DESCRIPTION (provided by applicant): The intestinal epithelium is a dynamic barrier that protects the body from the multitudes of bacteria that reside in the lumen. Yet multiple mechanisms exist allowing sampling of the lumen in order to promote tolerance and prevent inflammatory responses against innocuous dietary antigens. Through the use of two-photon (2p) microscopy, goblet cells (GC) were recently identified as a delivery mechanism for soluble antigen to the CD103+ dendritic cells residing in the villous lamina propria suggesting this mechanism promotes a tolerogenic immune response. We refer to these antigen delivery cells as Goblet Cell Associated Antigen Passages (GAPs). The formation of GAPs appears to be connected to the release of GC products during calcium- mediated compound exocytosis (CE), in response to acetylcholine through muscarinic receptors (mAchR). At steady-state, CE and GAP formation only occurs in the small intestine but not the colon of specific pathogen free-housed adult mice (SPF). However we did observe GAPs in the colon of germfree mice, neonatal SPF-housed mice, and Myd88-/- mice with altered microbial signaling. In this proposal, we hypothesize GAP formation and CE occurs through stimulation of the mAchR on GCs, and that this pathway is inhibited by TLR and NOD signaling in response to increased microbial growth or pathogenic infections. We propose to elucidate the details of how mAchR signaling leads to CE and where TLR and NOD intersect the signaling cascade. Through this grant, we hope to better understand how GAPs deliver antigen for the purpose of maintaining tolerogenic immune responses.