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Colitis-Associated Adipose Tissue Responses to Corticotropin-ReleasingHormone Family of NeuropeptidesChristopher Fink, Iordanis Karagiannidis, Kyriaki Bakirtzi, James M. Bugni, CharalabosPothoulakis

Background and Aims: Numerous studies have indicated that adipose tissue is an activeendocrine organ and several adipose-associated molecules have been implicated as potentialmediators of intestinal inflammation. Results from our group and others indicate that cortico-trophin releasing hormone (CRH or CRF), and its peptide family members urocortins (UCNs)participate in the pathophysiology of intestinal inflammation and inflammatory bowel disease(IBD). Recent evidence also indicates that CRH receptors 1 and 2 are expressed by humanadipocytes. Whether these ligands and receptors are expressed in the mesenteric fat and aremodulated during colitis has never been studied. Here we compared levels of expression ofthe CRH peptide family at in the mesenteric fat depots of mice before and after experimentalTNBS-induced colitis. We also exposed isolated human mesenteric preadipocytes to CRHand measured changes in cytokine production at the mRNA and protein levels. Methods:C57BL6 mice (n=10 per group) were injected intracolonically with either vehicle (40%ethanol, control) or TNBS (5 mg/20 grams) for 48 hr. Mesenteric adipose tissue was harvestedfrom the mice and mRNA was isolated and converted to cDNA for real time RT PCR. Humanmesenteric pre-adipocytes grown in culture were stimulated with 100nM CRH for 8 hours.Supernatants were collected for protein array and ELISA while mRNA was isolated forconversion to cDNA and use in real time RT PCR experiments. Data were analyzed usinga two tailedMannWhitney test. Results: CRHmRNAwas significantly increased inmesentericfat of TNBS-exposed mice compared to controls (by 112.9 fold, p<0.01). UCNs 2 and 3were also significantly increased in these tissues (by 30.7 and 9.2 fold, p< 0.0016 and p<0.0052, respectively). Protein arrays on CRH stimulated human mesenteric preadipocytesshowed increased secretion of TIMP-2 and Rantes (CCL5 gene product) and decreased MIF,MCP-3 and PAI-1 levels. Changes in released MIF were also confirmed by ELISA in themedium of preadipocytes (p< 0.01). Conclusions: Increased expression of CRH and urocort-ins in adipose tissue during colitis together with the ability of CRH to induce changes incytokine release from isolated adipocytes suggest that the CRH family of peptides maymediate inflammatory responses in mesenteric fat and affect the development of intestinalinflammation. These findings may represent a novel signaling pathway in colitis and couldhave important implications for inflammatory bowel disease treatment targets. ResearchSupport: NIH NIDDK T32 DK07180-36 Gastroenterology Training Grant and P01 DK 33506

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Epidermal Growth Factor Receptor Inhibits Colitis-Associated Cancer inInterleukin-10 Knockout MicePhilip E. Dubé, Kay Washington, D Brent Polk

Epidermal growth factor receptor (EGFR) enhances colon epithelial homeostasis and woundrepair and EGFR-directed therapies have been suggested for colitis. However, since EGFRis tumorigenic in the case of sporadic colon cancer, an important question is to what extentEGFR targeting is beneficial in treating colitis vs. harmful in promoting colitis-associatedcarcinoma. Previously, we showed that EGFR inhibition exacerbates spontaneous immune-mediated colitis in interleukin-10 knockout (IL-10-/-) mice. We hypothesized that EGFRinhibition protects IL-10-/- mice from colitis-associated cancer despite the fact that EGFRinhibition induces more severe colitis. METHODS: EGFR was inhibited in IL-10-/- mice bycrossing to mice with a dominant negative EGFR (IL-10-/-EGFRwa5). Controls were EGFR+/

+, IL-10-/- littermates. Mice were sacrificed at 8, 14, 28 or 52 wk (n=4-15). Colons wereexamined for the incidence of macroscopic tumors and sections were scored for tumorprogression from normal to dysplasia to adenoma, including multiplicity and depth ofinvolvement (score 0-8, most severe=8). Colitis was also scored based on hyperplasia,inflammation and injury (score 0-15, most severe=15). Proliferation (Ki67 and BrdU), E-cadherin (epithelial marker), DNA damage (phospho-H2A.X), β catenin and phospho-MAPKlocalization were assessed immunohistochemically. RESULTS: Surprisingly, IL-10-/-EGFRwa5

mice developed colon adenomas by 14 wk of age, with 100% incidence by 1 yr; in contrast,tumors were not observed in IL-10-/- mice prior to 1 yr (see table). Although by 1 yr tumorincidence and the level of invasion were not different between these genotypes, IL-10-/-

EGFRwa5 mice still had a higher tumor burden. Tumorigenesis in each case followed thedevelopment of colitis; however, this occurred much earlier in IL-10-/-EGFRwa5 mice. TheIL-10-/-EGFRwa5 epitheliumprior to tumor formationwas dysplastic and there were numerousproliferating surface epithelial cells and substantial DNA damage, presumably as a result ofinflammatory injury without normal epithelial repair. Of note, many of the surface epithelialcells and dysplastic lesions were positive for nuclear β catenin; however, at 1 yr, the adenomasfrom both genotypes did not stain for nuclear β catenin, but were instead positive forphospho-MAPK, suggesting that activating mutations in the MAPK pathway may be commonduring the progression of these tumors. CONCLUSIONS: EGFR signaling slows colonadenoma initiation and progression in IL-10-/- mice, likely by preventing colitis through themaintenance of epithelial integrity. This work demonstrates an important and paradoxicalrole of EGFR to slow tumorigenesis in chronic immune-mediated colitis vs. its tumorigenicrole in sporadic colon cancer and suggests that efficacious EGFR-directed colitis therapiesmay carry a low risk of colitis-associated cancer.Effect of EGFR inhibition on colon adenoma formation and progression in IL-10-/- mice.

S-27 AGA Abstracts

Data show mean (range); not significant (n.s.), not determined (n.d.).

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Protease Activated Receptor-2 (PAR2) Plays a Role in TH2-Mediated ProtectiveImmunity in Response to Enteric Nematode InfectionJennifer A. Stiltz, Luigi Notari, Rex Sun, Aiping Zhao, Sarah Netzel-Arnett, Toni M.Antalis, Joseph F. Urban, Terez Shea-Donohue

Introduction: Enteric nematode infection upregulates the Th2 cytokines, IL-4 and IL-13,which are critical for the smooth muscle hypercontractility observed during nematodeinfection that facilitates worm expulsion. We showed previously that PAR2 is upregulatedduring N. brasiliensis (Nb) infection by a STAT6-dependent mechanism. In addition, infectionresulted in enhanced smooth muscle response to PAR2 agonists that was dependent onenteric nerves. Aim: To determine the role of PAR2 in the development of host protectiveTh2 immune response and associated changes in intestinal smooth muscle function duringnematode infection. Methods: PAR2-/- or WT mice were inoculated subcutaneously with500 L3 Nb larvae and studied 11 days following infection. Small intestine segments weresuspended in organ baths and responses to nerve stimulation (EFS) were determined. Geneexpression of cytokines was determined using real-time PCR and expressed as fold changefrom respective control. Results: There was a significant upregulation of IL-13 during Nbinfection in WT (1±0.2 vs 52±14 fold) that was attenuated significantly in PAR2-/- mice(1±0.1vs 8.3±1.9 fold; p<0.01). In addition, the infection induced increase in IL-4 expressionin WT (1±0.2 vs 5 ±1 fold) was absent in PAR2-/- mice (1±0.2 vs 1.4±0.5 fold). In WTmice, the Th2 response was linked to a downregulation of inflammatory markers iNOS(1.1±0.2 vs 0.08±0.02 fold) and IL-12p40 (1.0±0.3 vs 0.4±0.2 fold.) while in PAR2-/- mice,expression of these markers was unchanged. In infected WT tissue there was a stereotypicalincrease in smooth muscle responses to EFS (Veh, 4.5±0.8 vs Nb , 10.4±2.0 N/cm2) thatwas abrogated in PAR2-/- infected mice (5.4±1.4 N/cm2). Conclusion: These data show thatPAR2 plays an important role in development of the Th2 protective immune response andinhibition of the Th1 response during nematode infection. In addition, PAR2 is involved inthe neurally-mediated hypercontractility of smoothmuscle in response to infection, consistentwith PAR2's role in the hypersensitivity of sensory afferents described in IBS.

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NOD2 Genotype is Associated With Shifts in Human Ileal Mucosa-AssociatedMicrobial CompositionEllen Li, Christina M. Hamm, Hongyan Chen, Xiao Wu, Tianyi Zhang, Wei Zhu, CharlesE. Robertson, Norman R. Pace, Ryan B. Sartor, Edgar C. Boedeker, Noam Harpaz, JeffreyYuan, Erica Sodergren, George Weinstock, Daniel Frank

Background and Aims: The aim of this project is to examine whether Crohn's disease (CD)risk alleles underlie changes in ileal-associated microbiota prior to the development ofmacroscopic disease. Methods: Sanger sequencing of the bacterial 16S ribosomal RNA(rRNA) gene and pyrosequencing of 16S rRNA gene hypervariable regions (V1-V3 and V3-V5), were conducted on macroscopically disease-unaffected ileal tissues collected from 52ileal CD, 58 colitis and 60 control patients without inflammatory bowel diseases (IBD)that were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R).Phylogenetic classification of the sequences was conducted using the Naïve Bayesian Classifierof the Ribosome Database Project and binned into eight phyla/subphyla categories. Theeffect of disease phenotype and NOD2 genotype were analyzed separately for each of thesequencing platforms using nonparametric MANCOVA for the overall composition andANCOVA for each bacterial category. Data from all three sequencing methods were alsojointly analyzed and compared through non-parametric repeated measures ANOVA. Results/Conclusion: Regardless of sequencing methodology, the MANCOVA results identified bothdisease phenotype and NOD2 genotype as having a significant effect on overall ileal microbialcompoistion. ANCOVA results confirmed that ileal CD phenotype was associated withdecreased relative frequency of the Clostridium group IV clade, with Faecalibacterium spp.representing a major subset. However, NOD2 genotype was associated with increased relativefrequency of this taxon in ileal CD patients. Repeated measures ANOVA confirmed theabove observations using data from all three sequencing methods. These results suggest thatthe effect of NOD2 genotype on ileal microbiota is not mediated by its association with ilealCD phenotype.

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Expression Changes of Xenobiotic Efflux Genes Are Associated With IlealInflammation in the Pelvic Pouch of Individuals With Ulcerative ColitisBoyko Kabakchiev, Andrea D. Tyler, Mark S. Silverberg

Background: As many as 50% of ulcerative colitis (UC) patients who have undergone ilealpouch anal anastomosis (IPAA) develop de-novo inflammation in the ileal pouch within 10years following surgery. With the use of microarray technology, we sought to investigatewhat gene expression changes occur in the pelvic pouch after surgery for UC and how these

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