Defining the role(s) of CGRPα during cutaneous antifungal immunity

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 immunity. Contribution to Training: This proposal combines rigorous training in neuroimmunology, systems immunology and clinical medicine to aid my development as a future physician-scientist at the forefronts of neuroimmunology.