217 Decreased Development of Experimental Necrotizing Enterocolitis in Apical Sodium-Dependent Bile Acid Transporter Knockout Mice

Total BA Kit. An adjacent section of distal ileum was evaluated for histological damage by a blinded observer using a scale of 0 (normal) - 4 (necrosis). For NEC incidence, animals with an ileal damage score of 2 or greater were considered to have developed NEC. Results: The incidence of NEC was significantly decreased in Asbt KO mice compared to WT (33 vs 75%, respectively). Median ileal damage was also decreased in Asbt KO mice (1.5 vs 2.5, respectively). Luminal total BA levels were significantly increased in Asbt KO mice, however intra-enterocyte levels were significantly decreased compared to WT. Conclusions: The inability to transport BAs across the apical membrane of ileal epithelial cells results in significantly less disease in mice subjected to the NEC protocol. These data show that intraenterocyte rather than luminal BAs contribute to ileal damage and that Asbt expression plays a crucial role in experimental NEC development in both rats and mice. Supported by NIH grant HD 054485. 218 The Regulation of the Intestinal Mucin MUC2 Expression By Short Chain Fatty Acids: Implications for Epithelial Protection Nanda Burger-van Paassen, Audrey Vincent, Patrycja Puiman, Maria van der Sluis, Janneke Bouma, Guenther Boehm, Hans van Goudoever, Isabelle VAN SEUNINGEN, Ingrid B Renes Introduction: Necrotizing enterocolitis (NEC) is a multifactorial gastrointestinal disease that particularly affects premature infants. Although the exact pathophysiology is unknown aberrant bacterial colonization is one of the known risk factors Type of feeding, i.e. human milk feeding versus formula feeding, determines bacterial colonization, and as such concentrations of short chain fatty acids (SCFAs: butyrate, propionate, acetate) that are produced by the intestinal microbiota. We hypothesize that SCFAs affect goblet cell-specific mucin MUC2 expression and, because MUC2 is the major structural component of the intestinal mucus layer, alter epithelial protection Aim: In the present study, we investigated the role of butyrate, as well as acetate and propionate on MUC2 expression, as there are distinct differences between luminal concentrations of these three SCFAs between formula fed infants and human milk fed infants.Methods: Human goblet cell-like LS174T cells were treated with SCFAs, after which MUC2 mRNA levels and stability and MUC2 protein expression were analyzed. SCFA-responsive regions and cis-elements within the MUC2 promoter were identified by transfection and gel shift assays. Effects of butyrate on histone H3/H4 status at the MUC2 promoter were established by chromatin immunoprecipitation. Results: One mM butyrate as well as propionate induced an increase in MUC2 mRNA levels. MUC2 mRNA levels returned to basal levels after incubation with 5-15 mM of butyrate. Interestingly, this decrease was not due to loss of RNA stability. In contrast, at concentrations of 5-15 mM of propionate MUC2 mRNA levels remained increased. Promoter regulation studies revealed an active butyrate-responsive region at -947/-371 within the MUC2 promoter. In this region we identified an active AP1 (c-Fos/c-Jun) cis-element at -818/-808 that mediates butyrate-induced activation of the promoter. Finally, MUC2 regulation by butyrate at 1015 mM was associated with increased acetylation of histone H3 and H4 and methylation of H3 at the MUC2 promoter. Conclusion: 1 mM of butyrate and 1-15 mM of propionate increase MUC2 expression. The effects of butyrate on MUC2 mRNA are mediated via AP1 and acetylation/methylation of histones at the MUC2 promoter. This demonstrates that SCFAs affect MUC2 expression and thereby intestinal protection.

216 MyD88/NF-κB Dependent Campylobacter Jejuni-Induced IL-12p40 Gene Expression Is Negatively Regulated By the AKT/GSK-3β Signaling Pathway in Murine Bone Marrow-Derived Dendritic Cells Xiaolun Sun, Brigitte Allard, Christian Jobin BACKGROUND: The enteric pathogen Campylobacter jejuni (C. jejuni) is one of the most prevalent causes of gastrointestinal illness in the world. Despite of its worldwide deleterious impact, the molecular mechanism responsible for C. jejuni-mediated pathogenesis is limited. AIM: Characterize the function of NF-κB and AKT/GSK-3β signaling in C. jejuni mediated innate host response. METHODS: In Vivo host response to C. jejuni infection (strain 81176) was determined by infecting germ-free IL-10-/-; NF-κB EGFP mice and control wildtype (WT) mice (10^7 CFU/mouse) for 14 days. Colonic EGFP expression was determined by macroimaging and confocal microscopy. Bone marrow-derived dendritic cells (BMDC) were generated from WT, IL-10-/-, or IL-10-/-; MyD88-/- mice and infected with C. jejuni (MOI 50) for various time periods. IL-12p40 and IL-6 mRNA accumulation was determined by real time PCR. Chromatin immunoprecipitation (ChIP) assay was used to measure NFκB and RNA polymerase II (RNApolII) binding to the IL-12p40 gene promoter. Phosphorylation of GSK-3β (S9), AKT (S473 and T308) and IκBα (S32/36) was determined by Western Blot analysis. Pharmacological inhibitors of PI3K (wortmannin and LY294002), AKT (AKTiII), and NF-κB (Bay 11-7082) were used between 1-5 μM to prevent activation of these pathways. RESULTS: EGFP expression was strongly increased in C. jejuni infected IL-10-/-; NF-κB EGFP mice compared to WT mice. EGFP positive cells consisted mainly of lamina propria monuclear immune cells. To study the impact of C. jejuni infection on immune cells, BMDC were generated from IL-10-/-; MyD88-/-, IL-10-/-, and WT mice. C. jejuni infected WT BMDC showed increased NF-κB and AKT/GSK-3β phosphorylation by 2-4h. C. jejuni induced IL-12p40 and IL-6 mRNA accumulation was reduced by 99% in IL10-/-; MyD88-/compared to IL10 -/- BMDC. In addition, C. jejuni induced IL-12p40 mRNA decreased by 80% in Bay 11-7082-treated WT BMDC. As opposed, blocking PI3K/AKT signaling with LY294002 or AKTiII enhanced C. jejuni induced IL-12p40 mRNA by 56 and 85%, respectively. Similarly, blocking PI3K signaling with wortmannin elevated C. jejuni induced IL12p40 mRNA by ten-fold. Interestingly, blocking PI3K/AKT signaling enhanced C. jejuni induced RNApolII binding to the NF-κB binding site of IL-12p40 gene promoter. CONCLUSION: C. jejuni induced gene expression is positively regulated by MyD88/NF-κB signaling in BMDC. However, the host utilizes the PI3K/AKT pathway to dampen C.jejuni-mediated proinflammatory effects. This negative regulation may be important to prevent the deleterious effect of constant innate immune activation on the host.

219 Prohibitin (PHB) Is a Novel Antioxidant That Attenuates Colonic Inflammation in Mice Arianne L. Theiss, Matam Vijay-Kumar, Tracy S. Obertone, Dean Jones, Jason Hansen, Andrew T. Gewirtz, Didier Merlin, Shanthi V. Sitaraman INTRODUCTION: Increased free radicals and/or impaired antioxidant defenses have been shown to play a pathogenetic role in human and animal models of inflammatory bowel disease. Our previous studies showed that prohibitin (PHB) levels are decreased during colitis and that cultured intestinal epithelial cells overexpressing PHB are protected from oxidative stress. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on oxidative stress associated with experimental colitis and the potential mechanism by which PHB functions as an antioxidant using PHB transgenic mice. METHODS: Colitis was induced using two established mouse models (Salmonella typhimurium and dextran sodium sulfate) in PHB transgenic mice and wild-type littermates. 6 week-old PHB transgenic mice and WT littermates were administered 3.0% DSS in their drinking water for 7 days; mice were infected with S. typhimurium or treated with vehicle after streptomycin treatment and killed 48 h after infection. Colitis was determined using established clinical (weight loss, diarrhea and fecal blood) and histological (crypt damage, ulcerations and inflammatory infiltration) scoring, and myleoperoxidase activity. Oxidative stress was determined by measuring glutathione and protein carbonyl levels in the cecum or colon. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of oxidant responses, expression and activation was assessed in colon mucosa and cultured intestinal epithelial cells overexpressing PHB. RESULTS: Cells overexpressing PHB showed sustained Nrf2 nuclear accumulation and DNA binding during oxidant stress. PHB transgenic mice exhibited decreased oxidative stress and colitis and increased Nrf2 mRNA expression, Nrf2 nuclear protein translocation and Nrf2/DNA binding compared to wild-type littermates during colitis. CONCLUSION: These results demonstrate that PHB is a regulator of Nrf2 expression in intestinal epithelial cells during oxidative conditions and prevents inflammation-associated oxidative stress and injury through sustained activation of Nrf2. Our data demonstrate that PHB is a novel antioxidant and suggest that restoration of PHB levels represents a potential therapeutic approach in inflammatory bowel disease.

217 Decreased Development of Experimental Necrotizing Enterocolitis in Apical Sodium-Dependent Bile Acid Transporter Knockout Mice Mitchell A. Thomas, Crystal Johnson, Ludmila Khailova, Kelly M. Arganbright, Bohuslav Dvorak, Melissa Halpern Background: Necrotizing Enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. We have previously shown that ileal bile acid (BA) accumulation contributes to ileal damage in a neonatal rat model of NEC. Ileal luminal BAs are transported across the apical membrane of enterocytes via the apical sodium-dependent bile acid transporter (Asbt). We have also shown that Asbt is significantly elevated in neonatal rats with NEC and intra-enterocyte BAs are also significantly increased. When neonatal rats subjected to the NEC protocol are given an Asbt inhibitor, they develop significantly less disease. These results suggest that intra-enterocyte rather than intra-luminal BAs contribute to ileal damage during development of experimental NEC in neonatal rats. However, the rat Asbt gene is not regulated via a negative feedback mechanism by BAs as is mouse (or human) Asbt. Objective: To determine if mice in which the Asbt gene has been knocked out will develop NEC. Methods: Newborn 129-Slc10a2 -/- (Asbt KO) and wild-type mice (WT) were hand-fed with formula using the Hoshiba Nipple Yajima-Style system (Meiji Dairies Corporation) and exposed to asphyxia and cold stress twice daily to develop NEC. After 3 days, animals were sacrificed and a 0.5 cm section of distal ileum from each animal was flushed with 100 ul sterile, cold PBS to evaluate total luminal BA levels. After flushing with an additional 200 ul PBS, this section of ileum was homogenized in 100 ul fresh PBS to determine intra-enterocyte BA levels. Total BA levels were determined using the Diazyme

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AGA Abstracts

AGA Abstracts

age to determine colitis onset. Mice were sacrificed at 8, 14 or 28wk (n=8-12). Whole colon sections were scored histologically for colitis severity (mucosal hyperplasia, lamina propria acute/chronic inflammation, crypt injury, and crypt inflammation; combined score 0-15, most severe=15). Proliferation (Ki67), and epithelial tight junction integrity (occludin and ZO-1) were assessed immunohistochemically. RESULTS: Colitis onset in IL-10-/-EGFRwa mice was 8wk as noted by endoscopy (mucosal thickening/granularity, loose stool), while disease in IL-10-/- mice developed >20wk. Rectal prolapse was prevalent in IL-10-/-EGFRwa mice (onset 9wk, 50% incidence by 14wk), but was less common in IL-10-/- mice (onset 27wk, 16% incidence by 28wk). IL-10-/-EGFRwa colons were 56±17% and 83±13% heavier (P<0.01) and 14±4% and 13±2% shorter (P<0.05) than IL-10-/- colons at 8 and 14wk, respectively. Colitis scores were greater in IL-10-/-EGFRwa mice at 8wk [median score 9 (range 4-10) vs. 1(0-3), P<0.01] and 14wk [12(9-13) vs. 0(0-13), P<0.001]. By 28wk, differences abated due to disease progression in IL-10-/- mice. Markedly increased epithelial hyperproliferation, dysplasia, and inflammatory cell infiltration were noted in 14wk IL-10-/EGFRwa colons. Unlike IL-10-/- colons, the epithelial proliferative zone in IL-10-/-EGFRwa colons was not restricted to the crypt base, extending to the surface epithelium. The 14wk IL-10-/- epithelium showed membrane occludin/ZO-1 staining; however, the IL-10-/-EGFRwa epithelium displayed diffuse intracellular staining, suggestive of a greater defect in tight junction integrity. Control mice were histologically normal. CONCLUSIONS: Loss of EGFR signaling accelerates colitis progression in IL-10-/- mice, possibly due to a defect in epithelial repair and barrier function with exacerbation of epithelial hyperplasia and dysplasia. This work demonstrates the important role of EGFR signaling to prevent disease progression in chronic colitis and suggests caution for use of EGFR inhibitors in colitis patients or for colitis-associated cancer.