammation. We analysed whether epigenetic mechanisms are involved in the upregulation of CEACAM6 expression in intestinal epithelial cells (IECs). DESIGN: Methylation of CEACAM6 promoter was analysed using bisulfite sequencing and site-specific methylation by SnapShot. pCpGfree reporter system was used to analyse CEACAM6 promoter activity. Transgenic mice expressing human CEACAM6 fed either standard food or a low-methyl diet (LMD) were orally challenged with 10(9) AIEC LF82. After 3 days, gut-associated AIEC and proinflammatory cytokines were quantified. RESULTS: Analysis of CEACAM6 gene promoter revealed potentially methylated dinucleotide CpGs within HIF-1-responsive elements (HREs). Methylation levels of CpG within CEACAM6 promoter were inversely correlated with CEACAM6 expression in IEC expressing various levels of CEACAM6. We show the critical role of HRE methylation and transcription factor HIF-1 in the regulation of CEACAM6 gene in IEC. This was confirmed in transgenic mice expressing human CEACAM6 fed a LMD. LMD-dependent HRE demethylation led to abnormal gut expression of CEACAM6, favouring AIEC colonisation and subsequent inflammation. CONCLUSIONS: HRE hypomethylation in CEACAM6 promoter correlates with high expression in IEC. Our findings suggest that abnormal DNA methylation leading to CEACAM6 increased expression and AIEC-mediated gut inflammation can be related to changes in nutritional habits, such as low intake in methyl donor molecules, leading to abnormal epigenetic marks in mouse model mimicking CD susceptibility.

Western diet induces dysbiosis with increased E coli in CEABAC10 mice, alters host barrier function favouring AIEC colonisation.

OBJECTIVE: Western diet is a risk factor for Crohn's disease (CD). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is abnormally expressed in CD patients. This allows adherent-invasive Escherichia coli (AIEC) to colonise the gut mucosa and leads to inflammation. We assessed the effects of a high fat/high sugar (HF/HS) Western diet on gut microbiota composition, barrier integrity and susceptibility to infection in transgenic CEABAC10 mice expressing human CEACAMs. DESIGN: Colonic microbiota composition and susceptibility of CEABAC10 mice to AIEC LF82 bacteria infection were determined in mice fed a conventional or HF/HS diet. Barrier function and inflammatory response were assessed by studying intestinal permeability, tight junction protein and mucin expression and localisation, and by determining histological score and levels of cytokine release. RESULTS: HF/HS diet led to dysbiosis in WT and transgenic CEABAC10 mice, with a particular increase in E coli population in HF/HS-fed CEABAC10 mice. These mice showed decreased mucus layer thickness, increased intestinal permeability, induction of Nod2 and Tlr5 gene transcription, and increased TNFα secretion. These modifications led to a higher ability of AIEC bacteria to colonise the gut mucosa and to induce inflammation. CONCLUSIONS: Western diet induces changes in gut microbiota composition, alters host homeostasis and promotes AIEC gut colonisation in genetically susceptible mice. These results support the multifactorial aetiology of CD and highlight the importance of diet in CD pathogenesis.

Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response.

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn's disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH(LF82) (expressing FimH with an AIEC-associated mutation) with fimH(K12) (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.

Adherent-invasive Escherichia coli induce claudin-2 expression and barrier defect in CEABAC10 mice and Crohn's disease patients.

BACKGROUND: Abnormal expression of CEACAM6 observed on the ileal epithelium in Crohn's disease (CD) patients allows adherent-invasive Escherichia coli (AIEC) to colonize gut mucosa. Since intestinal permeability is significantly increased in CD patients, we aimed at investigating whether and how AIEC alter barrier function. METHODS: Tissue microarray was performed on ileal biopsies from CD patients in quiescent and active phases. CEABAC10 or wildtype mice were orally challenged with 10(9) bacteria. Intestinal permeability was assessed by measuring 4 kDa dextran-FITC flux in serum, barrier integrity was analyzed using biotin tracer experiment, and claudin-2 protein immunostaining. Bacterial translocation was analyzed in Ussing chambers. RESULTS: Pore-forming tight junction protein claudin-2 is strongly expressed in the ileum of 51% patients in quiescent phase and in 49% of the patients with active CD. Infection of CEABAC10 transgenic mice expressing human CEACAMs with AIEC, but not with nonpathogenic E. coli, led to a significant 3.0-fold increase in intestinal permeability and to disruption of mucosal integrity in a type 1 pili-dependent mechanism. This is consistent with the claudin-2 abnormal expression at the plasma membrane of intestinal epithelial cells observed in AIEC-infected CEABAC10 mice. AIEC bacteria were able to translocate through CEABAC10 intestinal mucosa. CONCLUSIONS: These findings strongly support the hypothesis that AIEC type 1 pili-mediated interaction with CEACAM6 abnormally expressed in the quiescent phase of CD may disrupt intestinal barrier integrity before the onset of inflammation. Thus, therapeutic targeting claudin-2 induced by AIEC infection could be a new clinical strategy for preserving intestinal barrier function in CD patients.

Interactions with M cells and macrophages as key steps in the pathogenesis of enterohemorrhagic Escherichia coli infections.

Enterohemorrhagic Escherichia coli (EHEC) are food-borne pathogens that can cause serious infections ranging from diarrhea to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Translocation of Shiga-toxins (Stx) from the gut lumen to underlying tissues is a decisive step in the development of the infection, but the mechanisms involved remain unclear. Many bacterial pathogens target the follicle-associated epithelium, which overlies Peyer's patches (PPs), cross the intestinal barrier through M cells and are captured by mucosal macrophages. Here, translocation across M cells, as well as survival and proliferation of EHEC strains within THP-1 macrophages were investigated using EHEC O157:H7 reference strains, isogenic mutants, and 15 EHEC strains isolated from HC/HUS patients. We showed for the first time that E. coli O157:H7 strains are able to interact in vivo with murine PPs, to translocate ex vivo through murine ileal mucosa with PPs and across an in vitro human M cell model. EHEC strains are also able to survive and to produce Stx in macrophages, which induce cell apoptosis and Stx release. In conclusion, our results suggest that the uptake of EHEC by M cells and underlying macrophages in the PP may be a critical step in Stx translocation and release in vivo. A new model for EHEC infection in humans is proposed that could help in a fuller understanding of EHEC-associated diseases.