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• W2114863119 abstract "Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk. Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk. Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in individuals with genetic risk. Proteolytic-resistant gluten peptides are deamidated by transglutaminase 2 (TG2) in the small-intestinal lamina propria, increasing their binding affinity to the CD-associated HLA-DQ2 or DQ8 molecules, leading to T-cell activation.1Dieterich W. Ehnis T. Bauer M. Donner P. Volta U. Riecken E.O. Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease.Nat Med. 1997; 3: 797-801Crossref PubMed Scopus (1773) Google Scholar, 2Shan L. Molberg Ø. Parrot I. Hausch F. Filiz F. Gray G.M. Sollid L.M. Khosla C. Structural basis for gluten intolerance in celiac sprue.Science. 2002; 297: 2275-2279Crossref PubMed Scopus (1239) Google Scholar CD also requires an innate immune response, characterized by up-regulation of stress markers on epithelial cells as well as up-regulation and activation of intraepithelial lymphocytes (IELs).3Allegretti Y.L. Bondar C. Guzman L. Rua E.C. Chopita N. Fuertes M. Zwirner N.W. Chirdo F.G. Broad MICA/B expression in the small bowel mucosa: a link between cellular stress and celiac disease.PLoS One. 2013; 8: e73658Crossref PubMed Scopus (25) Google Scholar, 4Meresse B. Chen Z. Ciszewski C. Tretiakova M. Bhagat G. Krausz T.N. Raulet D.H. Lanier L.L. Groh V. Spies T. Ebert E.C. Green P.H. Jabri B. Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in celiac disease.Immunity. 2004; 21: 357-366Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar There has been a rapid rise in CD prevalence over the past 50 years.5Rubio-Tapia A. Kyle R.A. Kaplan E.L. Johnson D.R. Page W. Erdtmann F. Brantner T.L. Kim W. Phelps T.K. Lahr B.D. Zinsmeister A.R. Melton 3rd, L.J. Murray J.A. Increased prevalence and mortality in undiagnosed celiac disease.Gastroenterology. 2009; 137: 88-93Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar This, in conjunction with the fact that only 2% to 5% of genetically susceptible individuals will develop CD, argues for environmental modulators of CD expression.6Sollid L.M. Jabri B. Triggers and drivers of autoimmunity: lessons from coeliac disease.Nat Rev Immunol. 2013; 13: 294-302Crossref PubMed Scopus (229) Google ScholarThe intestinal microbiota plays an important role in mucosal immune maturation and homeostasis as evidenced from seminal studies using germ-free and gnotobiotic mice.7Geuking M.B. Cahenzli J. Lawson M.A. Ng D.C. Slack E. Hapfelmeier S. McCoy K.D. Macpherson A.J. Intestinal bacterial colonization induces mutualistic regulatory T cell responses.Immunity. 2011; 34: 794-806Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar, 8Round J.L. Mazmanian S.K. The gut microbiota shapes intestinal immune responses during health and disease.Nat Rev Immunol. 2009; 9: 313-323Crossref PubMed Scopus (3165) Google Scholar Clinical and animal studies also suggest that altered colonization early in life increases susceptibility to chronic inflammatory diseases and food sensitivities.9Shaw S.Y. Blanchard J.F. Bernstein C.N. Association between the use of antibiotics in the first year of life and pediatric inflammatory bowel disease.Am J Gastroenterol. 2010; 105: 2687-2692Crossref PubMed Scopus (306) Google Scholar, 10Stefka A.T. Feehley T. Tripathi P. Qiu J. McCoy K. Mazmanian S.K. Tjota M.Y. Seo G.Y. Cao S. Theriault B.R. Antonopoulos D.A. Zhou L. Chang E.B. Fu Y.X. Nagler C.R. Commensal bacteria protect against food allergen sensitization.Proc Natl Acad Sci U S A. 2014; 111: 13145-13150Crossref PubMed Scopus (489) Google Scholar, 11Candon S. Perez-Arroyo A. Marquet C. Valette F. Foray A.P. Pelletier B. Milani C. Ventura M. Bach J.F. Chatenoud L. Antibiotics in early life alter the gut microbiome and increase disease incidence in a spontaneous mouse model of autoimmune insulin-dependent diabetes.PLoS One. 2015; 10: e0125448Crossref Scopus (146) Google Scholar Indeed, alterations in intestinal microbial composition have been described in CD patients, some of which normalize after treatment with a gluten-free diet.12Sanz Y. Palma G.D. Laparra M. Unraveling the ties between celiac disease and intestinal microbiota.Int Rev Immunol. 2011; 30: 207-218Crossref PubMed Scopus (102) Google Scholar Clinical studies have also proposed a link between antibiotic use and elective caesarean section and CD development.13Mårild K. Stephansson O. Montgomery S. Murray J.A. Ludvigsson J.F. Pregnancy outcome and risk of celiac disease in offspring: a nationwide case-control study.Gastroenterology. 2012; 142: 39-45Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Canova C. Zabeo V. Pitter G. Romor P. Baldovin T. Zanotti R. Simonato L. Association of maternal education, early infections, and antibiotic use with celiac disease: a population-based birth cohort study in northeastern Italy.Am J Epidemiol. 2014; 180: 76-85Crossref PubMed Scopus (88) Google Scholar, 15Decker E. Engelmann G. Findeisen A. Gerner P. Laaβ M. Ney D. Posovszky C. Hoy L. Hornef M.W. Cesarean delivery is associated with celiac disease but not inflammatory bowel disease in children.Pediatrics. 2010; 125: e1433-e1440Crossref PubMed Scopus (191) Google Scholar However, recent studies in families with high genetic risk for CD (positive family history or homozygous for HLA-DQ2.5) have not been able to identify an environmental determinant, including the timing and dose of gluten introduction to an infant's diet.16Aronsson C.A. Lee H.-S. Liu E. Uusitalo U. Hummel S. Yang J. Hummel M. Rewers M. She J.-X. Simell O. Toppari J. Ziegler A.G. Krischer J. Virtanen S.M. Norris J.M. Agardh D. Teddy Study GroupAge at gluten introduction and risk of celiac disease.Pediatrics. 2015; 135: 239-245Crossref PubMed Scopus (91) Google Scholar, 17Vriezinga S.L. Auricchio R. Bravi E. Castillejo G. Chmielewska A. Crespo Escobar P. Kolaček S. Koletzko S. Korponay-Szabo I.R. Mummert E. Polanco I. Putter H. Ribes-Koninckx C. Shamir R. Szajewska H. Werkstetter K. Greco L. Gyimesi J. Hartman C. Hogen Esch C. Hopman E. Ivarsson A. Koltai T. Koning F. Martinez-Ojinaga E. te Marvelde C. Pavic A. Romanos J. Stoopman E. Villanacci V. Wijmenga C. Troncone R. Mearin M.L. Randomized feeding intervention in infants at high risk for celiac disease.N Engl J Med. 2014; 371: 1304-1315Crossref PubMed Scopus (317) Google Scholar Microbial factors were not directly investigated, and results may not apply to the general population or individuals with moderate genetic risk for CD (HLA-DQ2 heterozygous or HLA-DQ8).Whether altered colonization instigates CD in an individual at moderate risk of developing CD remains unclear.18Verdu E.F. Galipeau H.J. Jabri B. Novel players in coeliac disease pathogenesis: role of the gut microbiota.Nat Rev Gastroenterol Hepatol. 2015; 12: 497-506Crossref PubMed Scopus (146) Google Scholar Therefore, we investigated whether microbial colonization modulates host responses to gluten using transgenic HLA-DQ8 mice on the nonobese diabetic background (NOD/DQ8).19Galipeau H.J. Rulli N.E. Jury J. Huang X. Araya R. Murray J.A. David C.S. Chirdo F.G. McCoy K.D. Verdu E.F. Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.J Immunol. 2011; 187: 4338-4346Crossref PubMed Scopus (50) Google Scholar We used complementary strategies to investigate host responses to gluten under different microbial conditions. Clean specific-pathogen-free (SPF) mice, strictly monitored for the absence of a variety of pathobionts and Proteobacteria, were protected from gluten-induced immunopathology when compared to germ-free and conventional SPF mice. Perinatal disruption of the microbiota leading to Proteobacteria expansion in conventional SPF mice further enhanced severity of responses to gluten; whereas specific pathobiont supplementation to clean SPF mice reversed the protective effect of the benign microbiota.Materials and MethodsMice and Colonization ProceduresFemale and male germ-free, clean SPF and conventional SPF NOD AB° DQ8 (NOD/DQ8) transgenic mice maintained on a gluten-free diet were used for experiments.20Marietta E. Black K. Camilleri M. Krause P. Rogers 3rd, R.S. David C. Pittelkow M.R. Murray J.A. A new model for dermatitis herpetiformis that uses HLA-DQ8 transgenic NOD mice.J Clin Invest. 2004; 114: 1090-1097Crossref PubMed Scopus (122) Google Scholar Germ-free mice were generated by two-stage embryo transfer, as previously described,21Slack E. Hapfelmeier S. Stecher B. Velykoredko Y. Stoel M. Lawson M.A. Geuking M.B. Beutler B. Tedder T.F. Hardt W.D. Bercik P. Verdu E.F. McCoy K.D. Macpherson A.J. Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism.Science. 2009; 325: 617-620Crossref PubMed Scopus (378) Google Scholar and bred and maintained in flexible film isolators in McMaster's Axenic Gnotobiotic Unit. Clean SPF mice originated from germ-free mice that were naturally colonized by co-housing with female mouse colonizers harboring altered Schaedler flora and bred for three generations in individually ventilated cage racks. Pathogen contamination and microbiota diversification in mouse cecum contents of clean SPF mice was evaluated every 2 weeks in cage sentinels and at the end of the study in the experimental mice by PCR for Helicobacter bilis, H. ganmani, H. hepaticus, H. mastomyrinus, H. rodentium, Helicobacter spp., H. typhlonius, and Pneumocystis murina. Mouse serum was also tested for murine viral pathogens by multiplexed fluorometric immunoassay/enzyme-linked immunosorbent assay (ELISA)/indirect fluorescent antibody tests. Germ-free status was monitored in sentinels and, at the end of the study in the experimental mice, by immunofluorescence (SYTOX Green; Invitrogen, Burlington, ON, Canada), anaerobic and aerobic culture, as well as PCR technique.Additional experiments were performed in germ-free and clean SPF C57BL/6 mice. For pathobiont supplementation experiments, 8- to 12-week-old clean SPF NOD/DQ8 mice were orally fed with 108 colony-forming units of Escherichia coli ENT CAI:5, isolated from fecal microbiota of a CD patient,22Sánchez E. Nadal I. Donat E. Ribes-Koninckx C. Calabuig M. Sanz Y. Reduced diversity and increased virulence-gene carriage in intestinal enterobacteria of coeliac children.BMC Gastroenterol. 2008; 8: 50Crossref PubMed Scopus (64) Google Scholar three times a week, 1 week before the start of sensitization and once a week during the sensitization and challenge period. Conventional SPF mice were bred and maintained in a conventional SPF facility at McMaster University. All experiments were conducted with approval from the McMaster University Animal Care Committee.Gluten Sensitization and ChallengeNOD/DQ8 mice were sensitized with 500 μg of sterilized pepsin-trypsin digest of gliadin (PT-gliadin) and 25 μg of cholera toxin (Sigma-Aldrich, St. Louis, MO) by oral gavage once a week for 3 weeks, as previously described.19Galipeau H.J. Rulli N.E. Jury J. Huang X. Araya R. Murray J.A. David C.S. Chirdo F.G. McCoy K.D. Verdu E.F. Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.J Immunol. 2011; 187: 4338-4346Crossref PubMed Scopus (50) Google Scholar PT-gliadin was prepared as previously described.19Galipeau H.J. Rulli N.E. Jury J. Huang X. Araya R. Murray J.A. David C.S. Chirdo F.G. McCoy K.D. Verdu E.F. Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.J Immunol. 2011; 187: 4338-4346Crossref PubMed Scopus (50) Google Scholar In antibiotic experiments, mice were sensitized at 3 weeks of age, following weaning. For all other experiments, 8- to 12-week-old mice were used for sensitizations. Following PT-gliadin sensitization, gluten-treated mice were challenged by oral gavage with 2 mg of sterile gluten (Sigma-Aldrich) dissolved in acetic acid three times a week for 2 weeks. Nonsensitized control mice received cholera toxin alone during the sensitization phase and acetic acid alone during the challenge phase. NOD/DQ8 mice were weaned and maintained on a gluten-free diet. In additional experiments, C57BL/6 mice were sensitized with PT-zein and cholera toxin, once a week for 3 weeks, and challenged with zein dissolved in acetic acid three times a week for 2 weeks. All preparations were tested for lipopolysaccharide contamination using the E-Toxate kit (Sigma-Aldrich). Mice were sacrificed 18 to 24 hours following the final gluten or zein challenge.Microbial AnalysisFecal and cecal samples were collected and flash frozen on dry ice. DNA was extracted from samples as previously described.23Whelan F.J. Verschoor C.P. Stearns J.C. Rossi L. Luinstra K. Loeb M. Smieja M. Johnstone J. Surette M.G. Bowdish D.M. The loss of topography in the microbial communities of the upper respiratory tract in the elderly.An Am Thorac Soc. 2014; 11: 513-521Crossref PubMed Scopus (121) Google Scholar Extracted DNA underwent amplification for the hypervariable 16S rRNA gene v3 region and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA). Generated data were analyzed as described previously.23Whelan F.J. Verschoor C.P. Stearns J.C. Rossi L. Luinstra K. Loeb M. Smieja M. Johnstone J. Surette M.G. Bowdish D.M. The loss of topography in the microbial communities of the upper respiratory tract in the elderly.An Am Thorac Soc. 2014; 11: 513-521Crossref PubMed Scopus (121) Google Scholar Briefly, sequences were trimmed using Cutadapt software version 1.2.1,24Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads.EMBnet J. 2011; 17: 10-12Crossref Google Scholar aligned using PANDAseq software version 2.8,25Masella A.P. Bartram A.K. Truszkowski J.M. Brown D.G. Neufeld J.D. PANDAseq: paired-end assembler for illumina sequences.BMC Bioinformatics. 2012; 13: 31Crossref PubMed Scopus (1426) Google Scholar operational taxonomic units selected via AbundantOTU,26Ye Y. Identification and quantification of abundant species from pyrosequences of 16s rRNA by consensus alignment.Proceedings (IEEE Int Conf Bioinformatics Biomed). 2011; 2010: 153-157PubMed Google Scholar and taxonomy assigned against the Greengenes reference database.27DeSantis T.Z. Hugenholtz P. Larsen N. Rojas M. Brodie E.L. Keller K. Huber T. Dalevi D. Hu P. Andersen G.L. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.Appl Environ Microbiol. 2006; 72: 5069-5072Crossref PubMed Scopus (7572) Google Scholar α-Diversity was calculated using Quantitative Insights Into Microbial Ecology (QIIME),28Caporaso J.G. Kuczynski J. Stombaugh J. Bittinger K. Bushman F.D. Costello E.K. Fierer N. Pena A.G. Goodrich J.K. Gordon J.I. Huttley G.A. Kelley S.T. Knights D. Koenig J.E. Ley R.E. Lozupone C.A. McDonald D. Muegge B.D. Pirrung M. Reeder J. Sevinsky J.R. Turnbaugh P.J. Walters W.A. Widmann J. Yatsunenko T. Zaneveld J. Knight R. QIIME allows analysis of high-throughput community sequencing data.Nat Methods. 2010; 7: 335-336Crossref PubMed Scopus (23789) Google Scholar and heat maps were generated using R (R Foundation for Statistical Computing, Vienna, Austria), clustered based on Bray-Curtis dissimilarity.29McMurdie P.J. Holmes S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.PLoS One. 2013; 8: e61217Crossref PubMed Scopus (7277) Google ScholarHistology and ImmunohistochemistryCross sections of the jejunum were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin for histological evaluation by light microscopy (Olympus, Richmond Hill, ON, Canada) using Image-Pro Plus software version 6.3 (Media Cybernetics, Rockville, MD). Enteropathy was determined by measuring villus-to-crypt (V/C) ratios in a blinded fashion, as previously described.19Galipeau H.J. Rulli N.E. Jury J. Huang X. Araya R. Murray J.A. David C.S. Chirdo F.G. McCoy K.D. Verdu E.F. Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.J Immunol. 2011; 187: 4338-4346Crossref PubMed Scopus (50) Google Scholar Intraepithelial lymphocytosis was determined by counting CD3+ IELs per 20 enterocytes in five randomly chosen villus tips, as previously described, and expressed as IELs/100 enterocytes. CD3+ immunostaining was performed on paraffin-embedded sections of the jejunum as previously described.19Galipeau H.J. Rulli N.E. Jury J. Huang X. Araya R. Murray J.A. David C.S. Chirdo F.G. McCoy K.D. Verdu E.F. Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.J Immunol. 2011; 187: 4338-4346Crossref PubMed Scopus (50) Google ScholarEnterocyte cell death was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining using the ApopTag Peroxidase in Situ Apoptosis Detection Kit (Millipore, Billerica, MA) according to the manufacturer's instructions. Slides were viewed by light microscopy (Olympus). The percentage of TUNEL-positive enterocytes in 20 villi was determined for each mouse.Small-Intestinal Lamina Propria and IEL PreparationSmall intestines were removed from mice, and IELs and lamina propria lymphocytes isolated by established protocols. Briefly, small intestines from mice were flushed to remove intestinal contents, Peyer's patches and mesentery were removed, intestines opened longitudinally, and cut into 3- to 5-mm pieces. Intestinal pieces were incubated five to six times in EDTA/HEPES/Dulbecco's phosphate buffered saline for 15 minutes in a 37°C shaker. After each 15-minute incubation, intestines were vigorously vortexed and the IELs were collected by passing the supernatants through a 40-μm cell strainer. Intestinal pieces were further digested with DNase I (Roche, Mississuaga, ON, Canada) and Collagenase Type VIII (Sigma-Aldrich) to collect lamina propria lymphocytes. IELs and lamina propria lymphocytes were enriched on a Percoll gradient and resuspended in fluorescence-activated cell sorting buffer for cell staining.Single-cell suspensions of lamina propria preparations were stained with fluorochrome-labeled cell-surface antibodies including CD4-APC (RM4-5), CD8a-PerCP (53-6.7), and CD25-PE (7D4) purchased from BD Biosciences (San Jose, CA). IEL cell suspensions were stained with fluorochrome-labeled cell-surface antibodies including CD3-Alexa Fluor-700 (ebio500AZ; eBioscience, San Diego, CA), CD3-Pacific Blue (RM4-5; BD Biosciences), CD8-PerCP (53-6.7; BD Biosciences), β T-cell receptor (βTCR) (H57-597; eBioscience), TCRγδ-APC (eBioGL3; eBioscience), NKG2D-PE (CX5; eBioscience), and CD69-PE-CF594 (H1.2F3; BD Biosciences). For intracellular staining, cells were permeabilized using the Foxp3 staining buffer set (eBioscience). Lamina propria cells were incubated with fluorescein isothiocyanate–conjugated antibodies to Foxp3 (FJK-16s; eBioscience), and IELs were incubated with PE-Cy7–conjugated Granzyme-B (NGZB; eBioscience) for 90 minutes at 4°C. Stained cells were acquired using the LSR II (BD Biosciences) and analyzed with FlowJo software version 7.2.4 (TreeStar, Ashland, OR).T-Cell Proliferation and Cytokine AnalysisSingle-cell suspensions of mesenteric lymph nodes were prepared in RPMI 1640 (1% penicillin/streptomycin, 10% fetal calf serum, 2 mmol/L l-glutamine). CD4+ T cells were isolated from mesenteric lymph nodes using the EasySep Mouse CD4+ T cell Enrichment Kit (StemCell, Vancouver, BC, Canada), and labeled with carboxyfluorescein succinimidyl ester (CFSE; Life Technologies, Grand Island, NY). CD11c+ cells were isolated from spleens using the Easysep Mouse CD11c Selection Kit (StemCell). A total of 5 × 104 CD11c+ cells were co-cultured with 2 × 105 CD4+ T cells in the presence of 500 μg/mL PT-gliadin, 500 μg/mL PT-zein, or medium alone in a round-bottom 96-well plate for 3 days at 37°C, 5% CO2. Cells were resuspended in fluorescence-activated cell sorting buffer and stained with fluorochrome-conjugated antibodies to CD3 and CD4 and a viability stain. CFSE-labeled cells were acquired using the LSR II (BD Biosciences). Viable cells were gated on CD4+ T cells. CFSE intensity for this population was determined using FlowJo software (TreeStar) and the percentage of divided cells determined for each condition (PT-gliadin, PT-zein, medium). Proliferation of cells in response to PT-gliadin or PT-zein stimulation were normalized to the proliferation of medium alone and expressed as a proliferation index.Anti-Gliadin ELISASerum IgA and IgG antibodies to gliadin were measured by ELISA as previously described,30Lau N.M. Green P.H. Taylor A.K. Hellberg D. Ajamian M. Tan C.Z. Kosofsky B.E. Higgins J.J. Rajadhyaksha A.M. Alaedini A. Markers of celiac disease and gluten sensitivity in children with autism.PLoS One. 2013; 8: e66155Crossref PubMed Scopus (71) Google Scholar, 31Moeller S. Canetta P.A. Taylor A.K. Arguelles-Grande C. Snyder H. Green P.H. Kiryluk K. Alaedini A. Lack of serologic evidence to link IgA nephropathy with celiac disease or immune reactivity to gluten.PLoS One. 2014; 9: e94677Crossref PubMed Scopus (20) Google Scholar with minor modifications. In addition, intestinal wash IgA antibodies to gliadin were measured similarly. Intestinal wash IgG antibody reactivity was too low to be detected reliably and was not measured. One hundred mg of the US hard red spring wheat Triticum aestivum cv Butte 86 variety flour was suspended in 1 mL of phosphate-buffered saline and mixed for 1 hour at 4°C. The suspension was centrifuged at 10,000 × g for 20 minutes. The supernatant containing mostly non-gluten proteins, was removed. The pellet was washed with phosphate-buffered saline, resuspended in 50% isopropanol, and mixed for 1 hour at room temperature. The suspension was centrifuged at 10,000 × g for 20 minutes, and the supernatant, containing gliadin and glutenin proteins, was stored at −20°C. The 96-well Maxisorp round-bottom polystyrene plates (Nunc, Roskilde, Denmark) were coated with 50 μL/well of a 0.01 mg/mL solution of the gliadin gluten extract in 0.1 mol/L carbonate buffer (pH 9.6) or were left uncoated to serve as control wells. Wells were blocked by incubation with 1% bovine serum albumin. Serum samples were diluted at 1:100, whereas intestinal wash samples were diluted at 1:10. The samples were added at 50 μL per well in duplicates and incubated for 1 hour. Each plate contained a positive control sample. After washing the wells, they were incubated with a 1:2000 dilution of either horseradish peroxidase–conjugated anti-mouse IgG (GE Healthcare, Piscataway, NJ) or IgA (Abcam, Cambridge, MA) secondary antibodies. The plates were washed, and 50 μL of developing solution was added to each well; absorbance was measured at 450 nm after 20 minutes. Absorbance values were corrected for nonspecific binding by subtraction of the mean absorbance of the associated bovine serum albumin–coated control wells. The corrected values were first normalized according to the mean value of the positive control duplicate on each plate. The mean antibody level for the clean SPF control group was then set as 1.0 arbitrary units, and all other results were normalized accordingly.Anti-Gliadin Western BlotsAntibody reactivity to gluten in sera was confirmed by Western blot. The Butte 86 gluten extract was dissolved in sample buffer, heated for 10 minutes at 75°C, and separated by SDS-PAGE (0.66 μg of protein per lane) using NuPAGE 4% to 20% bis-tris gels (Life Technologies). Protein transfer onto nitrocellulose membranes was performed with the iBlot Dry Blotting System (Life Technologies). The membrane was incubated for 2 hours in blocking solution (5% milk + 0.5% bovine serum albumin) in tris-buffered saline containing 0.05% Tween-20 (TBS-T). Serum specimens (1:500) were incubated in dilution buffer (10% blocking solution + 10% fetal bovine serum in TBS-T) for 1 hour. The secondary antibody used was horseradish peroxidase–conjugated anti-mouse IgG or IgA. Bound antibodies were detected by the ECL system (Millipore) and autoradiography film (Fuji, Valhalla, NY).Cytokine MeasurementSections of the jejunum were collected 18 to 24 hours following the final gluten challenge, homogenized, and tissue supernatants collected. Supernatants were also collected from T-cell proliferation assays after 3 days of stimulation. Cytokines were measured in tissue supernatants and cell culture supernatants using the Mouse Inflammatory CBA kit (BD Biosciences), and then analyzed using FACSarray Bioanalyzer System (BD Biosciences).Antibiotic TreatmentPregnant conventional SPF NOD/DQ8 mice were placed on 200 mg/L vancomycin (Sigma-Aldrich) in sterile drinking water and continued after birth until pups were weaned at 3 weeks of age. Vancomycin-containing water was replaced every 3 days. Fecal pellets were collected at 3 weeks of age for microbial analysis by 16S rRNA gene sequencing. Additional mice originating from non–antibiotic-treated NOD/DQ8 mice served as controls.StatisticsData were evaluated by analysis of variance with the Bonferroni post-hoc test for multiple comparisons when comparing more than two groups. Unpaired t-test was used to compare two groups. For microbial analysis, the U-test was used. P < 0.05 was considered statistically significant. All statistical analysis were performed in GraphPad Prism software version 6 (GraphPad Software, San Diego CA).ResultsColonization with a Clean Microbiota Attenuates Gluten-Induced Markers of IEL Cytotoxicity and Enterocyte Cell DeathProliferation and activation of IELs is a hallmark of CD.32Abadie V. Discepolo V. Jabri B. Intraepithelial lymphocytes in celiac disease immunopathology.Semin Immunopathol. 2012; 34: 551-566Crossref PubMed Scopus (114) Google Scholar To test the hypothesis that the background microbiota modulates host responses to gluten, we first compared IEL numbers and phenotype in germ-free and clean SPF NOD/DQ8 mice following gluten sensitization and challenge (gluten treatment). The microbiota of clean SPF mice was dominated by members of the Bacteroidetes and Firmicutes phyla, and was free from any pathogens, opportunistic bacteria, or bacteria from the Proteobacteria phylum (Table 1). IEL counts increased following gluten treatment in germ-free, but not in clean SPF mice, and were greater in gluten-treated germ-free versus clean SPF mice (Figure 1, A and B). To test whether the germ-free condition enhances mucosal sensitivity to cholera toxin or to other non-gluten antigens such as zein, we de" @default.
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• W2114863119 date "2015-11-01" @default.
• W2114863119 modified "2023-10-01" @default.
• W2114863119 title "Intestinal Microbiota Modulates Gluten-Induced Immunopathology in Humanized Mice" @default.
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