Host Responses to Intestinal Microbial Antigens in Gluten-Sensitive Mice Jane M. Natividad 1 , Xianxi Huang1, Emma Slack1, Jennifer Jury1, Yolanda Sanz2, Chella David3, Emmanuel Denou 1 , Pinchang Yang4, Joseph Murray5, Kathy D. McCoy1., Elena F. Verdu´ 1.* 1 Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Canada, 2 Institute of Agrochemistry and Food Technology (IATA), Spanish National Research Council (CSIC), Valencia, Spain, 3 Department of Immunology, Mayo Clinic, Rochester, Minnesota, United States of America, 4 Department of Pathology, McMaster University, Hamilton, Canada, 5 Division of Gastroenterology, Mayo Clinic, Rochester, Minnesota, United States of America Abstract Background and Aims: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. Methodology/Principal Findings: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T- cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-c in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. Conclusion: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-c production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota. Citation: Natividad JM, Huang X, Slack E, Jury J, Sanz Y, et al. (2009) Host Responses to Intestinal Microbial Antigens in Gluten-Sensitive Mice. PLoS ONE 4(7): e6472. doi:10.1371/journal.pone.0006472 Editor: Wen-Liang Zhou, Sun Yat-Sen University, China Received May 6, 2009; Accepted June 30, 2009; Published July 31, 2009 Copyright: 2009 Natividad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant by the Canadian Association of Gastroenterology (CAG)/Canadian Institute of Health Research (CIHR) and by the Canadian Celiac Association New Investigator Award (E. Verdu). E. Verdu holds a McMaster University Dep. of Medicine Internal Career Research Award. Dr K. McCoy holds a Canada Research Chair in Gastrointestinal Immunology; Drs. Murray and David were supported in part by R01 DK71003. Competing Interests: The authors have declared that no competing interests exist. * E-mail: verdue@mcmaster.ca . These authors contributed equally to this work. Introduction Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of gluten containing cereals, and in particular gliadin, the storage protein in wheat. It has recently been recognized that both the pathology and the clinical spectrum of CD varies considerably from severe to subtle, and that the clinical expression is not restricted to the presence of mucosal atrophy [1,2]. The concept of gluten sensitivity (GS) incorporates a variety of pathologic, immunological, and clinical scenarios that may, or may not, form part of the ‘‘celiac’’ spectrum such as gluten-sensitive diarrhea, immunological mucosal response to gluten in family members of celiac disease, persistent positive specific serology for celiac disease in the absence of defined enteropathy, and subtle immunopathological changes in the intestine exposed to gluten. Typically, these disorders occur in individuals who carry the same HLA genotypes associated with celiac disease-DQ2 and DQ8 [3–7]. This has led to the development of animal models of gluten-sensitivity that mimic certain aspects of gluten-induced pathogenesis [8]. HLA-DQ8/ HCD4 or single HLA-DQ8 transgenic mice that are sensitized with gluten develop an immune response to gliadin that involves both the adaptive and innate immune system [8–11]. Although these gluten-sensitive mice do not spontaneously develop intestinal atrophy, they exhibit gluten-dependent changes in gut neuromus- cular and epithelial secretory function [11]. This model has proven useful for the preclinical testing of novel experimental therapies designed to block gluten-induced mucosal pathology [12]. The presence of HLA-DQ2/DQ8 genes are necessary but not sufficient for the development of CD [13], as up to 25–40% of general populations in United States carry these genes and eat gluten, but do not develop a celiac lesion [2,13], thus raising the possibility of contributing environmental and genetic risk factors yet to be identified [14]. The net availability of gliadin to the lamina propria seems to be an important factor in the inflammatory response of celiac patients. The immobilization and haptenation of PLoS ONE | www.plosone.org 1 July 2009 | Volume 4 | Issue 7 | e6472