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International audience; 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-alpha 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.

© The Author(s). 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data., 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., This work was supported by BBSRC Institute Strategic Programme Grants (BBS/E/B/000C0404, BBS/E/B/000C0405), a MRC project grant to PVW and MV (MR/N009398/1), a BBSRC-Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP-Brazil Pump-priming award (BB/N013565/1,FAPESP#2015/50379-1), and a Newton Advanced Fellowship from the Royal Society to MARV and PVW (NAF\R1\180116).

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.

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., Histone post-translational modifications are known key regulators of gene expression. Here, the authors characterize histone crotonylation at histone H3 lysine 18 in intestinal epithelia and find that it is a highly dynamic cell cycle regulated mark under the regulation of the HDAC deacetylases.

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. Electronic supplementary material The online version of this article (10.1186/s13059-018-1489-y) contains supplementary material, which is available to authorized users.

International audience; The pathogenesis of Crohn's disease (CD) is multifactorial and involves genetic susceptibility, environmental triggers and intestinal microbiota. Adherent-invasiveEscherichia 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 commensalE. coli(HS) and AIEC (LF82) strains: flagellum expression by AIEC bacteria, in contrast to that of commensalE. 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 layerin vitroandin 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-Delta fliC) 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-Delta flgM) 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.

Many advances have been made in the understanding of Crohn's disease (CD) pathogenesis over the last decade. In CD patients abnormal ileal bacterial colonization could be linked to inappropriate innate immune responses to invasive bacteria. Adherent and invasive Escherichia coli strains have been isolated from CD patients and are able to adhere to and to invade intestinal epithelial cells and to induce colitis in transgenic mice expressing the human CEACAM6 molecule. In this review, we report recent advances concerning the involvement of adherent-invasive E. coli in the aetiology of CD and analyze how they can initiate inflammation of the gut mucosa in individuals with genetic predisposition.

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 20th 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.

International audience; 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 109 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.

International audience; 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-1, 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.

International audience; 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 TNF 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.

International audience; Inflammatory bowel diseases (IBD) provide a complex model of host-microbe interactions underpinning disease pathogenesis. Although there is no widespread agreement on the aetiology of IBD, there is evidence that microorganisms lead to the often severe inflammatory response characteristic of the disease. IBD is thought to result from an inappropriate and continuing inflammatory response to pathobionts microbes in a genetically susceptible host. In this review, we discuss the complex microbial ecosystem of the mammalian gut, the underlying genetic factors that predispose to IBD, and how these gene variants may alter host-microbe interactions and propagate inflammation. Incentive should be given to research that will promote a better understanding of host-microbial interactions in the intestine and lay the foundations for new therapeutic approaches to both treat and prevent onset and relapse of intestinal inflammation in genetically susceptible hosts.

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 109 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. (Inflamm Bowel Dis 2011;)

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.

s of the 9th Congress of ECCO the European Crohn’s and Colitis Organisation S11 unknown function comprising 53% of nonpolar residues. This last feature has prevented the characterization of the putative anti-inflammatory activity of MAM-derived peptides. The gene transfection of MAM (MAM+) in epithelial cells (HEK293T, HT29) led to a significant decrease in the activation of the NF uB pathway with a dose-dependent effect (24±3 × AU activation in MAM cells vs. 4±1 × AU activation in MAM+ cells). This decrease in the activity of NF uB was also observed in the MAM+ cells after stimulation with IKKB (distal part of the NF uB pathway), confirming the results obtained with F. prausnitzii supernatant. Finally, immunofluorescence experiments showed that after expression in epithelial cells, MAM was localized in the perinuclear zone and colocalized with IKKB. Conclusions: We identified a 15 kDa protein with antiinflammatory properties produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein and/or derived peptides involved in the anti-inflammatory effect are able to inhibit NF uB pathway in intestinal epithelial cells. OP019 Hypoxia responsive elements methylation status governs adherent-invasive Escherichia coli-receptor CEACAM6 expression in Crohn’s disease J. Denizot1, A. Agus1, A. Desrichard2, N. Uhrhammer2, A. Darfeuille-Michaud1, N. Barnich1 *. 1Universite d’Auvergne, M2iSH, UMR INSERM/Universite d’Auvergne U1071; Unite Sous Contrat Institut National de la Recherche Agronomique 2018, Clermont-Ferrand, France, 2Universite d’Auvergne, Department of Oncogenetics, Department of Breast Oncology, Centre Jean Perrin, Clermont-Ferrand, France Background: Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn’s disease (CD) patients. This allows Adherent-Invasive Escherichia coli (AIEC) to colonize gut mucosa, leading to development of inflammation. Our aim was to understand the molecular mechanisms involved in the abnormal expression of CEACAM6 in the mucosa of CD patients. Since changes in DNAmethylation patterns were reported in CD patients, we analyzed whether epigenetic mechanisms are involved in the up-regulation of CEACAM6 expression in intestinal epithelial cells. Methods: Global methylation status of CEACAM6 promoter was analyzed using bisulfite sequencing and site-specific methylation level was quantified using snap-shot approach in intestinal epithelial cells. Protein expression and localization were performed using immunofluorescence staining and Westernblot analysis. Chromatin immunoprecipitation was used to determine HIF-1 transcription factor binding on CEACAM6 gene promoter. pCpG free SEAP reporter system was used to analyze CEACAM6 promoter activity. Transgenic CEABAC10 mice expressing human CEACAM6 fed either standard food (Normal Diet) or a Low-Methyl-Diet (LMD) composed with lower quantities of B12 vitamins, folic acid and choline, and were orally challenged with 109 AIEC LF82 bacteria. At 3 days postinfection, gut-associated AIEC and pro-inflammatory cytokines were quantified. Results: Analysis of CEACAM6 gene promoter revealed potentially methylated dinucleotide CpGs within Hypoxia Inducible Factor-1 (HIF-1) Responsive Elements (HREs). Methylation levels of these CpG were inversely correlated with CEACAM6 expression in intestinal epithelial cells (IEC) expressing various levels of CEACAM6. We also demonstrated the importance of HRE methylation levels and transcription factor HIF-1 in the regulation of CEACAM6 gene in IEC using pCpG free SEAP promoter reporter system. This was confirmed in vivo in CEABAC10 mouse model under a methyl donor-deficient diet. Low methyl diet-dependent HRE demethylation led to abnormal gut expression of CEACAM6, favoring AIEC colonization and subsequent inflammation. Conclusions: A CEACAM6 promoter hypomethylation on HREcontaining CpG correlates with higher CEACAM6 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 Crohn’s disease patients. OP020 16S rRNA gene pyrosequencing indicate that siblings of Crohn’s disease patients exhibit a biologically relevant dysbiosis in the mucosal microbiota communities C. Hedin1 *, C. van der Gast2, G. Rogers3, S. McCartney4, A.J. Stagg5, J.O. Lindsay6, K. Whelan1. 1King’s College London, School of Medicine, Diabetes and Nutritional Sciences Division, London, United Kingdom, 2NERC, Centre for Ecology & Hydrology, Wallingford, Oxfordshire, United Kingdom, 3University of Queensland, Translational Research Institute, Immunity, Infection, and Inflammation Program, Mater Research Institute, Woolloongabba, Australia, 4University College London, Centre for Gastroenterology and Nutrition, London, United Kingdom, 5Blizard Institute, Queen Mary University of London, Centre for Immunology and Infectious Disease, London, United Kingdom, 6Barts and the London NHS Trust, Gastroenterology Division, London, United Kingdom Background: Reduced mucosal concentrations of Faecalibacterium prausnitzii predict disease recurrence in patients with Crohn’s disease (CD). Siblings of CD patients have elevated risk of developing disease and share aspects of disease phenotype compared with healthy controls (HC), including a dysbiosis in the faecal microbiota [1]. No study has compared the mucosal microbiota in CD siblings to unrelated healthy controls. Aim: To apply 16S rRNA gene pyrosequencing in order to determine whether dysbiosis is present in the mucosal microbiota of siblings of CD patients with reference to HC, and to accomplish a more comprehensive characterisation of that dysbiosis. Methods: Rectal biopsies were taken from 21 patients with quiescent CD, 17 of their healthy siblings and 19 unrelated healthy controls (HC). Total DNA was extracted using a phenol/ chloroform based method. PCR amplification of the V1 to V3 region of the bacterial 16S ribosomal RNA gene was performed, and microbiota composition resolved by 454 pyrosequencing. Results: For each group, resulting species in the microbiota were classified into those that were common and abundant among similar subjects (core) versus infrequent and rare. [2] In terms of both microbial diversity (measured by both the Shannon Wiener and Simpson’s indexes of diversity) and species richness, the core mucosal microbiota of both siblings and CD patients were significantly less diverse than HC. Although the diversity of the rare microbiota was lower in CD compared with HC, there were no differences in diversity of rare microbiota between siblings and HC. Metacommunity profiling using the Bray Curtis (SBC) index of similarity with unweighted pair group averages showed that the core microbial metacommunity of siblings was more similar to CD patients (SBC=0.70) than to HC, whereas the rare microbial metacommunity of siblings was more similar to HC (SBC=0.42). As in patients with CD, the species that contributed most to the dissimilarity between healthy siblings and HC was F. prausnitzii, Table 1. Conclusions: This is the first in depth case control study of the mucosal microbiota in the siblings of CD patients. We report a dysbiosis that is characterised by reduced diversity of core microbiota and lower abundance of F. prausnitzii. Given that siblings of CD patients have elevated risk of developing CD, this dysbiosis in otherwise healthy people implicates microbiological processes in CD pathogenesis and risk. by gest on Feruary 3, 2016 http://eccoxfordjournals.org/ D ow nladed from

Background: The impact of antibiotics on colonic microbiota is poorly characterized. Methods: Three groups of women (N=10 each) treated for bacterial vaginosis were investigated . Metronidazole 3x400mg/day + ciprofloxacin 2x500mg/day were given for 2 weeks. Group I received antibiotics only, Gr.II received Saccharomyces boulardii(Sb) concomitant to, and Gr.III received Sb subsequent to antibiotic therapy. A 250 mg capsule Sb Perenterol® Biocodex was given 3xdaily for two weeks. Microbiota were investigated using structure functional FISH analysis of Carnoy fixated stool cylinders. Stools were collected at days -90,-60,-30,7,14,28,42,56, and 70 as related to the start of antibiotic therapy. 250 bacterial FISH probes were tested and 82 were selected for longitudinal analysis. Results: Bacteroides, F. prausnitzii and Roseburia (habitual groups) were present in all patients (10-30% of the fecal mass each). The occurrence of other bacteria was occasional. The composition of microbiota prior to antibiotics was stable in repeated investigations of the same patient, despite high interindividual variability. Antibiotic therapy suppressed all bacterial groups, and some of the microbiota previously not detected, emerged in marginal concentrations (C.viridae, Streptococcus, Staphylococcus, Bif. longum ). Bifidobacteriaceae, Enterobacteriacae or C.difficile groups increased in the first two to four weeks after the end of the therapy (p ,0.01) and returned to initial levels with recovery of the habitual bacterial groups. Sb concomitant with antibiotics reduced the antibiotic associated suppression of bacteria. After one week of antibiotic therapy, concentrations of F.prausnitzii dropped in Gr.I, II, and III, respectively, from a median of 13.6±3.7 to 3.1±2.0, 8±4.1, and 3±2.1 x 109bacteria/ml. The decrease in microbial concentrations in Gr. II receiving Sb concomitant to antibiotics was moderate and differed significantly from the decrease in Gr.I and III (p,0.001). The microbiota recovered quicker in groups receiving Sb. The habitual bacteria reached initial values 4 weeks after the end of antibiotics in all groups treated with Sb In Group I, F. prausnitzi was still reduced to 9.8±2.8 x 109bacteria/ml and was significantly lower than prior to antibiotics (p=0.04). The composition of microbiota returned quicker to the individual initial profiles in the groups receiving Sb In regard to the 82 investigated bacterial groups, the median number of mismatches in composition of microbiota prior to and after antibiotic therapy was 8.7; 5.2 (p=0.05) and 2.4 (p,0.001) in Gr.I, II and III, respectively. The difference between preand postantibiotic period was significantly higher in Gr.I, receiving no Sb. Conclusions: S. boulardii prevents/reduces effectively the antibiotic-associated changes in colonic microbiota, when given concomitant/subsequent to antibiotic therapy.

P454 A novel methylation panel for the early detection of neoplasia in high risk ulcerative colitis and Crohn’s colitis patients T. Lobaton Ortega1 *, D. Azuara2, F. Rodriguez-Moranta1, J. de Oca3, J. Guardiola1, A. Soriano-Izquierdo1, D. Montfort4, J. Boadas4, M. Piqueras5, S. Galter4, M. Estell6, V. Moreno2, G. Capella2. 1Hospital Universitari Bellvitage, Gastroenterology, Barcelona, Spain, 2Institut Catala de Oncologia, Translational Research Laboratory, Barcelona, Spain, 3Hospital Universitari Bellvitge, Surgery, Barcelona, Spain, 4Consorci Sanitari Terrasa, Gastroenterology, Barcelona, Spain, 5Consorcio Sanitari Terrasa, Gastroenterology, Barcelona, Spain, 6Institut Catala de Oncologia, Cancer Epigenetics and Biology Program, Barcelona, Spain

Crohn’s disease (CD) is an inflammatory bowel disease (IBD), evolving in a relapsing and remitting manner. No curative treatment exists for this debilitating disease, only symptomatic treatments are available to limit the frequency and intensity of the inflammatory flares. Different factors are involved in the development of CD such as genetic susceptibilities, environmental factors and microbiota composition. The consumption of a “western diet”, characterized by a high intake in fats, sugar and processed foods and a reduced quantity of fibers, is one of the major environmental factors involved in the development of CD. The adherent-invasive Escherichia coli (AIEC) pathotype abnormally colonizes the ileal mucosa of CD patients through mechanisms which have not been fully characterized. Recently, a renewed focus has been carried on the role of epigenetic in the pathogenesis of CD. Notably, alterations in the acetylation levels of histones proteins or a global decrease of the expression of histones deacetylases (HDAC) have been reported in CD patients. Hence, the main goal of my thesis was to better understand the AIEC-intestinal epithelium interaction by focusing on epigenetic and environmental factors.In vitro studies as well as validations in mice models and in a large cohort of CD patients revealed HDAC1 and HDAC5 as central and antagonistic actors of the regulation of Enterobacteria and AIEC intestinal colonization. Transcriptomic analyses performed on cells transfected with siRNAs directed against HDAC1 and HDAC5 unveiled the role of HDAC1 in the control of the inflammatory response and in the formation of actin cytoskeleton, and the involvement of HDAC5 in paracellular permeability. In addition, a reduced global HDAC activity and an increased HDAC5 expression have been observed in mice fed a high-fat (HF) diet, these conditions promoting the AIEC entry within intestinal epithelial cells. Following a new transcriptomic analysis, a repression of genes implicated in the host response to bacterium was highlighted in HF-fed mice, suggesting that HF diet could create a specific microenvironment favorable to AIEC establishment in the ileal mucosa. It was also demonstrated that AIEC bacteria were able, in that context, to modulate the host epigenome to their advantage in order to promote their own colonization. Thus, the data suggest that HDAC5 targeting, as well as a food rebalancing, could represent interesting strategies to limit AIEC intestinal colonization in CD patients.During my thesis, I also worked on the development of a new mouse model for the study of the AIEC-intestinal epithelium interaction. It was the Vill-hCC6 model, which reproduces the abnormal ileal expression of CEACAM6 receptor, on which AIEC bacteria particularly attach. AIEC bacteria preferentially persisted in the gut of transgenic mice demonstrating that this model could represent a robust preclinical model of ileal CD. In the future, it will enable to better understand the role of AIEC in CD but also to develop and test new molecules and strategies to specifically target AIEC bacteria established in the ileal mucosa of patients.; La maladie de Crohn (MC) est une maladie inflammatoire chronique de l’intestin (MICI), très invalidante, évoluant par poussées d’intensités et de localisations variables entrecoupées de périodes de rémission clinique. Cette maladie est incurable, seuls des traitements symptomatiques sont disponibles à ce jour pour soulager les patients. Concernant l’étiologie de la MC, elle est multifactorielle, résultant de l’interaction complexe entre des prédispositions génétiques, des facteurs environnementaux et des altérations de la composition du microbiote. Le régime alimentaire de type occidental, enrichi en sucres, en graisses, en aliments transformés et pauvre en fibres, est un des principaux facteurs de l’environnement mis en cause dans le développement de la MC. De plus, des souches particulières d’Escherichia coli, les E. coli adhérents et invasifs (AIEC), colonisent anormalement la muqueuse iléale des patients atteints de MC via des mécanismes qui n’ont pas été totalement caractérisés. Depuis quelques années, un intérêt nouveau s’est porté sur l’implication des facteurs épigénétiques dans la pathogenèse de la MC. Des altérations des niveaux d’acétylation des histones ou encore une diminution de l’expression des histones désacétylases (HDACs) ont notamment été rapportées chez les patients atteints de MC. Ainsi, l’objectif principal de cette thèse était de mieux comprendre l’interaction AIEC-épithélium intestinal en se concentrant sur des facteurs épigénétiques et environnementaux. Grâce à des études in vitro ainsi qu’à des validations in vivo et dans une large cohorte de patients atteints de MC, HDAC1 et HDAC5 ont été identifiées comme des acteurs centraux et antagonistes de la régulation de la colonisation intestinale par les Entérobactéries et les AIEC. Des analyses transcriptomiques réalisées sur des cellules transfectées avec des siARNs dirigés contre HDAC1 et HDAC5 ont révélé le rôle de HDAC1 dans le contrôle de la réponse inflammatoire et dans la formation du cytosquelette d’actine ainsi que celui de HDAC5 dans la perméabilité paracellulaire. Suite à la consommation d’un régime enrichi en graisses (HF), il a été observé une diminution de l’activité HDAC globale et une augmentation de l’expression de HDAC5, qui sont des conditions favorisant l’entrée des AIEC dans les cellules épithéliales intestinales. Aussi, suite à une nouvelle analyse transcriptomique, une répression des gènes impliqués dans la reconnaissance et la prise en charge des bactéries a été mise en évidence chez les souris nourries avec le régime enrichi, suggérant que le régime HF créerait un microenvironnement spécifique favorable à l’implantation des bactéries AIEC au niveau de la muqueuse iléale. Il a également été montré que les bactéries AIEC étaient capables, dans ce contexte, de moduler l’épigénome à leur avantage, afin de promouvoir leur propre colonisation. Ainsi, l’ensemble des données obtenues suggèrent que le ciblage de HDAC5, tout comme un rééquilibrage alimentaire, pourraient être des stratégies intéressantes pour limiter la colonisation intestinale par les bactéries AIEC chez les patients atteints de MC.Au cours de ma thèse, j’ai également travaillé sur la mise au point d’un nouveau modèle murin pour l’étude de l’interaction AIEC-épithélium intestinal : le modèle Vill-hCC6, qui permet de reproduire l’expression iléale anormale du récepteur CEACAM6, sur lequel les AIEC se fixent majoritairement. Grâce notamment à l’observation que les bactéries AIEC persistaient préférentiellement dans l’intestin des animaux transgéniques, nous avons montré que le modèle Vill-hCC6 pouvait constituer un bon modèle préclinique de MC iléale. À l’avenir, il pourra permettre de mieux comprendre le rôle des AIEC dans la MC mais également de développer et tester de nouvelles molécules et stratégies pour le ciblage spécifique des bactéries AIEC implantées dans la muqueuse iléale des patients.

Crohn’s Disease (CD) is an inflammatory bowel disease with a complex etiology including genetic, infectious, environmental and epigenetic factors. Modifications of DNA methylation profiles, and methyl donor molecules (folate and B12 vitamin) deficiencies have been observed in CD patients. The aim of this study was to investigate the impact of a high methyl diet (HMD), enriched in methyl donor group molecules involved in DNA methylation metabolism (folate and B12 vitamin), on intestinal physiology and gene expression. In this study, we showed that HMD favors bacterial species having beneficial effects on gut physiology. Furthermore, this diet modifies DNA methylation level and expression of various genes. An increase in DNA methylation was observed on the CEACAM6 gene promotor (involved in adherent and invasive Escherichia coli (AIEC) adhesion in CD patients) leading to a decrease of its expression. In conclusion, this work suggests that a methyl donor group supplementation could modulate microbiota composition and expression of genes involved in development and maintenance of inflammation in CD.; La maladie de Crohn (MC) est une maladie inflammatoire chronique de l’intestin dont l’étiologie complexe comprend des facteurs génétiques, infectieux, environnementaux ainsi qu’une composante épigénétique. Il a été montré des profils de méthylation de l’ADN modifiés chez les patients MC ainsi que des carences en molécules donneuses de groupements méthyles (folate et vitamine B12). L’objectif de ce projet était d’étudier l’impact d’un régime alimentaire enrichi en molécules donneuses de groupements méthyles (HMD), participant au métabolisme de la méthylation de l’ADN, sur la physiologie intestinale et l’expression des gènes. Nous avons montré qu’un régime HMD favorise le développement de certaines populations bactériennes ayant des effets bénéfiques sur la physiologie intestinale. De plus, ce régime modifie les niveaux de méthylation de l’ADN et l’expression de nombreux gènes. En particulier, l’augmentation du niveau de méthylation du promoteur du gène CEACAM6 (impliqué dans l’adhésion des Escherichia coli adhérents et invasifs (AIEC) chez les patients MC) ainsi qu’une diminution de son expression ont été observées. En conclusion, une supplémentation en molécules donneuses de méthyles pourrait permettre de réguler la composition du microbiote intestinal et l’expression de gènes participant au déclenchement et au maintien de l’inflammation intestinale dans la MC.