gluten components to the extracellular matrix proteins by tissue transglutaminase aids and allows reservoirs of antigenically potentiated gluten components to reach increased concentrations in vivo, and may even induce a widespread mucosal response against auto-antigens [15]. Indeed, celiac patients have been shown to increase systemic titres of IgA antibodies against collagen [15]. Under normal conditions, the intestinal epithelium acts as a protective barrier restricting transport of luminal antigens, and only allows small and selective quantities to permeate the mucosa [16–18]. In contrast, increased intestinal permeability has been demonstrated in patients with active CD [19,20] and their healthy relatives, suggesting that in a proportion of cases, intestinal barrier abnormalities may predate overt inflammation [21]. Altered barrier function could be a critical step in facilitating the host responses that contribute to the clinical expression of gluten sensitivity. Thus, the present study was designed to investigate whether alteration of intestinal barrier function using the non- steroidal anti-inflammatory drug (NSAID), indomethacin, enhanc- es gluten-induced epithelial injury and influences subsequent host responses to gut luminal antigens. Our results show that indomethacin enhances gluten-induced changes in the mucosa leading to increased IFN-c release by gliadin-stimulated spleno- cytes and to systemic priming against intestinal microbiota antigens. In genetically predisposed hosts with long standing barrier abnormalities, this mechanism may lower the threshold of inflammatory responses to specific antigens. Results Gluten sensitization and indomethacin treatment led to retardation of weight gain Gluten sensitized mice and non-sensitized mice treated with indomethacin exhibited a mild retardation of weight gain after 7 weeks compared to non-sensitized controls. Gluten-sensitized mice NSAID and Gluten Sensitivity treated with indomethacin exhibited a more severe retardation of weight gain after 7 weeks, compared to all groups (Figure S1). These results suggest delayed thriving in mice treated with both gluten and indomethacin. Indomethacin increased tissue conductance and permeation of macromolecules across epithelium in gluten-sensitized mice In order to determine the effects of gluten sensitization and indomethacin treatment on intestinal permeability, tissue conduc- tance and HRP flux were measured in segments of small intestine. Gluten-sensitized mice treated with indomethacin exhibited a significant increase in small intestinal tissue conductance com- pared to non-sensitized controls and indomethacin alone-treated mice (Figure 1A). HRP flux, a measurement of transcellular macromolecular transport, was elevated in all groups compared to non-sensitized controls. Gluten sensitization and indomethacin treatment, however, led to the highest increase in HRP flux with approximately 2.5 fold increase compared non-sensitized controls (Figure 1B). The potentiation of intestinal permeability changes by indomethacin was not observed in C57BL/6 mice sensitized with gluten, stressing the importance of the DQ8 transgene in the model (Figure S2). Indomethacin led to epithelial ultra-structural damage in gluten-sensitized mice Intestinal morphology was analyzed using electron microscopy. No mitochondrial abnormalities were detected in non-sensitized controls or in gluten-sensitized mice without indomethacin (Figure 2A,B). Mitochondrial abnormalities were observed in mice treated with indomethacin alone (Figure 2C) and in gluten-sensitized mice treated with indomethacin (Figure 2D). We quantified the proportion of mitochondria with disrupted cristae in a defined area with Figure 1. Intestinal barrier measurements. Ussing-chamber experiments were performed on jejunum from all four groups 24 hours after the last gluten challenge. (A) Gluten-sensitized mice treated with indomethacin showed a significant increase in tissue conductance. (B) HRP flux (transcellular permeability) increased significantly in all treatment groups compared to non-sensitized controls, however the highest values were observed in gluten plus indomethacin treated mice. Data represent the means6SEM of 10 mice/group. doi:10.1371/journal.pone.0006472.g001 PLoS ONE | www.plosone.org 2 July 2009 | Volume 4 | Issue 7 | e6472