priately filtered light at green (FL1, 525 nm) and red-orange (FL3, 620 nm) wavelengths. The event rate was kept at the lowest setting (200–300 events per second) to avoid cell coincidence. A total of 15, 000 events were recorded in a list mode file and analyzed with the System II V.3 software (Beckman Coulter). Statistical Analysis Statistical analysis was performed using ANOVA with post-hoc test for simple and multiple comparisons, respectively. Nonpara- metric statistical significance of relative RNA expression was calculated with REST software [53] by a pairwise fixed reallocation randomization test with 50,000 repeats. Data were presented as means6standard error (SEM). Supporting Information Figure S1 Weight gain over 7-week period. Both gluten- sensitized and indomethacin treated mice exhibited a decreased rate of weight gain compared to non-sensitized controls. Gluten- sensitized mice treated with indomethacin exhibited more pro- nounced weight gain retardation compared to controls and to gluten-sensitized and indomethacin treated mice. Data represent the means6SEM of 10 mice/group. Found at: doi:10.1371/journal.pone.0006472.s001 (0.08 MB TIF) Figure S2 Conductance and HRP flux in C57Bl/6 mice. Gluten and/or indomethacin treatment did not lead to changes in tissue conductance. HRP flux was increased in indomethacin treated mice, but not in gluten sensitized mice without indomethacin. Data represent the means6SEM of 10 mice/group. Found at: doi:10.1371/journal.pone.0006472.s002 (0.09 MB TIF) Figure S3 ZO-1 RNA expression relative to non-sensitized controls. No significant differences were seen when RNA expression for each treatment group was analyzed relative to non-sensitized controls. Data represent the means6SEM of 6 mice/group. Found at: doi:10.1371/journal.pone.0006472.s003 (3.00 MB TIF) Figure S4 Splenocyte proliferation after incubation with PT- gliadin and/or indomethacin. Stimulation with indomethacin alone did not increase splenocyte proliferation in gluten-sensitized mice. In-vitro stimulation with both PT-gliadin and indomethacin, did not further enhance cell proliferation compared to PT-gliadin alone. Data represent the means6SEM of 6 mice/group. Found at: doi:10.1371/journal.pone.0006472.s004 (0.07 MB TIF) Figure S5 IFN-c levels in supernatant of cultured splenocytes after incubation with PT-gliadin and/or indomethacin. Stimula- tion with indomethacin alone did not increase IFN-c production in gluten-sensitized mice. In-vitro stimulation with PT-gliadin and indomethacin did not increase IFN-c levels compared to PT References 1. Cronin CC, Shanahan F (2003) Exploring the iceberg-the spectrum of celiac disease. Am J Gastroenterol 98: 518–520. 2. Rostom A, Murray JA, Kagnoff MF (2006) American Gastroenterological Association (AGA) Institute technical review on the diagnosis and management of celiac disease. Gastroenterology 131: 1981–2002. 3. Troncone R, Franzese A, Mazzarella G, Paparo F, Auricchio R, et al. (2003) Gluten sensitivity in a subset of children with insulin dependent diabetes mellitus. Am J Gastroenterol 98: 590–595. 4. Marsh MN (1992) Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue’). Gastroenterology 102: 330–54. 5. Kaukinen K, Maki M, Partanen J, Sievanen H, Collin P (2001) Celiac disease without villous atrophy: revision of criteria called for. Dig Dis Sci 46: 879–887. 6. Mino M, Lauwers GY (2003) Role of lymphocytic immunophenotyping in the diagnosis of gluten-sensitive enteropathy with preserved villous architecture. Am J Surg Pathol 27: 1237–1242. NSAID and Gluten Sensitivity gliadin alone. Data represent the means6SEM of 6 mice/group. ND = not detectable. Found at: doi:10.1371/journal.pone.0006472.s005 (0.07 MB TIF) Figure S6 Systemic antibodies against commensals. Gluten- sensitized plus indomethacin-treated mice exhibited increased serum antibodies against aerobic and anaerobic bacteria as assessed by median fluorescent intensity signal of APC-labelled anti-IgM (1:20 serum dilution). Negative controls include serum (2): no serum and bacteria (2): no bacteria. Data represent the means6SEM of 6 mice/group. Found at: doi:10.1371/journal.pone.0006472.s006 (0.11 MB TIF) Figure S7 Positive and negative systemic antibodies against commensals. (A) Salmonella M557, which is a pathogen not present in our HLA-DQ8/HCD4 mice colony, was stained with serum antibodies from indomethacin plus gluten treated mice. Results show the absence of positive antibodies against Salmonella, thus the specificity of the technique. (B) Salmonella M557 was stained with serum antibodies from Salmonella M557 infected mice. Results show the absence of positive antibodies against Salmonella. Found at: doi:10.1371/journal.pone.0006472.s007 (0.09 MB TIF) Figure S8 Immunohistochemistry for F4/80+ cells. Staining for F4/80+ was increased in gluten sensitized mice. Infiltration of F4/ 80+ cells was most marked in gluten-sensitized mice treated with indomethacin. Data represent the means6SEM of 6 mice/group. Representative picture of macrophage infiltration in the lamina propria from (A) control mice (B) gluten-sensitized mice (C) indomethacin-treated mice (D) gluten-sensitized plus indometha- cin treated mice. Found at: doi:10.1371/journal.pone.0006472.s008 (0.47 MB TIF) Table S1 Oligonucleotide probes and hybridization conditions used in FCM-FISH analysis of intestinal bacteria. Found at: doi:10.1371/journal.pone.0006472.s009 (0.06 MB DOC) Immunohistochemistry for macrophages. doi:10.1371/journal.pone.0006472.s010 (0.03 MB Protocol S1 Found at: DOC) Acknowledgments We acknowledge Markus Geuking, Julia Cahenzli and Giada de Palma for their excellent assistance. Author Contributions Conceived and designed the experiments: EFV. Performed the experi- ments: JMN XH ES JJ YS EFV. Analyzed the data: JMN XH ES JJ YS ED PY KDM EFV. Contributed reagents/materials/analysis tools: YS CD JM KDM EFV. Wrote the paper: JMN JM KDM EFV. 7. Verdu´ EF, Armstrong D, Murray JA (2009) Between celiac disease and irritable bowel syndrome: The ‘No Man’s Land’ of gluten sensitivity. Am J Gastroenterol 104: 1587–1594. 8. Black KE, Murray JA, David CS (2002) HLA-DQ Determines the Response to Exogenous Wheat Proteins: A Model of Gluten Sensitivity in Transgenic Knockout Mice. J Immunol 169: 5595–5600. 9. Marietta E, Black K, Camilleri M, Krause P, Rogers RS 3rd, et al. (2004) A new model for dermatitis herpetiformis that uses HLA-DQ8 transgenic NOD mice. J Clin Invest 114: 1090–1097. 10. Senger S, Maurano F, Mazzeo MF, Gaita M, Fierro O, et al. (2005) Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization. J Immunol 175: 8087–8095. 11. Verdu EF, Huang X, Natividad J, Lu J, Blennerhassett PA, et al. (2008) Gliadin- dependent neuromuscular and epithelial secretory responses in gluten-sensitive HLA-DQ8 transgenic mice. Am J Physiol Gastrointest Liver Physiol 294: G217–25. PLoS ONE | www.plosone.org 10 July 2009 | Volume 4 | Issue 7 | e6472