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sthe waaWVL operon is not present in non pathogenic E. coli strains but is present in mostAIEC from our collection and in two other sequenced AIEC strains NRG 857C and UM146.CONCLUSION: The presence of waaWVL operon is essential for AIEC bacteria to formbiofilm at the surface of the intestinal mucosa. The search for the presence of the waaWVLoperon could represent a useful molecular tool to identify pathogenic AIEC and targetingwaaWVL operon expression could be a very potent therapeutic strategy to interfere withthe ability of AIEC to form biofilm on the gut mucosa of Crohn's disease patients.

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Induction of Bacterial Antigen-Specific Colitis by a Simplified HumanMicrobiota Consortium in Gnotobiotic IL-10-/- MiceChang Soo Eun, Yoshiyuki Mishima, Bo Liu, Ryan B. Sartor

Background: We previously demonstrated that a simplified human microbiota consortium(SIHUMI) forms a stable community in mouse intestine that is maintained under gnotobioticconditions. Aims: To evaluate whether SIHUMI induces colitis in genetically susceptiblegerm-free (GF) IL-10-/- mice on both 129S6/SvEv (129) and C57BL/6 (B6) strains, determinebackground strain effects on colitis and microbiota, and examine the effects of inflammationon relative bacterial composition of ex-GF 129 and B6 IL-10-/- mice. Methods: GF wild-type (WT) and IL-10-/- mice on both 129 and B6 strains were colonized with 7 human-derived IBD-related gut bacteria that include E. coli LF82, Enterococcus faecalis OG1RF,Ruminococcus gnavus ATCC 29149, Bacteroides vulgatus ATCC 8482, Faecalibacteriumprausnitzii A2-165, Lactobacillus plantarum WCFS1 and Bifidobacterium longum subsp.longum ATCC 15707, by oral gavage and maintained in gnotobiotic conditions for 12 weeks.Bacterial quantification of feces at different time-points, intestinal contents and mucosaltissues of cecum, colon, and ileum were performed using 16S rRNA gene-based quantitativePCR. After 6 and 12 weeks, colonic segments were scored histologically, and IFN- γ, IL-12p40, and IL-17 levels were measured by ELISA in the supernatants of unstimulated colonictissue explants and mesenteric lymph node (MLN) cells cultured with each bacterial lysateor combination lysate. Results: These seven human bacteria formed a stable communityunder gnotobiotic conditions in the intestine of WT and IL-10-/- mice on both 129 and B6strains. Relative bacterial abundances in feces changed over time and were significantlydifferent between 129 and B6 strains, and were also different between WT and IL-10-/-mice. Differences in bacterial species concentrations existed between intestinal contents andmucosal tissues, and between colonic contents and ileal contents. SIHUMI induced colitisin IL-10-/- mice of both 129 and B6 strains with aggressiveness of colitis and MLN cellsactivation greater in 129 than B6 mouse strains. E. coli and R. gnavus lysates inducedpredominant effector ex-vivo MLN T cell responses with highest secretion of IL-12p40 byE. coli and predominant secretion of IL-17 by R. gnavus lysates. Conclusions: A humanbacterial consortium, SIHUMI, causes mild to moderate colitis in ex-GF IL-10-/- mice:aggressiveness of colitis depends on the strain of IL-10-/- mice. Concentrations of individualSIHUMI species are determined by host genetic background and are related to colonicinflammation. Human enteric bacterial strains differentially stimulate murine immuneresponses with E. coli and R. gnavus preferentially inducing effector immune responses inIL-10-/- mice. This humanized gnotobiotic model with SIHUMI is a valuable tool for studyingthe inflammatory and protective roles of individual bacterial strains in IBD.

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Acid-Tolerance Genes Gadab in Commensal Escherichia coli Impair LuminalBacterial Growth, Alter Bacteria-Epithelial Cell Interactions and AttenuateExperimental ColitisSandrine Y. Tchaptchet, Ting-Jia Fan, Laura E. Goeser, Alexi A. Schoenborn, Ajay S.Gulati, Ryan B. Sartor, Jonathan J. Hansen

BACKGROUND: Inflammatory bowel diseases (IBDs) are due in part to uncontrolled immuneresponses to commensal bacteria in genetically predisposed individuals. However, the effectsof inflammation on commensal gut microbial function and colitogenic potential are onlybeginning to be elucidated. Colitis in long-term monoassociated Il-10-/- (KO) mice is associ-ated with upregulated stress response genes, including gadA and gadB (gadAB), in luminalEscherichia coli. GadAB protect E. coli from low pH and fermentation acids, factors presentin the intestinal lumen in active IBDs. HYPOTHESIS: Upregulation of gadAB in commensalE. coli during inflammation enhances bacterial survival and virulence. METHODS: Wild-type (WT) and KO germ-free mice were monoassociated with the commensal murine E.coli isolate NC101 or NC101 lacking gadAB genes (NC101 ΔgadAB) for up to 10 wks.Luminal bacterial viability and gene expression were determined using quantitative cultureand real-time PCR, respectively. Inflammation was measured using blinded histologicalscoring and ELISA for pro-inflammatory cytokines secreted by colonic explant cultures. Invitro bacterial killing by the recombinant antimicrobial peptide, cryptdin-4, and invasionand translocation across polarized T84 intestinal epithelial cell monolayers were measuredusing quantitative culture. RESULTS: Expression of gadAB in luminal E. coli increasesproportionately with intestinal inflammation in KO mice. Contrary to our hypothesis, gadABexpression is associated with decreased luminal bacterial densities in vivo (3.8x10^9 vs.7.1x10^9 colony forming units (CFU)/g cecal contents in NC101 vs. NC101ΔgadAB 5 week-monoassociated mice; p,0.01) and increased killing by cryptdin-4 in vitro. Consistent withthe decreased survival of NC101, histological colon inflammation scores (7.1 ± 0.3 vs. 11.4± 0.1, p,0.001) and IL-12/23p40 secretion in colon explant cultures (61.9 vs. 78.5 ng/mgtissue; p,0.05) were reduced in NC101- compared to NC101ΔgadAB-monoassociated KOmice. NC101 invaded (1.1x10^3 vs. 8.0x10^3 CFU/mL, p,0.001) and translocated across(0.05x10^6 vs. 8.80x10^6; p,0.05) polarized T84 cell monolayers less efficiently comparedto NC101ΔgadAB. CONCLUSIONS: Chronic intestinal inflammation is associated withupregulation of an acid-tolerance pathway in commensal luminal E. coli that increasessusceptibility to killing by cryptdin-4, impairs migration across intestinal epithelial cells,and contrary to our original hypothesis, limits the colitogenic potential of E. coli. Elucidatingmechanisms of this host-microbial dialogue will provide insights into the pathogenesis ofIBDs and potentially reveal novel therapeutic targets.

S-66AGA Abstracts

317

Iron Supplemented Diet Protects Against Chronic Immune Mediated Colitis inIL-10 Deficient MiceMelissa Ellermann, Nitsan Maharshak, Raad Gharaibeh, Wei Sha, Ernesto Perez-Chanona,Kristi J. Whitehead, Christian Jobin, Anthony Fodor, Scott E. Plevy, Ryan B. Sartor

Iron deficiency is a common cause of anemia in IBD patients. While most patients receiveoral iron preparations to treat anemia, the effect of iron on enteric inflammation is somewhatcontroversial. In several animal models of IBD, an iron deficient diet is protective againstthe development of ileocolitis possibly through decreased oxidative stress and an alteredenteric microbiota. However, the clinical relevance of these studies is unclear because irondeficient diets are rarely encountered in Western society. Indeed, most human studies havefailed to show an increase in clinical indices or serum CRP in IBD patients treated with oraliron supplementation. We thus aimed to study the effects of clinically relevant doses ofdietary iron on inflammation in the IL-10-/- mouse model of chronic colitis. We hypothesizedthat iron supplementation will increase severity of colitis in a dose dependent mannerthrough its effects on enteric microbial populations. Methods: Two cohorts of IL-10-/- germ-free mice were inoculated with feces from a conventionally housed mouse and placed oniron deficient (supplemented by 5 μg/g IP iron weekly), iron sufficient or iron supplementedisocaloric diets (,10, 35 or 200 ppm iron/kg respectively) for four weeks. In cohort 1 (n=8/group), clinical severity score (0-3) was assessed. In cohort 2 (n=5/group), severity ofcolitis was assessed through clinical score, colon histology score (0-12) and IL-12 p40 levels.Bacterial 16S ribosomal RNA from stool was sequenced using the Illumina platform andbacterial specific qPCR were performed on stool and cecal mucosal samples. Results: Micemaintained on the iron supplemented diet developed significantly less severe colitis comparedwith mice on the iron sufficient diet as assessed through clinical score (0.33 vs 1.15, P ,0.01),histology (3.16 vs 6.28, P,0.05), serum IL-12 p40 levels (2181 vs 5483 pg/ml, P ,0.05)and colonic IL-12 p40 levels (1683 vs 3044 pg/100mg tissue, P=0.07). The iron deficientdiet mediated only mild and non-significant protection against colitis in the two cohorts.While compositional changes of the enteric bacteria did not explain the findings, targetedqPCR studies revealed a decreased total mucosal bacteria load (6.25 fold decrease, P ,0.05)and a higher proportion of mucosa associated Fecalibacterium prausnitzii (2.3 fold increase,P,0.05) in mice maintained on an iron supplemented diet compared to an iron sufficientdiet. Increased iron availability also enhanced in vitro growth of F. prausnitzii. Conclusion:The iron supplemented diet protected against colitis in mice. Protection may be mediatedthrough decreased mucosal bacterial load and increased proportions of F. prausnitzii, aprotective and iron responsive bacterium. These findings may be relevant to human studiesas iron supplementation has been assumed, perhaps mistakenly, to aggravate colitis.

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Central Muscarinic 1 Acetylcholine Receptor Activation Reduces the Severityof Experimental Colitis via the Splenic Nerve and the SpleenHong Ji, Mohammad F. Rabbi, Benoît Labis, Valentin A. Pavlov, Kevin J. Tracey, Jean-EricGhia

Background/Aim: Recent findings indicate that functional connection between the efferentvagus nerve and the splenic nerve importantly mediates the cholinergic anti-inflammatorypathway during endotoxemia. Central pharmacological activation of this pathway by muscar-inic acetylcholine receptor (mAChR) ligands results in lower pro-inflammatory cytokinelevels during lethal endotoxemia. . The aim of this study was to determine whether centralactivation of the cholinergic anti-inflammatory pathway alters disease severity and inflamma-tion in mice with colitis and the mediating role of splenic nerve and the spleen. Methods:C57Bl/6 mice received 5% dextran sulfate sodium (DSS) in drinking water for 5 days.Groups of mice were subjected to sham operation, splenic neurectomy (NRX) or splenectomy(SPX) and i.c.v. cannulation. I.c.v. infusion of the M1 mAChR agonist McN-A-343 or vehiclewere initiated 8 days later and one day before induction of colitis, and disease severity indexwas evaluated daily. Mice were euthanized and inflammation was evaluated clinically andby myeloperoxidase activity (MPO) in colonic tissue. Colonic tissue cytokines levels and C-reactive protein (CRP) serum levels were determined by ELISA. Splenic acetylcholine levelsby ELISA and IL-12p40 production by splenic dendritic cell were also determined. Results:McN-A-343 treatment reduced the onset of clinical disease as assessed by loose stools, weightloss and rectal bleeding. On day 3 after colitis induction, 13% and 26% of McN-A-343-DSS-treated mice had rectal bleeding and weight loss, respectively, compared to 44% and78% of DSS-treated mice (p,0.05). On day 5, macroscopic scores were 53% lower in McN-A-343-DSS group compared to DSS group (p,0.05). MPO and CRP activity decreased from2.6±0.3 to 1.4±0.3 U/mg/tissue and from 35.3±2.9 in DSS group to 25.4±4.3 pg/ml in McN-A-343-DSS group (p,0.05) respectively. Colonic levels of IL-1β and IL-6 were reduced inMcN-A-343-DSS group (p,0.05) and no effect was evident on IL-10 levels. Dendritic cellisolated from McN-A-343-DSS group showed a significant decrease of IL-12p40 release (-44%) as compared to the vehicle-DSS-group. The attenuation of colitis and IL-12p40 byMcN-A-343 treatment was not evident in NRX mice. Splenic level of acetylcholine wassignificantly increased following treatment with McN-A-343 and NRX abolished it. Attenua-tion of colitis by McN-A-343 treatment was not observed in SPX mice. Conclusions: Theseresults indicate that central stimulation of the cholinergic anti-inflammatory pathway by aM1 mAChR agonist controls intestinal inflammation in a murine model of colitis via thesplenic nerve and the spleen. These findings provide a rationale for further studies on therole of the brain to spleen cholinergic circuit in the context of colitis that may lead to noveltherapeutic strategies in IBD.