Genetic dissection of defensin signaling in urinary tract infections - PROJECT SUMMARY Urinary tract infections (UTIs) are commonly caused by uropathogenic Escherichia coli (UPEC) and affect millions of patients, especially elderly women. Antibiotics do not reliably eliminate uropathogens and there is an urgent need for more effective treatments to improve UTI patient outcomes. Thorough understanding of microbe- host interactions in the urinary system will be key to the development of next generation immune therapies. Antimicrobial peptides (AMPs) are a key component of innate immunity, but the functions of these peptides have been very difficult to study due to the lack of animal models. The largest mammalian AMP family, the defensins, has >50 members in mice, and deleting any single gene does not result in a strong phenotype. In my postdoctoral work, I solved this problem by generating a Defensin cluster knockout (Def cKO) mouse line removing all Def genes in select tissues. My previous work focused on skin immunity and established the necessity of defensins and novel defensin receptors in anti-bacterial immunity. In my independent lab, I will shift my focus from the skin to a niche that is entirely new to me – the urinary tract. In this Stephen J. Katz application, I propose to utilize unique and innovative genetic tools to analyze the functions of defensins and defensin receptors in a mouse model of UTI. We hypothesize that defensins are important for anti-UTI immunity, but are also a main cause of UTI-mediated pelvic pain. We have three independent aims: Aim 1 will examine whether defensins synthesized and secreted by bladder epithelial cells contribute to the epithelium’s immunity against UPEC invasion. We will study defensin interactions with both intracellular and extracellular bacteria. Aim 2 will systematically characterize the defensin-related immune response in UTI using single cell RNA sequencing (scRNAseq) analyses and compare infected bladder tissues of wild type (WT) and Def cKO animals. This will allow us to identify all cell populations whose numbers and transcriptomes are altered by the lack of defensins. We will then focus on a few innate immune cell types with known defensin receptors, neutrophils, mast cells and dendritic cells, and explore the significance of defensin-immune cell signaling in urinary immunity. Aim 3 will test the hypothesis that defensins released in the bladder during UTI activate urinary nociceptive neurons and contribute to UTI-mediated pelvic pain. We will measure pelvic pain behaviors and urination frequencies of UPEC-infected WT and Def cKO mice as well as mice whose defensin-responsive neurons are genetically removed. The proposed project will be the first comprehensive loss-of-function analysis of defensins in the urinary system. This will also be one of the first surveys of neuroimmune crosstalk in the urinary tract. The immediate goal for this project is to understand the cell types and cell surface receptors that respond to defensins in UTI. The ultimate goal of my research program is to develop better treatments for UTI by targeting specific immune defensin receptors to enhance innate immunity while limiting painful side effects caused by neuronal activation.
