ABSTRACT The GI tract hosts as many neurons (enteric-associated neurons, EANs) as the spinal cord and more immune cells than all other compartments together. Bidirectional interactions between immune and neuronal cells have been documented during steady state and are proposed to be part of several disease processes, ranging from multiple sclerosis to irritable bowel syndrome (IBS), and food allergy. With complementary expertise using orthogonal approaches, our individual groups within this program and collaboration between our labs have substantially contributed to the neuroimmune field by uncovering sensing mechanisms from the immune and nervous systems that monitor perturbations at the luminal surface. In Project 3, we will investigate whether physical interactions between enteric neurons, immune cells, and enteroendocrine cells (EEC) play major roles in sensitization to food antigen (sensitization phase) or during allergic response (effector phase), dictating the severity of food allergy. The experiments proposed here will complement Project 1 by defining cellular partners in the gut epithelium, including EEC-neurons and EEC-immune cells, at different phases of allergic responses. Project 3 will also complement studies performed by the Liberles lab in Project 2 by defining functional changes in local enteric neurons and epithelial cells contacting vagal sensory neurons. In Aim 1, we will define transcriptional changes in enteric neurons following allergic sensitization in mice (with Bioinformatics Core). We will also characterize morphological or structural changes in enteric neurons during allergic responses utilizing 3D-tissue clearing imaging tools (interface with Project 1). In Aims 2&3, we will take advantage of uLIPSTIC, recently developed in collaboration with G. Victora (Rockefeller). This new mouse genetic approach allows for contact-dependent labeling of cell-cell interactions in specific cell types. To identify epithelial-neuron-immune partners, we will use neuronal uLIPSTIC, allowing in vivo labeling of non-neuronal cells via neuronal-restricted sortase expression upon cell-cell contact. Using a combination of imaging and gene-reporter mouse strains, we will define whether neuron-immune cell interactions change spatially under allergic versus non-allergic conditions. Finally, we will use genetically engineered mouse strains and AAV constructs using our Transgenic Core to target or modulate specific immune or neuronal pathways in the context of allergic sensitization (interface with Projects 1 and 2). In Aim 3, we will identify EEC-interacting partners during physiology, allergic sensitization and effector phases, in collaboration with the Liberles lab. We will use mouse strains restricting LIPSTIC sortase expression to EECs in the small and large intestine. With the Bioinformatics and Transgenic Cores, we will transcriptionally profile FACS-sorted LIPSTIC labelled and unlabeled cells during steady state or food allergy, characteriz...