Project Summary Chronic rhinosinusitis represents a significant healthcare burden, yet the cellular and molecular mechanisms driving disease are unclear in part, from a lack of clinically relevant mouse models. Allergic fungal rhinosinusitis (AFRS) is characterized by exacerbated sneezing, type 2 inflammatory response, eosinophilia and high numbers of specialized epithelial cells called sinonasal tuft cells (STCs) in response to fungal allergen exposure. It is currently not known how allergens are detected and inflammation is initiated in sinonasal mucosa. Trigeminal (TG) sensory neurons that innervate the respiratory epithelium sit in close apposition to STCs. TG neurons that express the Transient Receptor Potential Channel Vanilloid 1 (TRPV1) are required for sneezing, but it is not known if they become sensitized by allergen and progressively increase sneezing. To address these knowledge gaps, we have developed a mouse model of AFRS using chronic intranasal (i.n.) administration of a fungal allergen mix (FAM) that recapitulates all the cellular and cytokine-associated features of AFRS. Preliminary data show that ablation of intranasal TRPV1+ neurons, and perhaps other cell types, blocks STC expansion and eosinophil recruitment and reduces sneezing. Intranasal treatment of mice with the neuropeptide substance SP alone induces STC expansion and sneezing and cultured TG neurons exposed to fungal allergen secrete SP in and adaptive manner; showing greater secretion of SP when previously exposed to fungal allergen. Curiously, the adaptive increase in allergen-induced SP was lost in mice with a genetic deficiency in the alarmin cytokine interleukin (IL)-33. Taken together, we hypothesize that sinonasal TRPV1+ neurons respond to fungal allergens by releasing SP, which in turn, directs STC expansion from progenitor cells, leading to allergic inflammation, and excessive sneezing. We will test this hypothesis in two complementary, but independent aims. Aim 1 will identify the SP-responsive cells that promote STC expansion and allergic inflammation. This will be done in three sub-aims using transgenic mice that allow either gain (1A) or loss (1B) of function approach to either activate or ablate epithelial and immune cells receiving the SP signal and evaluate the cellular profile and sneezing behavior associated with those treatments. In 1C, we will perform single cell RNA sequencing in progenitor cells to establish how SP and fungal allergen instruct their differentiation into STC. Aim 2 seeks to define the neuron-specific contribution to fungal allergen-induced inflammatory responses and test whether selective loss of IL-33 responsive nociceptive neurons blocks allergic disease pathophysiology. In 2A we will use bone marrow chimeras of TRPV1+cre-DTR mice to determine the relative contribution of different TRPV1+ cell types to our previous findings. In 2B we will test whether mice with a selective loss of IL-33 responsiveness in nociceptive neurons are ...