Tu1750 Microbial, Metabolomic, and Immunologic Dynamics During Disease Onset, Remission, and Relapse in a Mouse Model of Colitis Involving a Genetically Impaired Mucus Barrier

Abstract

Introduction: Disturbances of the intestinal microbiota due to enhanced hygienic conditions or antibiotic use have been linked to the increasing prevalence of obesity, allergy, autoimmunity, and functional and inflammatory disorders of the gut, including celiac disease (CD). It is unknown whether perturbation of normal gut colonization plays a causal role in subsequent development of immune responses to gluten. Thus, our aim was to determine and characterize whether absence of gut commensals modifies the response to gluten using germ-free (GF), specific pathogen free (SPF) and altered Schaedler flora (ASF) colonized NOD/DQ8 mice. Methods: GF, SPF, and ASF 8-10 week old male and female NOD/DQ8 mice were mucosally sensitized with peptic tryptic (PT) gliadin (500μg) and cholera toxin (CT; 25μg) by oral gavage once a week for three weeks. Control mice were sensitized with CT alone. Following sensitization, mice were challenged three times a week for two weeks with 2mg of gluten by oral gavage. All materials for GF experiments were aseptically prepared and administered to mice housed in gnotobiotic isolators. Twenty-four hours following the final gluten challenge enteropathy was evaluated by intraepithelial lymphocyte (IEL) counts and villus/crypt (V/C) ratios. Anti-gliadin IgA antibodies (AGA) in intestinal washes were determined as markers of sensitization and specificity towards gliadin was tested by Western blot. Gliadin-specific T cell responses were tested using in vitro proliferation assays. Results: V/C ratios in GF NOD/DQ8 mice decreased by 52% following sensitization, which was significantly greater than SPF (40%) andASF (38%) gliadin-sensitizedmice (p<0.01). Gliadinsensitized GF mice also had the greatest increase in IELs compared to gliadin-sensitized SPF and ASF mice (p<0.01). No control GF, SPF or ASF mouse had a positive AGA result. In gliadin-sensitized mice, positive AGA was detected in 75% of GF mice, 33% of SPF mice and no ASF mice. The specificity of AGA towards gliadin was confirmed by Western blot. T cells isolated from mesenteric lymph nodes of GF gliadin-sensitized mice responded to PT-gliadin stimulation in vitro, but not to PT-zein stimulation. T cells from non-sensitized mice did not respond to PT-gliadin stimulation. Conclusions: Gluten-induced enteropathy and development of gliadin-specific AGA was enhanced under germ-free conditions in NOD/ DQ8 mice and T cell responses are gliadin-specific. These results suggest that the commensal intestinal microbiota may have a protective effect during gliadin-sensitization and challenge in NOD/DQ8mice. Themechanisms leading to this protective effect are under current investigation.