Regulation of T cell responses to oral antigens

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Immune responses triggered by oral antigens can have significant clinical consequences. Dendritic cells (DCs) are among the most important cellular determinants of oral tolerance, as they are tasked, in a mucosal environment that also contains antigens and pattern stimuli derived from vast commensal microbiota, with deciding whether adaptive immunity should be mobilized or tolerized to given antigens. We have recently generated a DC-specific mouse model of spontaneous Th2-associated disease of the small intestine (SI) called TRAF6ΔDC, which initially appeared to be microbiota-dependent because antibiotic treatment is ameliorative. However, germ-free (GF) TRAF6ΔDC mice surprisingly exhibit exacerbated disease compared to mice housed under specific pathogen-free (SPF) conditions, suggesting a trigger other than microbiota. We now have preliminary findings that TRAF6ΔDC disease is completely ameliorated in mice fed antigen-free (AF) diet, suggesting the driving stimulus of disease is food antigen. The TRAF6ΔDC model may be employed to reveal novel mechanisms underpinning DC-mediated oral tolerance, and thus we propose the following specific aims: 1. Investigate the immunological mechanisms of TRAF6ΔDC oral antigen sensitivity. We propose to further characterize the immunologic abnormalities of TRAF6ΔDC mice in the context of oral antigen sensitivity to elucidate novel immunologic mechanisms linking development and maintenance of oral tolerance to TRAF6-mediated signals in DCs. In this respect, we will examine the relevant cellular actors, as well as regulatory and effector molecules to determine how homeostasis is disrupted in the presence of oral antigen. We will use diphtheria toxin receptor (DTR) transgenic mice to examine the effects of temporal ablation of various types of DCs in the context of both DC-specific TRAF6 deficiency and oral tolerance maintenance to begin to determine whether TRAF6 provides key pro-tolerance signals to certain cell types versus creating dysfunctional DCs in its absence. Model oral antigen will also be utilized in TRAF6ΔDC mice to better define the mechanism(s) of disease development. 2. Investigate the independent effects of microbiota and antibiotics on TRAF6ΔDC oral antigen sensitivity. Mitigation of TRAF6ΔDC disease by antibiotics in the absence of microbiota suggests potential biologically relevant direct effects of antibiotics on immune cells which we propose to investigate in the context of TRAF6ΔDC oral antigen sensitivity in the presence or absence of microbiota and/or food antigens. While we typically employ a broad-spectrum cocktail of four antibiotic drugs from different classes for the purpose of microflora depletion, our hypothesis in these studies will initially focus on the role of ampicillin, which belongs to the β-lactam class that has been previously reported to have direct biotic immunomodulatory activity. Additionally, we will examine the effects of antibiotics on oral antigen sensitivity in TRAF6ΔDC neonates under SPF and GF conditions to determine temporal relationships between antibiotic treatment, commensal microbiotic colonization, and oral antigen sensitivity.