NSAID and Gluten Sensitivity Figure 8. Systemic antibodies against commensals. Serum from indomethacin and gluten treated mice showed significant positive serum antibodies against their aerobic and anaerobic intestinal microbiota. (A) Representative FACS histogram from each treatment gated on IgM+ cells; (B) Proportion of IgM+ aerobic bacterial cells for each treatment groups; (C) Proportion of IgM+ anaerobic bacterial cells for each treatment groups. Data represent the means6SEM of 6 mice/group. doi:10.1371/journal.pone.0006472.g008 examination in gluten-sensitized HCD4/DQ8 mice treated with indomethacin revealed more structural abnormalities in the apical region of the epithelium compared to gluten alone-treated mice. Furthermore, RT-PCR analysis demonstrated reduced E-cadherin RNA levels in gluten-sensitized plus indomethacin treated mice. E- cadherin is required for TJ formation and there is growing evidence for its role as modulator of TJ and intestinal barrier function [30,31]. E-cadherin expression is reduced in children with CD and gliadin has been shown to alter its expression [32]. Our results support the hypothesis that both gluten and indomethacin play a role in the expression of E-cadherin, and that this effect is potentiated in a genetically susceptible host when both agents are administered together. Marked reduction of E- cadherin may constitute a mechanism for the enhanced barrier dysfunction observed in gluten-sensitized and indomethacin- treated HCD4/DQ8 mice. The marked changes in barrier function in gluten-sensitized plus indomethacin-treated mice were accompanied by increased IFN-c production in splenocyte cultures after incubation with PT-gliadin. These results suggest a shift towards a mild systemic pro-inflammatory state. Previous studies have shown that cyclooxygenase-2 (COX-2)- dependent arachidonic acid metabolites are important in the maintenance of intestinal immune homeostasis, particularly in the immunoregulation of dietary antigens [33]. Consequently, COX-2 inhibitors such as indomethacin may exacerbate the immune response to dietary antigens [33]. Our results using in vitro incubation of splenocytes with PT gliadin and indomethacin, however, do not support a direct effect of indomethacin on splenocyte proliferation and IFN-c release. Thus, we hypothesize that the shift in the immune response may be due to an enhanced uptake of luminal contents, including commensal bacteria, through a more structurally damaged and permeable epithelium. The intestinal epithelium regulates permeation of luminal antigens and excessive immune activation within the mucosa [16,34]. The marked barrier defect in gluten-sensitized mice treated with indomethacin may not only allow an increased influx of gliadin peptides across the epithelium but also of other luminal antigens such as intestinal microbiota with potential bystander, pro-inflammatory effects. Germ-free rats have been reported to have a higher threshold for intestinal damage after indomethacin administration compared to specific pathogen-free (SPF) rats [35]. Since inhibition of prostaglandins in the absence of an intestinal PLoS ONE | www.plosone.org 7 July 2009 | Volume 4 | Issue 7 | e6472