Characterization of basophils in urinary tract infections

About This Grant

PROJECT SUMMARY Basophils are the rarest immune cells and account for less than 1% of circulating white blood cells. Despite their rarity, basophils play key roles in allergies, inflammation and immunity in the skin, airways, and intestines. How- ever, their function in bladder immunity remains unexplored. Our lab recently discovered that basophils infiltrate the bladder epithelium after urinary tract infection (UTI). Preliminary data showed that genetic ablation of baso- phils, or deletion of a basophil-specific G protein-coupled receptor (GPCR), Mrgpra6, resulted in improved blad- der immunity and reduced colony forming units (CFUs) of uropathogenic Escherichia coli (UPEC) 24 hours post infection. We hypothesize that basophils are key contributors to Type 2 immunity in the bladder, and negatively regulate anti-UPEC immunity. We have developed novel genetic tools to visualize, analyze and ablate basophils. Using these new tools, we plan to systematically investigate the impact of these rare granulocytes on the bladder immune landscape. First, we will comprehensively analyze the numbers, location, morphology, and dynamics of basophils in the bladder before and after UPEC infection using flow cytometry and microscopy (Aim 1A). In collaboration with my colleague Dr. Nicole De Nisco and Dr. Douglas Strand at UTSW we will also examine the location of basophils in the bladder tissues of human UTI patients. Next, we will establish the roles of basophils in anti-UPEC immunity (Aim 1B). Mice whose basophils are ablated (using Mcpt8DtA and Mrgpra6DtA mice or anti-CD200R3 antibody) will be infected with UPEC and compared to wild-type (WT) controls. Bacterial CFU in the bladder, kidney and urine, neutrophils, eosinophils, M1 and M2 macrophages, Th1, 2, and 17 T cells, and innate lymphoid cells will be quantified at key time points. We will also perform bulk RNA sequencing (RNAseq) on WT and basophil-ablated bladders with and without UPEC infections to understand the impact of basophil loss-of-function on a whole-transcriptome level. Together, these experiments will build basic knowledge about the functions of basophils in bladder immunity. In Aim 2, we will focus on the novel basophil-specific GPCR Mrgpra6. We will perform immune phenotyping by comparing the anti-UPEC cellular and molecular immunity between WT and Mrgpra6 KO mice using CFU enumeration, flow cytometry, RNAseq, qPCR and ELISA (Aim 2A). We will also evaluate the basophil-specific functions of the putative human homologue of Mrgpra6, MRGPRX2, using a humanized mouse model Mcpt8MRGPRX2; Mrgpra6 KO. In Aim 2B, we will culture and purify bone marrow-derived basophils from WT and Mrgpra6 KO mice, and compare their responses to UPEC super- natants, host-derived immune factors and putative Mrgpra6 ligands. In summary, our study will fill in a key gap in bladder immunology by bringing basophil biology into UTI research. Successful completion of the proposed project will build basic knowledge about the immunological functions of basophils and novel basophil-specific GPCR Mrgpra6 in the bladder, allowing us to evaluate its putative human homologue MRGPRX2 as a therapeutic target in the future.