LF82 endocytosis led to decreased number of AIEC LF82 bacteria internalized withinmacrophages, but did not interfere with the secretion of TNF-α. Dose-dependent increasesin the number of intracellular AIEC LF82 bacteria were observed when infected macrophageswere stimulated with exogenous TNF-α. Conversely, neutralization of TNF-α secreted byAIEC infected macrophages using Infliximab induced significant dose-dependent decreasesin the numbers of intramacrophagic bacteria. Conclusion: Replication of AIEC bacteria withinmacrophages induces the release of TNF-α, which in turn increases the intramacrophagicreplication of AIEC. Infliximab is effective in controlling AIEC replication within macro-phages.

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Differences in Gut Microbial Metabolism are Responsible for ReducedHippurate Synthesis in Crohn's DiseaseHorace R. Williams, I Jane Cox, David G. Walker, Jeremy F. Cobbold, Simon D. Taylor-Robinson, Sara E. Marshall, Tim Orchard

INTRODUCTION: Certain urinary metabolites are the product of gut microbial or mamma-lian metabolism; others are mammalian-microbial ‘co-metabolites'. We have recently shownsignificantly lower levels (p<0.0001) of the urinary mammalian-microbial co-metabolite,hippurate, in Crohn's disease (CD) patients compared to healthy controls [1]. There aretwo stages in the biosynthesis of this metabolite: 1) the gut microbial metabolism of dietaryaromatic compounds to benzoate and 2) subsequent hepatorenal conjugation of benzoatewith glycine to form hippurate, which is excreted in the urine. AIMS & METHODS: It washypothesised that the reduced hippurate excretion in CD patients was due to alterations inthe gut microbiota, and not differences in dietary benzoate, nor defective enzymatic conjuga-tion of benzoate. 5 mg/kg sodium benzoate were administered to 16 CD patients and 16healthy control individuals who had taken a specifically-designed low benzoate diet. Baselineand peak urinary hippurate excretion (one hour post-ingestion) were measured using nuclearmagnetic resonance spectroscopy. RESULTS: Baseline hippurate levels were significantlylower in the CD patients (p=0.0009). Peak hippurate excretion after benzoate ingestionincreased more dramatically for the CD cohort. Indeed, the peak excretion values did notdiffer significantly between the CD patients and healthy control subjects; the relative increasein hippurate excretion was consequently significantly greater in the CD cohort (p=0.0007).CONCLUSION: Lower urinary hippurate in CD is not due to differences in dietary benzoate.After benzoate ingestion, peak levels of urinary hippurate were similar in the two cohorts;the increase was relatively greater in the CD population, whose baseline levels were lower.This excludes a defect in enzymatic conjugation in the CD cohort, strongly implicating analtered gut microbiota as the cause of decreased hippurate levels in CD. REFERENCE(S):1. Williams HRT et al. Characterization of inflammatory bowel disease with urinary metabolicprofiling. Am J Gastroenterol 2009; 104: 1435-1444.

T1789

Intestinal Epithelia Activate Anti-Viral Signaling by Intracellular Sensing ofRotavirus Structural ComponentsAmena H. Frias, Matam Vijay-Kumar, Rheinallt Jones, Jon R. Gentsch, Mary K. Estes,Andrew T. Gewirtz

Background: Rotaviruses (RV) are the leading cause of diarrhea in children. RV primarilyinfects intestinal epithelial cells (IEC) and causes self-limiting illness in which the virus iscleared via a mechanism that does not require adaptive immunity. We hypothesize suchinnate immune clearance of RV is mediated by epithelial antiviral gene expression involvingtype 1 interferon (IFN), and that these antiviral responses are induced upon pattern-recogni-tion receptor (PRR) detection of RV components. Thus, we sought to define anti-viralsignaling induced in epithelia by RV infection and determine the extent to which thisresponse was recapitulated by non-infectious RV components. Methods: Model humanintestinal epithelia (HT29 cells grown on permeable supports) were treated with RV (MOI0.5-10), RV in the presence of neutralizing antibodies to IFN-α/β, UV-inactivated RV (UV-RV) which was non-infectious but structurally intact, and RV and UV-RV that was eithertrypsinized or non-trypsinized prior to infection. In addition, epithelia were stimulated withrotaviral components including RV RNA (0.1-5 ug) and RV virus-like particles (VLPs) (0.5-5 ug). Viral protein expression and activation of antiviral markers (IRF 3/7, STAT1, PKR)was detected via immunostaining and/ or western blotting. Secretion of IFN-β and IL-8(pro-inflammatory marker) was measured via ELISA. Gene transcription was assessed usingmicroarray and qRT-PCR analysis. Results: RV infection robustly induced protein expressionof antiviral markers in a trypsin-dependent manner and via an apical pathway of infection,and this pattern was largely mimicked by UV-RV. Like RV, UV-RV significantly inducedtranscription of 1190 genes (> 1.3 fold change induction) at 24h poi, including antiviralmarkers associated with PRR-mediated and Type 1 IFN signaling. UV-RV also internalizedsimilarly to RV, efficiently entering cells as early as 1h poi. In contrast to UV-RV, however,potent induction of antiviral gene expression was not observed in epithelia treated with RVRNA or VLPs. Interestingly, while live viral replication was not a requirement for activatingtype 1 IFN-mediated responses, it was important for optimal production of proinflammatorycytokine IL-8. Conclusions: Overall, our results are in accord with a model whereby RVproteins mediate viral entry leading to intracellular detection of RV RNA that generates anantiviral response.

T1790

Adherent and Invasive E. coli (AIEC) From IBD Patients Regulates BiofilmFormation Through the AI-3/Epinephrine/Norepinephrine Quorum SensingSystemDenis O. Krause, Charles N. Bernstein, Juan D. Hernandez

Escherichia coli UM146 is an adherent and invasive E. coli (AIEC) strain linked with theonset of inflammatory bowel disease (IBD). This bacterium colonizes enterocytes in IBDpatients by means of type 1pili. E. coli uses different quorum sensing (QS) systems to regulate

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motility and flagella genes essential for biofilm formation (BF). In K-12 BF is controlled bythe autoinducer 2 (AI-2) QS mediated regulator (MqsR), but in O157:H7 the autoinducer3 (AI-3) QS system regulates motility and flagellar activities. The AI-3 system regulatesmotility and flagella genes via the qseBC operon in conjunction with the gut hormonesepinephrine (Epi) and norepinephrine (Ne). There are no reports that demonstrate the roleof AI-3 or AI-2 in AIEC. We hypothesized that AIEC control biofilm formation via AI-3/Epi/Ne and not the AI-2 QS system. Methodology: AIEC 146 was isolated from an IBDpatient in a previous study in our laboratory. In-silico analysis of 10 E. coli publicallyavailable genomes was conducted with reference to the AI-2 receptor genes (lsrA, lsrK andmqsR) using BLASTp. Data was confirmed in AIEC 146 with PCR. Mutants for luxS andfimH were constructed in AIEC 146 using the lambda (λ) red procedure. Mutants wereconfirmed by PCR and sequencing. Motility assays were conducted using soft agar plates.Biofilm formation assays were performed in 96 well PVC plates using two different media:M9 medium supplemented with 0.4%G plus 0.4% CAA, and M9 plus 50μM and 100 μMEpi. Bacteria were grown at 12, 24 and 48 hours and the adherent cells were quantified bycrystal violet staining then measuring absorbance at 595nm. Strains were assayed 6 timesin three different plates giving a total of 18 individual tests. Student t-test was performedwherever needed considering a P value of <0.05 as significant. Results: We demonstratedthat deletion mutants of luxS and fimH do not affect BF, but BF is stimulated by Epiindependent of the luxS and fimH mutations. Conclusion: this is the first demonstration ofthe presence of AI-3/Epi/Ne system in AIEC; and the AI-2 QS system appears not to beoperative in this bacterium.

T1791

Selective Colonization With Helicobacter bilis or Escherichia coli InducesDifferential Host Immune Responses Following a Colitic InsultAmanda E. Ramer-Tait, Abigail Henderson, Jesse Hostetter, Albert Jergens, Michael J.Wannemuehler

Aberrant mucosal immune responses to the resident microbiota are a significant cofactor inthe pathogenesis of inflammatory bowel diseases (IBD). Bacterial provocateurs have beenlinked to increased host susceptibility and may play a role in the multiple hit model of IBD.Immunocompetent, defined microbiota (DM) mice colonized with Helicobacter bilis showincreased host immune responses to the resident microbiota and disease susceptibility totyphlocolitis following an inflammatory insult in the form of low-dose (1.5%) dextransulfate sodium (DSS). Moreover, bacterial colonization alone was not sufficient to increasesusceptibility to a mild colitic insult, as colonization with Escherichia coli failed to affectdisease severity. Based on these observations, we examined host immune responses to thecommensal microbiota following selective colonization with either H. bilis or E. coli. DMmice harbor the altered Schaedler flora (ASF), a functional microbial community consistingof eight bacterial species that can all be independently cultured. DM mice colonized witheither H. bilis or E. coli developed ASF-antigen-responsive CD4+ T cells. DM mice colonizedwith H. bilis also developed a predominant IgG2a serum antibody response against all eightmembers of the ASF. Conversely, E. coli-colonized mice failed to develop ASF-specificantibody responses despite demonstrating serum antibody against E. coli-derived antigens.Following DSS treatment, increased IL-17A mRNA expression was observed in the proximalcolon of E. coli-colonized as compared to H. bilis-colonized mice at 3 weeks post-infection(wpi). This trend was reversed at 12 wpi. Similarly, CD4+ T cells derived from the mesentericlymph nodes of E. coli-colonized mice produced more IL-17A following ASF antigen restimu-lation than T cells isolated from H. bilis-colonized mice at 3 wpi. By 12 wpi, lamina proprialcells from DSS treated, H. bilis-colonized DF mice demonstrated enhanced secretion of IL-17A in response to ASF-antigen stimulation. Colonization with H. bilis alone failed to inducean IL-17A response but instead primed infected mice to secrete IL-17A following a DSSinsult. Acute induction of IL-17A in E. coli-colonized mice appears protective to the hostwhile the immune profile associated with long-term H. bilis-colonization, including elevatedIL-17A, increases the risk for development of colitis following an inflammatory insult.Collectively, these data indicate that bacterial provocateurs differentially influence the profileof host responses against the commensal microbiota andmodulate the impact of an inflammat-ory insult and subsequent disease susceptibility.

T1792

Helicobacter is Required for Colitis in WASP-Deficient Mice and InducesColon CancerDeanna D. Nguyen, Michelle A. Eston, Sureshkumar Muthupalani, Melissa W. Mobley,Amanda F. Potter, Nancy S. Taylor, Scott B. Snapper, James G. Fox

Background & Aims: Wiskott-Aldrich Syndrome protein (WASP) is an intracellular signalingmolecule involved in actin polymerization; its absence leads to the development of colitisin WASP KO (WKO) mice associated with elevated levels of IL-4 and IFN-γ by 4 monthsof age when Helicobacter-infected mice are housed in SPF animal facilities. Onset of diseaseis accelerated by sublethal radiation. We determined whether Helicobacter is required forspontaneous as well as radiation-induced disease in WKO mice. Methods: Helicobacter-free WKO mice were re-derived by embryo transfer at two facilities (MGH, MIT). Mice weremonitored for clinical signs, and colons were harvested at 5, 6-12, and >12 months forhistological analyses. Four to 6-month-old sublethally irradiated (700rad) re-derived WKOmice were also analyzed for colitis. Re-derived weaning WKO mice were orally dosed with0.2ml of 108 CFU H. bilis (n=33) or sham (n=20) over 5 consecutive days. Animals 7-9months post-infection (mpi) were analyzed for Treg proportions, lower bowel histology,and cytokine analysis of isolated colonic lamina propria cells. Results: At MGH, no Helicob-acter-free WKO mice (n=23) aged four to 15 months demonstrated clinical signs of diseaseor gross colonic thickening. No mice (n=5) at 7 months and 2 out of 5 mice at 10-15months (average score: 0.4 out of 8) had minimal evidence of histological inflammation.Four to six-month-old Helicobacter-free irradiated mice (n=12) developed mild diseasecompared to non-irradiated counterparts (n=10) (average scores: 1.4 vs 0.17, p = 0.001).H. bilis-infected mice had significant typhlitis index scores compared to uninfected controls(Hb vs Control: at 7mpi = 8.5 vs 2.7, p= 0.018; at 8-9mpi = 7.7 vs 2.33, p= 0.0008). Therealso was a trend towards more severe colitis index scores (Hb vs Control: at 7mpi = 6.0 vs

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