gene per locus, 24 genes (57%) showed high-level expression (p<0.009, FDR<0.07). Nineof these (CPEB4, CCL2, ICAM3, RTL1, LTB, TNFSF8, CREM, LRRK2 and PTPN2) correspondto those previously reported as associated with CD in the meta-analysis, and 15 werenew additional candidate genes (LHX9, C11ORF10, PPP1R15A, SDF2L1, LIF, APOBEC3C,PHTF1, ARHGAP30, GMPPB, ANXA6, TRIB1, EGR2, CYLD, PTRF and C19ORF23). Of the18 genes (43%) showing lower-level expression (p<0.008, FDR<0.07), two are the same ofthe previously defined significant hits (SULT1A2 and SLC22A5), and 16 are new additionalgenes (PER3, ZBTB7B, PUS10, SLC9A2, TMEM171, CAST, SLC22A23, PLCB3, GPATCH1,CEBPA, SERBP1, AK3, GOT1, TSKU, THRA and PFKL). Conclusions: Sixty-seven deregulatedgenes in 71 loci were identified analyzing the gene expression in the colonic mucosa of CDpatients. Out of the 42 hit genes identified, 31 were new additional candidate deregulatedgenes in inflamed colonic mucosa and were reported as associated with active phase CD.Extensive resequencing, large-scale fine mapping and functional studies of these loci willbe required to elucidate the pathogenic mechanisms of CD.

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Autophagy Induces Immune Tolerance by Regulating Interactions BetweenDendritic Cells-Epithelial Cell in the GutCaterina Strisciuglio, Marjolijn Duijvestein, Anne Christine W. Vos, Auke Verhaar, ErasmoMiele, Riccardo Troncone, Gijs R. van den Brink, Daniel W. Hommes, Manon E.Wildenberg

Introduction Recently, various single nucleotide polymorphisms(SNPs)in the autophagyrelated genes ATG16L1 and IRGM have been associated with the development of Crohn'sdisease. These SNPs lead to decreased autophagic activity, suggesting a regulatory role forthis mechanism in immunity. In the intestine, DC are capable of sampling luminal antigenby protruding dendrites through the epithelial cell layer, while maintaining barrier integrity.It has been shown that the proper sampling of antigen, as well as the interactions betweenDC and the epithelium is crucial for maintenance of intestinal tolerance. We have previouslyshown that autophagy contributes to regulation of cell-cell interactions between dendriticcells (DC) and T cells and therefore, the aim of this study was to elucidate the role ofautophagy in the regulation of cell-cell interactions between DC and intestinal epithelium.Materials and methods An In Vitro model system for luminal sampling by DC was set up,in which an epithelial colon cancer cell line was cultured on one side of a transwell insert,and human dendritic cells (DC) on the other side of the insert, at the basolateral side ofthe epithelium. Modulation of autophagy was achieved using siRNA. In this assay, DCphenotype and luminal antigen sampling were measured by flow cytometry. The capacityof DC to form transepithelial protrusions was determined by confocal microscopy. ResultsIn the gut DC-epithelial interactions are characterized by the presence of tight junctionsand adhesion molecules. Interestingly, the adhesion molecule E-Cadherin partly localizedto autophagosomes and decreased autophagy resulted in increased levels of this protein,suggesting autophagy is involved in proper DC-epithelial interactions. Indeed, reducedautophagy in either DC, epithelial cells or both resulted in the decreased formation oftransepithelial protusions by DC as well as a reduction in antigen sampling. Moreover, whenautophagy was inhibited in either DC or epithelial cells in the co-culture model, DC expressedincreased levels of HLA-DR and costimulatory molecules. Furthermore, decreased levels ofautophagy resulted in an altered cytokine profile of DC and these cells induced significantlymore T-cell proliferation in an allogenic mixed lymphocyte reaction. This data suggest thatdecreased autophagy is also involved in increased maturation and increased immunoreactiv-ity. Conclusions Decreased autophagy is involved in the formation of proper interactionsbetween DC and intestinal epithelium. Autophagy deficiency leads to aberrant interactionsresulting in decreased antigen sampling, increased DCmaturation and a more pro-inflammat-ory type of DC. Therefore, autophagy-related SNPs may contribute to CD pathogenesisthrough dysregulation of the interactions between DC and epithelial cells resulting in a lackof immune tolerance.

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Human IL23R R381Q Genetic Variant Implicated in Crohn's DiseasePathogenesis is Hypomorphic Resulting in Reduced Function of the ReceptorHilary Clark, Svetlana Pidasheva, Sara Trifari, Jason Hackney, Yan Ma, Ashley Smith, SueSohn, Hergen Spits, Randall Little, Timothy Behrens, Lee Honigberg, Nico Ghilardi

The biggest challenge of the genome-wide association studies (GWAS) era is conductingfollow-up studies to identify and functionally analyze causal variants that are associated withcomplex diseases. GWAS in several populations have demonstrated significant associationsof the interleukin 23 receptor (IL23R) gene with Crohn's disease (CD) and psoriasis, sug-gesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiologyof these diseases. One particular variant, R381Q (rs11209026), confers strong protectionagainst development of CD, but the mechanism by which IL23R Q381 confers protectionis unresolved and remains a question of key importance. We investigated the effects of theR381Q IL23R variant in transformed cells and primary T cells from IL23R R381 and IL23RQ381 positive donors. We believe that the functional validation of GWAS findings is acritical step in the translation of genetics into novel therapeutics. Our study is the first toshow the alterations in function of IL23R Q381 allele, further strengthening the implicationfrom GWAS results. These data provide an explanation for the protective role of R381Q inCD and may lead to the development of personalized therapeutics for autoimmune disorderslike CD.

S-273 AGA Abstracts

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Identification of Serum and Tissue Micro-RNA Expression Profiles in DifferentStages of the Inflammatory Bowel DiseaseMarisa Iborra, Francesca Bernuzzi, Belen Beltran, Antonino Spinelli, Pietro Invernizzi,Silvio Danese

Background: Inflammatory Bowel Disease (IBD), Crohn`s disease (CD) and ulcerative colitis(UC), is associated with the differential expression of genes involved in inflammation andimmune functions. MicroRNAs (miRNAs) are a class of small non-coding RNAs involvedin the control of gene expression at post-transcriptional level and are involved in theregulation of biological processes, as well as in the induction of chronic inflammatory diseasesand autoimmune diseases. Recently, the altered expression of miRNA has been associatedwith IBD. Aims: To establish specific miRNAs expression patterns in serum and mucosa ofCD and UC patients at different stages of the disease and to compare with healthy patients.Methods: Samples of serum and biopsies were obtained from 36 IBD patients (9 active CD(aCD), 9 inactive CD (iCD), 9 active UC (aUC) and 9 inactive UC (iUC)), and from 9healthy subjects (H). Blood samples were drawn at the time of the planed endoscopicprocedure. Three pools of 3 serum and biopsies samples were analysed for each group. TheRNA fraction highly enriched for small RNA species was isolated by the mirVanaTMPARISTMkit, according to the manufacturer's protocol. The small RNA fraction was reverse transcribedusing the miRNA Megaplex RT primers and TaqMan® microRNA Reverse Transcriptionkit. The cDNA obtained was amplified using to TaqMan® PreAmp Master Mix and MegaplexPreAmp Primers. Up to 700 miRNAs were evaluated by TaqManR Human miRNA Array.The values were calculated as DCt and U6 was the housekeeper gene. P<0.05 was consideredsignificant. Results: In aCD 21 serum miRNAs were exclusively regulated (H vs aCD). IniCD 85 serum miRNAs were exclusively regulated (H vs iCD). There were 37 serum miRNAscommonly expressed in the 3 groups (aCD-iCD-H). In the mucosa of aCD 8 miRNAs wereexclusively regulated (H vs aCD). In mucosa of iCD 11 miRNAs were exclusively regulated(H vs iCD). There were 6 tissue miRNAs commonly expressed in the 3 groups (aCD-iCD-H). In aUC 19 serum miRNAs were exclusively regulated (H vs aUC). In iUC 91 serummiRNAs were exclusively regulated (H vs iUC). There were 58 serum miRNAs commonlyexpressed in the 3 groups (aUC-iUC-H). In mucosa of aUC 38 miRNAs were exclusivelyregulated (H vs aUC). In mucosa of iUC 11 miRNAs were exclusively regulated (H vs iUC).There were 4 tissue miRNAs commonly expressed in the 3 groups (aUC-iUC-H). Conclusion:Our results suggest the existence of specific miRNA expression patterns associated to IBDand to their different stages. MiRNAs could be useful to distinguish IBD from healthycontrols. The different expression among CD, UC and H support the utility of miRNA aspossible biomarkers. It is necessary that our results are confirmed in larger samples andfurther studies based in the miRNA as therapeutic targets.

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Forward Genetic Analysis of Gut Homeostasis Using a Sensitized Screen inMiceKatharina Brandl, Wataru Tomisato, Bruce Beutler

Background: The epithelium of the gastrointestinal tract is occasionally subject to injury bytoxigenic enteric pathogens, and perhaps on occasion by microscopic particulates capableof wounding cells. However, homeostatic mechanisms normally restore the status quo ante,so that dysfunction is short-lived. These homeostatic mechanisms remain largely obscure.Their failure may lead to a chronic disease state, recognized clinically as inflammatory boweldisease (IBD). Methods: By exposing randomly mutagenized mice to brief treatments withdextran sodium sulfate (DSS) at a concentration that is harmless to wild type animals, wehave sought to identify genes with non-redundant function in homeostasis. A total of 5846mice have been screened by placing them on 1% DSS in drinking water for a period of 7days. During this time period all animals are weighed daily, and extreme outliers are retrievedfor further study. Results and Conclusion: A total of 17 transmissible mutations causinghypersensitivity to DSS have been detected. Of these, 6 have been identified, and fall into5 genes. Failure of epithelial integrity is sensed by Toll-like receptors (TLR) on epithelialcells, which signal to cause expression of epidermal growth factor ligands amphiregulin andepiregulin, promoting epithelial cell growth. Wound closure also requires water transportvia aquaporin 3, and an intact unfolded protein response, which depends on Mbtps1, alsoknown as the site-1 protease. Relatively common mutations affecting the structure of Mucin-2 enhance susceptibility to colitis, evidently by increasing ER stress. In addition, genes withunknown functions have been found to cause increased susceptibility to DSS colitis. Acoherent picture of the essential events required for homeostasis has begun to emerge.

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Variants in the PTPN2 Gene Are Associated With Susceptibility to BothCrohn's Disease and Ulcerative Colitis in the German PopulationJohanna Wagner, Jürgen Glas, Julia Seiderer, Burkhard Göke, Thomas Ochsenkühn, JuliaDiegelmann, Stephan Brand

Introduction: Genome-wide association studies identified PTPN2 (protein tyrosine phosphat-ase, non-receptor type 2) as susceptibility gene for inflammatory bowel diseases (IBD).However, the exact role of PTPN2 in Crohn's disease (CD) and ulcerative colitis (UC) isstill controversial. Given different results of previous replication studies, we investigatedwhether genetic variants in this gene are associated with CD and UC in a large German IBDcohort. Methods: Genomic DNA from 2131 individuals of Caucasian origin including 906patients with CD, 318 patients with UC, and 907 healthy, unrelated controls was analyzedfor two SNPs in the PTPN2 region (rs2542151, rs7234029). From all study participants,blood samples were taken and genomic DNA was isolated from peripheral blood leukocytesusing the DNAbloodmini kit fromQiagen (Hilden, Germany) according to themanufacturer'sguidelines. Genotyping was performed by PCR and melting curve analysis using a pair offluorescence resonance energy transfer (FRET) probes in a LightCycler®480 Instrument(Roche Diagnostics, Mannheim, Germany). Results: Our analysis revealed a significant associ-ation of the PTPN2 SNP rs2542151 with susceptibility to both CD (p=1.95 x 10-5; OR

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