tions in the relative abundance of all bacterial groups analysed as compared with control mice (Figure 7). Indomethacin led to systemic priming against intestinal microbiota in gluten-sensitized mice Previous data suggests that bacterial translocation beyond the mucosal immune system is necessary for systemic priming to intestinal commensals [26]. To determine if the increased conductance and HRP flux induced by gluten-sensitization and indomethacin was accompanied by loss of the host’s normal systemic ignorance to the intestinal microbiota, we measured specific IgM antibody responses to culturable aerobic or anaerobic commensals. Non-sensitized mice showed no evidence of IgM specific antibodies against aerobic and anaerobic commensal flora as assessed by flow cytometric analysis of anti-bacterial IgM responses (Figure 8 & Figure S6). Treatment with either gluten or indomethacin alone led to the production of very low titres of IgM antibodies directed against a subset of culturable bacteria. Gluten sensitization plus indomethacin treatment, however, resulted in induction of strong specific IgM responses directed against 40–80% of culturable bacteria. The anti-bacterial IgM induced was specific to the commensal microflora of the host and did not bind to Salmonella, which these mice have never been exposed to (Figure S7). These data indicate that the combination of gluten-sensitization and increased intestinal permeability as induced by indomethacin treatment increased systemic priming to the commensal microflora. Discussion The aim of this study was to investigate whether modulation of the intestinal barrier by an environmental trigger can affect host NSAID and Gluten Sensitivity responses to luminal antigens in a genetically predisposed host. Our results demonstrate that gluten sensitization and long-term gluten challenge in HLA-DQ8/HCD4 mice alters intestinal permeability as assessed by increased transcellular macromolecu- lar transport and a tendency for higher conductance values (paracellular pathway). This is in agreement with previous reports showing that gluten peptides have the ability to rapidly disrupt the apical junctional structure [22–25], and can also be transported by an abnormal transcellular route [26]. Administration of indo- methacin to gluten-sensitized mice led to more pronounced barrier dysfunction, which was accompanied by a mild pro-inflammatory shift with marked changes in EC ultra-structure, reduced E- cadherin mRNA levels in the proximal small intestine and generation of systemic antibody responses to intestinal microbiota. Indomethacin has been used at higher doses as a model of inflammatory bowel disease (IBD) [27,28], and has been shown to compromise epithelial barrier integrity and function, induce motility changes, and promote small intestinal bacterial over- growth and translocation [27,29]. In the present manuscript, low- dose indomethacin induced permeability changes without pro- ducing macroscopic or microscopic ulcers. However, ultra- structural observations revealed epithelial cell abnormalities characterized by mitochondria with disrupted cristae in mice receiving indomethacin. Mucosal toxicity induced by gluten in HCD4/DQ8 mice was potentiated by indomethacin, as shown by more marked elevation of HRP flux and a significant increase in tissue conductance. After administration of indomethacin, C57BL/6 mice exhibited increased HRP flux, but no change in tissue conductance. Gluten sensitization however, did not induce barrier dysfunction in C57Bl6 mice, emphasizing the relevance of the DQ8 transgene in the model (Figure S2). Electron microscopy Figure 7. Microbiota composition. Using 9 different oligonucleotide probes and fluorescent in situ hybridization (FISH), microbial profile was investigated in the distal jejunum of sensitized mice with and without indomethacin. The results indicate a significant perturbation in the proportions of microbiota investigated in all 3 treatment-groups when compared to non-sensitized controls, and remarkably in the gluten-sensitized plus indomethacin group. These differences achieve statistical significance in Bifidobacteria (*p = 0.04 vs controls, +p = 0.03 vs gluten) and Clostridium Leptum cluster (both *p = 0.02 vs controls and gluten sensitized, **p = 0.04 vs indomethacin) compared to gluten-sensitized alone. Data represent the means6SEM of 6 mice/group. doi:10.1371/journal.pone.0006472.g007 PLoS ONE | www.plosone.org 6 July 2009 | Volume 4 | Issue 7 | e6472