Intestinal epithelial cells (IEC), the first line of defense within the gastrointestinal tract, exist in a dichotomous state, and participate in either nutrient absorption or immune activation1. As immune-educators, IECs express antigen presentation molecules, co-stimulatory molecules, and adhesion molecules important for lymphocyte trafficking, as well as wide range of soluble mediators (cytokines)(24-35), and thus have the capacity to regulate intestinal immune populations2,3. IECs participate in microbial sensing through Toll-like receptors (TLRs)4, and this TLR-mediated dialogue confers intestinal barrier integrity and mucosal homeostasis in health5. Defects in TLR-mediated microbial sensing mechanisms have been demonstrated in both murine models of T1D and in humans6. In addition, previous data from my mentor’s lab has demonstrated oral epithelial-specific TLR hyper-reactivity in T1D7,8, but whether these aberrant sensing mechanisms also occur at the intestinal interface is not yet clear. My mentor’s laboratory proposes that in T1D, inappropriate innate immune signaling within the IEC results in activation and induction of gastrointestinal (GI) inflammation. We propose that either directly (through APC function) or indirectly (through soluble mediator production), IECs drive highly-inflammatory, non-tolerizing conditions that can result in a loss of tolerogenic immune phenotypes while concomitantly increasing the survival of and/or transition to effector cells which in turn are at least in part responsible for the leukocyte driven destruction of the pancreatic β-cell (Fig. 1). The first step in deciphering these mechanisms is to evaluate the IEC innate immune responsiveness under conditions of health and autoimmunity. Thus, the purpose of this project is to evaluate the TLR-induced effects on IEC barrier function, activation and soluble mediator expression. To date, the intestinal biology field has relied on IEC lines, which often exhibit defects in innate sensing9-11. Here we will utilize novel primary IEC cultures derived from human T1D cadaveric donors, wherein we have already established novel culture techniques to characterize innate immune function of IEC under the condition of T1D12. IECs will be grown on transwell inserts and then stimulated with 1ug/ml of TLR agonists after which barrier integrity will be evaluated using a voltmeter and dextran leakage assays, while the soluble mediator profiles will be characterized my multiplex technologies. In addition changes in TLR expression and IEC activation will be evaluated by qPCR, flow cytometery and confocal microscopy. Data generated here will characterize the innate immune function of IECs under conditions of health and autoimmunity. 1. Shulzhenko, N., Morgun, A., Hsiao, W., Battle, M., Yao, M., Gavrilova, O., Orandle, M., Mayer, L., Macpherson, A. J., McCoy, K. D., Fraser-Liggett, C., Matziner, P. Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut. Nat Med 17, 1585-1593 (2011). 2. Mayer, L. Mucosal immunity. Immunological Reviews 206(2005). 3. Yamamoto, M., Fujihashi, K., Kawabata, K., McGhee, J.R. & Kiyono, H. A mucosal intranet: intestinal epithelial cells down-regulate intraepithelial, but not peripheral, T lymphocytes. Journal of immunology 160, 2188-2196 (1998). 4. Fukata, M. & Arditi, M. The role of pattern recognition receptors in intestinal inflammation. Mucosal immunology 6, 451-463 (2013). 5. Abreu, M.T. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function. Nature reviews. Immunology 10, 131-144 (2010). 6. Zipris, D. Toll-like receptors and type 1 diabetes. Advances in experimental medicine and biology 654, 585-610 (2010). 7. Wallet, S.M., Alonso, T., Lalane, C., Catalfamo, D., Neiva, K. Altered oral mucosal homeostasis in Type 1 diabetes. Immunology 186 (Meeting Abstract Supplement), 162.119 (2011). 8. Neiva, K.G., Calderon, N.L., Alonso, T.R., Panagakos, F. & Wallet, S.M. Type 1 Diabetes-associated TLR Responsiveness of Oral Epithelial Cells. Journal of dental research 93, 169-174 (2014). 9. Sakamoto, K. & Maeda, S. Targeting NF-kappaB for colorectal cancer. Expert opinion on therapeutic targets 14, 593-601 (2010). 10. Ben-Neriah, Y. & Karin, M. Inflammation meets cancer, with NF-kappaB as the matchmaker. Nature immunology 12, 715-723 (2011). 11. Karin, M. NF-kappaB as a critical link between inflammation and cancer. Cold Spring Harbor perspectives in biology 1, a000141 (2009). 12. Graves, C.L., et al. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function. Journal of immunological methods 414, 20-31 (2014).