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smice infected with H.pylori for 12 months revealed that there was significantly greaternumber of atypical glands in NOD1-deficient mice compared to NOD1-intact mice, althoughthere was no significant difference in inflammation. In order to investigate the mechanismsinvolved in the formation of this dysplasia, we established two murine gastric epithelial celllines that express excess and reduced amount of NOD1. The microarray analysis suggestedthat PI3K-Akt-GSK3 pathway is activated when NOD1 is suppressed. Indeed H. pylori-induced phosphorylation of PI3K, Akt and GSK3β was increased in NOD1 knocked-downcells, and these cells exhibited increased nuclear and intracellular accumulation of β-catenin,as well as expression of its target genes c-Myc and cyclinD1. Moreover, TCF reporter assayrevealed the enhanced β-catenin signaling in H. pylori infected NOD1 knocked-down cells.These results suggested that the absence of NOD1 enhances β-catenin signaling. In addition,studies using Akt inhibitor LY294002 and TRAF3 siRNA suggested that this enhanced β-catenin signaling in NOD1 knocked-down cells were due to the H. pylori induced phosphory-lation of Akt and GSK3β, and that TRAF3, the down-stream molecule of NOD1, is affectingthe phosphorylation of Akt. Furthermore, intracellular accumulation of β-catenin and nuclearexpression of c-Myc was observed in the cells that constitute atypical glands in H. pyloriinfected NOD1-deficient mice. Conclusion Our study reveled that innate immunity relatedmolecule NOD1 in the gastric epithelial cells is negatively regulating β-catenin signalingthrough PI3K-Akt-GSK3 pathway. In the situation where the expression of NOD1 is insuffi-cient, the resulting enhancement of β-catenin signaling may lead to gastric carcinogenesisthrough the increased expression of oncogenes such as c-Myc and cyclinD1.

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NOD2 Plays a Pro-Inflammatory Role in the Chronic Phase of Crohn's-LikeIleitis by a Mechanism Involving Th2 CytokinesDaniele Corridoni, Davide Pietropaoli, Gabriella Di Stefano, Alexander Rodriguez-Palacios, Mitchell Guanzon, Paola Menghini, Wei Xin, Theresa T. Pizarro, Fabio Cominelli

Background & Aims: Nucleotide-binding oligomerization domain-containing 2 (NOD2) isan intracellular receptor that has been implicated in the pathogenesis of different auto-inflammatory diseases including IBD. In fact, loss of function NOD2 mutations have beengenetically-associated with Crohn's disease (CD). However, the role of wild type (WT) NOD2during chronic intestinal inflammation has not been fully elucidated. We evaluated theeffects of NOD2 genetic deletion in SAMP1Yit/Fc (SAMP) mice, a murine model of CD-likeileitis. Methods: SAMPxNOD2-/- mice were generated through speed congenic methodolo-gies. Ilea were directly examined using a stereomicroscopic technique to calculate the percent-ages of abnormal mucosa. Ileal total inflammatory scores (based on histologic indices ofchronic, active, and villous distortion) were determined at 4, 10, 20 and 30 wks of age.Mesenteric lymph node (MLN) cells were cultured in the presence of anti-CD3/CD28 andsupernatants analyzed for cytokine secretion. Using stereomicroscopy, abnormal mucosa(involved areas) were isolated and compared to areas exhibiting normal morphology (unin-volved areas). For each section, severity of inflammation was assessed using MPO activityand gene expression of pro-inflammatory cytokines by qPCR. Results: Evaluation of ilea bystereomicroscopy demonstrated that SAMPxNOD2-/- mice had lower percentages of abnor-mal gut mucosa (21.2±3.0 vs 32.5±4.2, n=18; p<0.04), and histologically exhibited lowertotal inflammatory scores (6.4±0.6 vs 9.1±0.9, n=15; p<0.03) compared to WT SAMPlittermates during the chronic phase of ileitis (20-30 wks). No differences were detected inthe early phase of inflammation (4-10 wks). Production of IL-4, IL-5, and IL-13 fromcultured MLN cells was reduced by 3.8, 2.3, and 2.0-fold in SAMPxNOD2-/- compared toWT SAMP mice at 30 wks of age. Involved areas of mucosa showed lower MPO activity inSAMPxNOD2-/- compared to SAMP WT mice (606.6±50.3 vs 822.3±36.3 U/g, n=7; p<0.05);consistently, MPO activity in total ileal mucosa from SAMPxNOD2-/- was significantly lowercompared to WT SAMP littermates (221.8±68.9 vs 503.4±103.2 U/g, n=6; p<0.05). Involvedareas from SAMP WT mice exhibited elevated mRNA levels of pro-inflammatory cytokinescompared to uninvolved areas. In contrast, cytokine expression in SAMPxNOD2-/- micewas significantly decreased and comparable to mRNA levels in uninvolved areas, with specificattenuation of Th2 cytokines, including IL-4, IL-5, IL-13 and IL-33 by 2.9, 2.8, 4.0 and2.0-fold, respectively. Conclusions: Our data demonstrate that genetic deletion of NOD2 inSAMP mice is associated with decreased severity of chronic ileitis. The pro-inflammatoryrole of NOD2 in exacerbating disease may be mediated by Th2 cytokine production, whichis characteristic of the chronic phase of SAMP ileitis.

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High Dose Vitamin D Supplementation Stimulates Innate Cytokine Responsesin Patients With Inflammatory Bowel Disease on InfliximabKrista Reich, Karen Madsen, Rae R. Foshaug, Richard N. Fedorak, Karen I. Kroeker

Background: Vitamin D deficiency is highly prevalent among patients with inflammatorybowel disease (IBD). In that vitamin D is an important immunomodulator of both the innateand adaptive immune system, it has been suggested that vitamin D deficiency may have a rolein the development of IBD as well as influencing disease severity. Therefore, understanding theimpact of vitamin D supplementation on the inflammatory cytokine cascade in IBD patientswill be critical for improving clinical care of these patients. Aim: The primary objective ofthis study was to characterize the serum cytokine profile in vitamin D sufficient and deficientIBD patients on maintenance infliximab therapy and to examine changes in these profilesin vitamin D deficient IBD patients following supplementation with vitamin D. Methods:Adult Crohn's disease and ulcerative colitis patients on maintenance (q8w) infliximab therapywere invited to participate. Serum vitamin D and cytokine (IL-1β, IL-2, IL-8, IL-12, GM-CSF, IFN-γ, IL-6, IL-10, and TNF-α) levels were measured immediately before their inflixi-mab infusion. Vitamin D deficient patients (serum 25(OH)D levels <75 nmol/L) were thenadministered a high dose (250,000-500,000 IU) of vitamin D intramuscularly within 2weeks following their infusion. Vitamin D sufficient patients were not supplemented. Mea-surements of serum vitamin D and cytokine levels were repeated 8 weeks later just priorto their next infusion. Serum cytokines were measured using the Meso Scale Discoveryplatform. Mann-Whitney U Tests and Wilcoxon Signed-Rank Tests were performed andresults are reported as the median and interquartile range (IQR) of the levels. Results:Twenty patients in stable clinical remission were recruited. At baseline, 8/20 (40%) patients

S-48AGA Abstracts

were vitamin D deficient. There were no significant differences in baseline cytokine levelsbetween the groups (data not shown). However, after vitamin D supplementation, serumlevels of IL-1β, IL-8, GM-CSF, and IL-6 significantly increased in the supplemented vitaminD deficient group compared to the non-supplemented group (Table 1). Comparison of thecytokine levels in the supplemented vitamin D deficient group showed a significant increasein IL-1β, IL-8, GM-CSF, and IL-6, with no difference in IL-2, IL-12, IFN-γ, IL-10, or TNF-α levels post supplementation (data not shown). Conclusion: The increase in IL-1β, IL-8,GM-CSF, and IL-6 following vitamin D supplementation in vitamin D deficient IBD patientssupports a role for vitamin D as a stimulator of the innate immune response. Ongoingstudies will examine the role of vitamin D supplementation as an adjuvant treatment duringimmunosuppressive and biologic therapy.Table 1. Cytokine levels at follow up, stratified by vitamin D group

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The Epithelial IL-23R Mediates Intestinal Immune Defense and EpithelialRegenerationKonrad Aden, Ateequr Rehman, Robert Haesler, Florian Tran, Steffen Pfeuffer, SusanneBillmann, Maren Paulsen, Simone Lipinski, Arthur Kaser, Stefan Schreiber, Philip C.Rosenstiel

BACKGROUND & AIMS: The identification of the IL23R as a genetic risc factor in inflamma-tory bowel disease (IBD) has highlighted the role of IL-23 signaling in the intestinal immuneresponse. However, the impact of IL-23 on the immune regulation is ambigious. WhereasIL-23 induced Th-17 polarization contributes to pathogenesis of IBD5, IL-23 induced IL-22 in Thy-1+ innate lymphoid cells is indispensable in the innate immune response tobacterial pathogens and experimental colitis. This study aimed to describe the role of theIl23R in the intestinal epithelium. METHODS: Conditional knockout of the Il23R in theintestinal epithelium was established by crossing VillinCre mice with Il23Rfl/fl mice, resultingin IL23RIEC-KO or IL23Rfl. For chronic colitis induction, mice were supplied with 2% ofDSS dissolved in drinking water for 5 days followed by 5 days of regular drinking waterwith total 3 cycle repetitions. Diseased intestines were subjected to gene expression analysiswas performed using custom made TaqMan probes and post-mortem histopathologicalanalysis. Lumina faeces were subjected to pyrosequencing of bacterial DNA and sequenceswith at least 97% similarity were clustered in to species level operational taxonomical units(OTUs). RESULTS: Here we show that IL23R is expressed in intestinal epithelial cellsand profoundly affects the intestinal immune defense. IL23RIEC-KO mice produce lessantimicrobial peptides, have a disturbed colonic microflora and succumb to experimentalcolitis. IL23RIEC-KO intestinal lamina propria cells contain less immune cells and produceless IL-22 in response to IL-23 or Flagellin stimulation. Lastly, we could show, that IL-22therapy fully restores epithelial immune defense in IL23RIEC-KO. CONCLUSIONS: Thesedata contribute to the understanding of the IL-23 axis in primary immune response anddescribes a so far unknown role of IL23R signaling in the intestinal epithelium.