NSAID and Gluten Sensitivity Figure 2. Evaluation of mitochondrial disruption. Mitochondrial ultra-structure was assessed by electron microscopy. Indomethacin increased the fraction of disrupted mitochondria. Gluten sensitization plus indomethacin treatment further increased the proportion of altered mitochondria. Data represent the means6SEM of 6 mice/group. Reperesentative pictures from (A) Control mouse, arrow: normal mitochondria; (B) Gluten- sensitized mouse; (C) Indomethacin-treated mouse, arrowhead: mitochondria with disrupted cristae; (D) Indomethacin-treated plus gluten-sensitized mouse, arrowhead: mitochondria with disrupted cristae. doi:10.1371/journal.pone.0006472.g002 approximately the same number of mitochondria (Figure 2E). Gluten-sensitized mice treated with indomethacin, had a higher proportion of damaged mitochondria than gluten-sensitized mice without indomethacin. Epithelial cell edema and disrupted microvilli were observed in tissues obtained from gluten-sensitized plus indomethacin treated mice but not from the rest of the groups (Figure 3). These marked ultra-structural changes were not observed in gluten-sensitized (Figure 3C) and indomethacin alone-treated mice (Figure 3D). Altered junctional ultra-structure was more pronounced in tissues from gluten-sensitized plus indomethacin-treated mice (Figure 3E–G) compared to non-sensitized (A–B), gluten-sensitized (C) and indomethacin alone-treated mice (D). Concomitant treatment with indomethacin and gluten led to reduction of E-cadherin mRNA expression The changes in the ultra-structure of the tight junctions prompted us to investigate whether there were alterations in RNA expression of epithelial adherens and tight junctional proteins. Gluten-sensitized and indomethacin alone-treated mice showed reduced E-cadherin RNA expression by a mean factor of 0.762 and 0.533 respectively, but this did not achieve statistical difference relative to non-sensitized controls (Figure 4). In contrast, expression of E-cadherin RNA was markedly down regulated by 2.75 fold compared to non-sensitized mice, in gluten-sensitized plus indomethacin-treated mice. Gluten sensitization and indo- methacin did not affect significantly the relative RNA expression of tight junction ZO-1 (Figure S3). Indomethacin treatment affected the release of IFN-c by splenocytes from gluten-sensitized mice after in vitro challenge with PT-gliadin In order to assess whether the increase in permeability and the damage to the intestinal structure in gluten-senstized mice after treatment with indomethacin led to an increase in the systemic immune response to gliadin, we analyzed antigen-specific proliferation and cytokine production of splenocytes. Increased T cell proliferation after incubation with PT-gliadin was observed in gluten-sensitized mice, but not in non-sensitized controls (Figure 5). Differences in proliferation did not reflect cell death or an inability to proliferate as polyclonal stimulation with ConA led to equal responses in all groups (data not shown). Surprisingly, indomethacin-treatment of gluten-sensitized mice did not exhibit higher levels of antigen-specific proliferation compared to gluten- sensitized mice that were not given indomethacin (Figure 5). In- vitro incubation of splenocytes from gluten-sensitized mice with indomethacin did not increase cell proliferation (Figure S4). To further assess the systemic immune response IL-12, IFN-c and IL-10 levels in the supernatant of the PT-gliadin stimulated splenocytes cultures were determined (Figure 6). Whilst IL-12 was not induced above media alone, IL-10 levels were slightly increased in the culture supernatant of splenocytes from gluten-sensitized and indomethacin- treated gluten-sensitized splenocytes after in vitro stimulation with PT- gliadin, although the increases were not statistically significant. In contrast, indomethacin treatment of gluten-sensitized mice led to a significant increase in IFN-c production in response to PT-gliadin stimulation. In-vitro incubation of splenocytes from gluten-sensitized mice with indomethacin did not increase IFN-c production (Figure S5). Gluten and indomethacin led to changes in intestinal microbiota composition We next analyzed whether gluten sensitization or indomethacin treatment could lead to changes in the composition of the intestinal microflora. Gluten-sensitized mice showed a significant decrease of gut bacterial proportions of E. coli and E. rectale- Clostridium groups, as compared to control mice. Indomethacin- treated mice also showed reductions in E. coli proportions, but increases in those of Bacteroides-Prevotella group. Gluten-sensitized mice treated with indomethacin showed the most remarkable alterations in the intestinal microbiota, characterized by reduc- PLoS ONE | www.plosone.org 3 July 2009 | Volume 4 | Issue 7 | e6472