The intestinal epithelium represents a primary physical barrier, and is the first line of defense against ingested toxins and bacterial products. In addition, a growing body of evidence suggests that intestinal epithelial cells (IEC) actively participate in gut inflammation. Dysregulation of epithelial function can disrupt the homeostasis of normal mucosal immunity and drive adaptive immune responses to a pathogenic disease state. In the last funding period, we demonstrated that epithelial phenotypic alterations and increased small intestinal permeability occur early, before the onset of inflammation in SAMP1/YitFc (SAMP) mice. In addition, bone marrow chimeras (BMCs) of irradiated SAMP mice reconstituted with control AKR bone marrow exhibit significant ileitis, suggesting that an innate defect in epithelial function may represent the primary source of disease susceptibility in SAMP mice. The central hypothesis of Project 5 is that dysregulation of intestinal epithelial barrier function leads to aberrant innate immune responses that drive the activation of pathogenic effector cells, resulting in chronic intestinal inflammation similar to that observed in Crohn's disease (CD). To test this hypothesis, we will perform three specific aims: 1) Determine if the increase in small intestinal permeability of SAMP mice is a consequence of environmental factors or genetic predisposition. In vivo and in vitro analyses of epithelial permeability will compare SAMP mice raised under germ-free conditions to SPF-raised mice. We will also determine whether a genetic component contributes to the susceptibility of increased small intestinal permeability, utilizing consomic mice generated by Project 2 that carry BL76 chromosomal intervals associated with disease severity on the SAMP background. Finally, to facilitate fine gene mapping of disease susceptibility genes, Affymetrix chip technology will be used to compare the relative expression of epithelial-derived genes from isolated IEC of consomic and native SAMP mice. 2) Characterize the relative contribution of epithelial tight junction (TJ) and adherens junction (AJ) proteins comprising the apical junctional complex (AJC) in SAMP mice, and if alteration in their expression affects ileitis. Based on our preliminary findings, we will characterize the TJ proteins ZO-1, claudins 1-4 and occludin, and the AJ proteins E-cadherin and beta-catenin, using real-time RT-PCR, Western blotting and immunohistochemical techniques. In addition, consomic mice that possess BL/6 chromosomal intervals containing genes of the AJC will be evaluated for expression of TJ and AJ proteins, as well as the state of intestinal inflammation. 3) Evaluate the specific role of hematopoietic cells in the development of ileitis. in the presence of epithelial barrier dysfunction. Phenotypic and functional characterization will be performed on immune cells from BMCs, and the ability of specific T/B cell populations to adoptively transfer disease will be determined. Finally, we will investigate whether ileitis and activation of pathogenic adaptive immune responses are a consequence of uncontrolled translocation of microbial products across a leaky epithelial barrier. The overall goal of Project 5 is to elucidate the precise role of the epithelium and barrier dysfunction in the pathogenesis of chronic ileitis, and to facilitate the design of future therapeutic strategies based on manipulation of the intestinal epithelial barrier.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Program Projects (P01)
Project #
5P01DK057880-10
Application #
7932778
Study Section
Special Emphasis Panel (ZDK1)
Project Start
Project End
Budget Start
2009-09-01
Budget End
2010-08-31
Support Year
10
Fiscal Year
2009
Total Cost
$214,115
Indirect Cost
Name
University of Virginia
Department
Type
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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Lou, Yuefen; Lu, Xiaojiong; Dang, Xitong (2012) FOXO1 Up-Regulates Human L-selectin Expression Through Binding to a Consensus FOXO1 Motif. Gene Regul Syst Bio 6:139-49
Reuter, Brian K; Pastorelli, Luca; Brogi, Marco et al. (2011) Spontaneous, immune-mediated gastric inflammation in SAMP1/YitFc mice, a model of Crohn's-like gastritis. Gastroenterology 141:1709-19
Pizarro, Theresa T; Pastorelli, Luca; Bamias, Giorgos et al. (2011) SAMP1/YitFc mouse strain: a spontaneous model of Crohn's disease-like ileitis. Inflamm Bowel Dis 17:2566-84
Gorfu, Gezahegn; Rivera-Nieves, Jesus; Hoang, Sharon et al. (2010) Beta7 integrin deficiency suppresses B cell homing and attenuates chronic ileitis in SAMP1/YitFc mice. J Immunol 185:5561-8
Shanahan, Michael T; Vidrich, Alda; Shirafuji, Yoshinori et al. (2010) Elevated expression of Paneth cell CRS4C in ileitis-prone SAMP1/YitFc mice: regional distribution, subcellular localization, and mechanism of action. J Biol Chem 285:7493-504
Pastorelli, Luca; Garg, Rekha R; Hoang, Sharon B et al. (2010) Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis. Proc Natl Acad Sci U S A 107:8017-22
Gorfu, G; Rivera-Nieves, J; Ley, K (2009) Role of beta7 integrins in intestinal lymphocyte homing and retention. Curr Mol Med 9:836-50
Vidrich, Alda; Buzan, Jenny M; Brodrick, Brooks et al. (2009) Fibroblast growth factor receptor-3 regulates Paneth cell lineage allocation and accrual of epithelial stem cells during murine intestinal development. Am J Physiol Gastrointest Liver Physiol 297:G168-78
Reuter, Brian K; Pizarro, Theresa T (2009) Mechanisms of tight junction dysregulation in the SAMP1/YitFc model of Crohn's disease-like ileitis. Ann N Y Acad Sci 1165:301-7

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