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Pathobionts employ unique metabolic adaptation mechanisms to maximize their growth in disease conditions. Adherent–invasive Escherichia coli (AIEC), a pathobiont enriched in the gut mucosa of patients with inflammatory bowel disease (IBD), utilizes diet-derived L-serine to adapt to the inflamed gut. Therefore, the restriction of dietary L-serine starves AIEC and limits its fitness advantage. Here, we find that AIEC can overcome this nutrient limitation by switching the nutrient source from the diet to the host cells in the presence of mucolytic bacteria. During diet-derived L-serine restriction, the mucolytic symbiont Akkermansia muciniphila promotes the encroachment of AIEC to the epithelial niche by degrading the mucus layer. In the epithelial niche, AIEC acquires L-serine from the colonic epithelium and thus proliferates. Our work suggests that the indirect metabolic network between pathobionts and commensal symbionts enables pathobionts to overcome nutritional restriction and thrive in the gut.

Background: Crohn's disease displays heterogeneity in terms of disease location, progression and response to treatment. Most patients require an intestinal resection due to complicated disease, and a majority of them will experience disease recurrence. We aimed to characterize biological pathways driving postoperative recurrence and to identify predictors which could guide therapeutic management. Methods: In this multicentre prospective study, we conducted transcriptome analysis on ileal mucosa of CD patients undergoing ileocolonic resection. Samples were collected from the most inflamed part of the ileum (n=200), from the ileal resection margin (n=149) and in the neo-terminal ileum 6 months after surgery (n=122). The primary endpoint was postoperative endoscopic recurrence at month 6. We applied a regression model to identify gene signatures predicting for endoscopic recurrence. Findings: Molecular patterns of early endoscopic recurrence significantly differed from those observed in chronic inflammation. Gene expression analysis of the inflamed area of the surgical specimens identified clusters of CD patients. However, pathway gene signatures at the ileal margin were superior to those of the inflamed area to predict postoperative outcome. Several pathways, including JAK/STAT signalling, cell adhesion molecules and extracellular matrix protein were associated with a higher risk of endoscopic recurrence. JAK/STAT pathway activation at the ileal margin predicted early postoperative endoscopic recurrence (AUC of 0*74) and was associated with a higher of clinical relapse on the long term. A significant increase of p-STAT3 levels at the ileal margin was associated with severe endoscopic recurrence (i3, i4) compared to no recurrence (i0) (p=0*02) and moderate recurrence (i1, i2) (p=0*04). Interpretation: Gene expression analysis at the ileal margin of the surgical specimen may strongly predict postoperative outcome. The inflammatory processes associated with postoperative recurrence differ from those engaged in chronic inflammation. Activation of JAK/STAT signalling at the ileal margin of the surgical specimen is predictive of postoperative recurrence and should be specifically targeted. Trial Registration: ClinicalTrials.gov (NCT03458195). Funding Statement: This work was supported by Helmsley Charitable Trust and the Institut national de sante et de la recherche biomedical (INSERM). Declaration of Interests: SN received honoraria from MSD, Abbvie, Takeda, Janssen, HAC Pharma, Tillots, Ferring, and Novartis. BP received honoraria from AbbVie, MSD, Takeda, Janssen, Bioagaran, and Ferring. AB received honoraria from MSD, Abbvie, Ferring, Takeda, Vifor Pharma, Sanofi-Aventis, Hospira, and Janssen. XT received honoraria from Abbvie, MSD, Takeda, Ferring, Norgine, and Janssen. MF received honoraria from AbbVie, MSD, Takeda, Janssen, Pfizer, Ferring, and Boehringer. PS received honoraria from Takeda, MSD, Biocodex, Ferring and Abbvie and financial support from Takeda. MA received honoraria from Janssen, Takeda, Pfizer, MSD, Abbvie, Ferring, Amgen, Biogen, Celgene, and Genentech/Roche. All other authors have no conflict of interest regarding this study. Ethics Approval Statement: All patients provided an informed written consent. The study was approved by AFFSAPS (IDRCB: 2009-A00205-52) and the French Ethic Committee (CPP 2009/17).

Introduction Increasing evidence link the immune microenvironment, microbiota and colorectal cancer (CRC). E. coli pks + are more frequently detected on CRC patient mucosa and exhibit procarcinogenic properties in CRC murine models. Aim of this work was to evaluate the impact of chronical infection by colibactin-producing E. coli on immune cells in APC min/+ mice colon. Material and methods Min mice were per os inoculated with a CRC E. coli pks + strain (11 G5), non-pathogenic E. coli (K-12) or PBS. Induction of oxidative stress and inflammation during the infection was evaluated using optical in vivo imaging (IVIS spectrum). After 7 weeks, number and volume of polyps were evaluated and colonic samples were histologically analysed. Immunofluorescent staining of immune cells (neutrophils and T cell populations) was performed to quantify positive cells in three interesting colon areas: mucosa, lymphoid follicle and tumour using an innovative algorithm created with Tissue Studio software. In parallel, quantification of immune cells (lymphoid populations) was performed in mesenteric lymph node (MLNs) by flow cytometry. Results and discussions Using optical imaging, we detected a significant increase of oxidative stress and inflammation in the gut after thirty days of 11 G5 infection. However, using our specific algorithm and CD45 immunostaining, we observed a significant increase of lymphoid follicle size in the gut of mice infected with the 11 G5 strains. Interestingly, follicle size was positively correlated with tumour volume suggesting an association between pro-carcinogenic properties of 11 G5 strain and gut immune response. In addition, we observed an increase of neutrophils on the mucosa and lymphoid follicle of 11 G5 infected mice. These results can be linked to our in vivo optical imaging observations. Moreover we observed a trend to decrease of CD3 + T cells on mucosa and tumour for the 11 G5 group, suggesting a possible link between E. coli pks +and anti-tumour T cells. Finally we noticed a significant decrease of CD4 + CD25 + anti-inflammatory T cells in MLNs of 11 G5 infected mice, negatively correlated with gut bacteria colonisation. Conclusion Here we report a potential immunomodulatory effect on gut microenvironment by pks+E. coli, which could be associated with its carcinogenic effect. Our results suggest that gut lymphoid follicle and immune cells such as neutrophils and T cells could be involved in this process. This work shows a link between immune microenvironment, pathogenic E. coli and tumour development.

Background and aim: Contaminant molecules could play a key role in the initiation and/or promotion of colorectal cancer (CRC) which is the second leading cause of cancer worldwide. Among the different pollutants, titanium dioxide (TiO2) is a white natural metal oxide which is particularly monitored. It is one of the most widely used engineered nanomaterials in daily consumer products, including food. Long-term inhalation studies with TiO2 nanoparticles (NPs) in rats reported development of lung tumors leading to classify this compound as a possible carcinogen for humans by International Agency for Research on Cancer. The food additive TiO2, referred to as E171 in the European Union, is commonly used as a whitening and brightening agent in sugar confectionary, sauces, ice creams and pastries. The gastro-intestinal tract may be an important absorption route for TiO2 NPs and additive E171 could be involved in the CRC etiology. Thus, this project aimed to assess the impact of chronic exposure of food additive E171 in CRC development using APCmin/+ mouse model which spontaneously develop intestinal tumors. Because intestinal microbiota play a role in colorectal carcinogenesis, carcinogenic properties of TiO2 will be evaluated in association with microbiota dysbiosis. Methods: Additive E171 composition was characterized by X-ray diffraction (XRD) and thermogravimetric analysis (TGA). APCmin/+ mice were chronically exposed (twice per week) to E171 (1 and 10 mg/kg of body weight (BW)) or vehicle (water) for 28 days via intragastric gavage. Micro-inflammation was assessed by the quantification of fecal lipocalin-2 (LCN-2) marker. At the end of the experimental protocol, macroscopic and histologic examinations of colon were performed and colonic mucosa-associated microbiota was analyzed by quantitative PCR. Results: As shown by the TGA analysis, the E171 compound is purely mineral and the size of NPs administrated to mice in this study did not exceed 28 nm (XRD analysis). No significant body weight loss was observed between mice groups suggesting no toxicity in our experimental conditions. However macroscopic and histological observations showed a more advanced tumor development in mice receiving the additive E171 (at a dose of 10 mg/kg of BW) compared to control animals. At the end of the experiment, high level of fecal LCN-2 concentration correlated with increase of tumoral volume. Mice with adenocarcinoma within this group also exhibit an increase of Bacteroides and Akkermansia as well as a higher prevalence of Escherichia coli and a decrease of Bifidobacterium. Conclusion: Additive E171 promotes colonic tumorigenesis and induces change in gut microbiota composition. Underlying carcinogenic mechanisms focusing on microbiota dysbiosis implication are in progress. This study supports the carcinogenic properties of TiO2 in the context of colorectal cancer.

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.

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-kappaB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.

Background: Fecal microbiota transplant (FMT) is fast emerging as a promising therapy for recurrent clostridium difficile colitis in patients not responding to antibiotic therapy. However all patients for FMT need a fecal sample from a healthy donor. However the effect of age on the fecal microbiome of the healthy donor and its clinical efficacy, has not been examined previously. Aim: To examine fecal samples of healthy human subjects of various ages, volunteering to donate their samples for FMT by genomic analysis and clinical outcom. Methods: All healthy subjects who were rigorously screened for infectious disease and selected as donors for FMT were included in the study. Fecal samples were processed and analyzed using 16S rRNA gene amplicon sequencing. Microbiota compositions were studied using standard 16S rRNA analysis tools (CloVR-16S). Differences in bacterial phylum abundance and diversity (Shannon index) of the donor fecal microbiota were analyzed using three different cutoff for age of 50 years, 60 years and 70 years. Effect of fecal microbiota from donors of different age groups on efficacy of FMT in patients with RCDI was monitored in the GI clinic. Results: Fecal microbiota of 30 healthy donors was analyzed. The mean age of the donors was 50±15.3 years and ranged between 20 years to 82 years. Of these 30 donors, 18 (60%) were males and 26 (87%) were related to the recipients. The Shannon index did demonstrate significant increase in the fecal microbiome diversity in 70+ year old donors, as demonstrated in Figure 1. Althoughmicrobiome dissimilarity between the younger donors groups was relatively larger than the older donors groups on weighted UniFrac metric analysis, the association was not statistically significant. At phylum level, while the relative abundance of Firmicutes was higher in older groups (p

G A A b st ra ct s unknown. Hence, we investigated here whether the gut microbiota can affect colonic miRNAs secretion and profile. Results: High throughput technology screening the expression levels of 752 known mouse miRNAs revealed that miRNA profiles were different in the feces of GF mice compared to conventionalized mice, with 13 miRNAs deregulated in the absence of a microbiota. In order to investigate if the microbiota composition play a role in fecal miRNA profiles, germ-free C57/Bl6 mice were "conventionalized" with microbiota of WT mice, non colitic IL10-/mice or colitic IL10-/mice, and fecal miRNAs profiles were analyzed. Microbiota composition analysis confirmed that we successfully transferred the difference in composition, with clear clustering according to the microbiota transferred. Mice receiving microbiota from colitic IL10-/mice harbor a proinflammatory microbiota 49 days post conventionalization, as assayed by fecal LPS and flagellin loads, and proinflammatory potential of mice receiving microbiota from non-colitic IL10-/mice were found to be moderately elevated compare to mice receiving microbiota from WT mice. Intestinal inflammatory status of mice from the three groups was unchanged, as assessed by MPO. However, mice colonized with colitic IL10-/microbiota displayed a slight but significant increase in blood glucose level, as well as other parameters of low-grade intestinal inflammation, such as fecal Lcn-2. miRNA profile was analyzed and Principal component analysis showed a clear clustering between the 3 groups at 49 days post conventionalization, that was not observed at day 0 (before conventionalization). Interestingly, the level of some miRNAs correlate with the level of some members of the gut microbiota found to drive the difference in the microbiota composition previously observed. Conclusion: Altogether, these data showed that the microbiota composition induces drastic changes in the dynamic of fecal miRNA. Fecal miRNA signature could be used as tools for assessing the gut microbiota composition and its inflammatory potential.

Introduction: Mucosal healing has become the treatment goal in patients with ulcerative colitis (UC) and Crohn's disease (CD). Whether low fecal calprotectin levels and histological healing combined with mucosal healing is associated with a further reduced risk of relapses is currently unknown. Methods: Patients with CD, UC or IBD-unclassified (IBD-U) scheduled for a surveillance colonoscopy were asked to collect a stool sample prior to the start of bowel cleansing. Only patients with mucosal healing (MAYO endoscopic score of 0) and no inflammation in the terminal ileum (CD patients) were included. Fecal calprotectin was measured using a quantitative enzyme-linked immunosorbent assay (R-Biopharm, Germany). Biopsies were obtained from four colonic segments and the terminal ileum (CD patients) and histological disease severity was assessed using the Geboes scoring system by a single expert pathologist. Patients were followed until the last outpatient clinic visit or the development of a relapse, which was defined as IBD-related hospitalization, surgery or step-up in IBD medication. ROC statistics were used to determine the optimal cut-off value for calprotectin. Results: Of the 164 patients undergoing surveillance colonoscopy, 67 patients were excluded due to active inflammation and 25 patients because biopsies were missing or because intubation of the terminal ileum was not performed (CD patients). Of the remaining 72 patients (20 CD, 52 UC or IBD-U), six patients (8%) relapsed after a median follow-up of 11 months (range 5 15 months). Median fecal calprotectin levels at baseline were significantly higher for patients who relapsed compared to patients who maintained remission (284 mg/kg vs. 37 mg/kg. p 3.1) was not associated with an increased risk of relapse (p=0.94). A higher Geboes score was not associated with higher levels of calprotectin either (p=0.27). Fecal calprotectin above 56mg/kg predicted relapse during follow-up with 100% sensitivity and 64% specificity, whereas active histological inflammation (Geboes score >3.1) predicted relapse with 33% sensitivity and 68% specificity. Conclusion: Elevated fecal calprotectin levels reliably predict relapses in IBD patients with mucosal healing.

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.

response to ATP (5 mM) than control group after 3h (291 ± 4.6 versus 55.85 ± 1.9, respectively, p < 0.0001). IL-β secretion was completely abrogated when ATP was preincubated with cIAP (200 units/ml) for 2 h. Conclusions: We hypothesize that IAP could play a role in protecting the host against excessive gut inflammation induced by extracellular danger signals like ATP. These processes are critical to the fundamental understanding of the gut mucosal enzyme defense system which maintains gut homeostasis. IAP could represent a novel therapy for human IBD.