PROJECT SUMMARY/ABSTRACT Invasive Candidiasis (IC) is a life-threatening fungal infection that occurs when Candida spp. invade into deep tissues and circulation. In patients with an intact immune system, Candida infections are self-limiting. However, IC presents a serious threat to hospitalized and immunocompromised patients, resulting in ~350,000 deaths globally each year. The growth of these vulnerable populations and emergence of treatment-resistant Candida spp. have resulted in an urgent need for novel therapies to improve patient mortality. Recently, it has been appreciated that nociceptors (pain-sensing neurons) and the neuropeptide Calcitonin gene-related peptide alpha (CGRP) potentiate antifungal immunity in the skin by a mechanism yet to be defined. Therefore, an improved mechanistic understanding of CGRP may expand the prophylactic and therapeutic armamentarium for IC. Based on our preliminary studies, we hypothesize that CGRP is released by nociceptors in the skin and directly acts on local Dendritic Cells (DC) to induce IL-23 production and Type-17 inflammation. Here we propose 2 specific aims to directly test our hypothesis and characterize the cell-specific direct and secondary effects of CGRP on antifungal immunity. Aim 1: Determine the obligate source(s) and target(s) of CGRP required for robust antifungal immunity using a Candida albicans infection model. Subaim 1A: We will ablate CGRP in cutaneous nociceptors in order to test the requirement of nociceptor-derived CGRP for robust antifungal Type-17 immunity. Subaim 1B: We will ablate the CGRP-specific receptor subunit (RAMP1) in DCs in order to test the requirement of CGRP acting on DCs for robust antifungal Type-17 immunity. Through these subaims we will delineate a cellular circuit responsible for CGRP-mediated antifungal immunity. Aim 2: Test the functional requirement of CGRP signaling for cellular and transcriptional responses during a C. albicans infection model by two complementary subaims of single-cell transcriptomics. Subaim 2A: We will generate chimeric mice reconstituted with congenically distinct RAMP1KO and WT bone marrow progenitors and perform single-cell RNA-sequencing (scRNA-seq) on immune cells isolated from infected skin. By differential analyses of RAMP1KO and WT effector populations from the same infection, we will isolate transcriptional programs downstream of CGRP/RAMP1 signaling. Subaim 2B: We will perform spatial multi-omics analyses on infected skin from CGRP receptor antagonist- and vehicle-treated mice to test the requirement of CGRP/RAMP1 signaling for downstream transcriptional, cellular and microenvironment phenotypic changes during antifungal immunity. We will use single-cell transcriptomics with spatial context to define transcriptional signatures in situ and cell-cell interactions/communities that depend on CGRP/RAMP1 signaling. Through these subaims we will characterize the direct and secondary effects of CGRP during antifungal ...