A major gap exists in our understanding of how neuroendocrine mediators impact urothelial innate immune regulation to increase the risk for urinary tract infection (UTI) in females. During UTI, epithelial Toll-like Receptors (TLRs) detect microbial components to induce antimicrobial peptides (AMPs), innate immune factors that exhibit direct microbicidal activity and stimulate subsequent immune responses. We previously determined that psychological stress and the loss of the neuroendocrine protein, chromogranin A (CHGA), increased the susceptibility to skin infection by decreasing AMP responses. Our objective is to expand this area of urinary clinical research by identifying a role for psychological stress and CHGA in modulating urothelial AMP responses. Our long-term goal is to identify the mechanisms by which CHGA modulates TLR- dependent urothelial AMP responses, and relate these changes to UTI risk in humans. The objective here is to characterize CHGA-mediated AMP dysfunction in human urothelial cells and in a mouse model of Escherichia coli (UPEC) infection. Our hypothesis is that aberrant production of CHGA/Cst contributes to UPEC susceptibility by compromising inherent urothelial AMP responses. Our hypothesis was formulated based on observations from our lab that the loss of CHGA increases the susceptibility to skin bacterial infection in mice, and that catestatin, a CHGA-derived peptide released in response to stress, modulates epithelial AMP responses. Our rationale is that no one has investigated the role of CHGA in TLR-dependent urothelial AMP responses in the context of UTI, even though activation of the neuroendocrine system is impaired in several disorders of the female urinary tract. Driven by compelling published data in skin and pilot data in urothelial cells, our hypothesis will be evaluated by addressing two Specific Aims: 1.) We will use pharmacologic approaches to assess the effect of CHGA and catestatin peptides (wild-type and mutant) on AMP production and activity in primary human urothelial cells (HUCs) and urothelial organotypic rafts 2.) We will determine the role of CHGA in the urothelial AMP response to UPEC infection in vivo using mice deficient in urothelial CHGA. Our approach is innovative by using high-throughput molecular and proteomic approaches and tissue-specific knock-out mice to establish a role for CHGA in modulating urothelial AMP responses during UPEC infection. The proposed research is significant, because it will begin to dissect the mechanisms of urothelial AMP dysregulation caused by CHGA deficiency, in a controlled setting, using novel human urothelial cell and genetic mouse models in parallel, which will provide the foundation for future R01 applications and human clinical trial networks focused on UTIs. Our study results are anticipated to greatly improve our knowledge of urothelial AMP regulation by CHGA, which may lead to novel strategies to manipulate CHGA-mediated AMP responses to ameliorate the frequency or severity of UTIs in females.
The proposed investigations are relevant to improving overall human health, since they will establish a role for chromogranin A (CHGA) and its proteolytic fragment, catestatin, in regulating antimicrobial responses in the context of female urinary tract infection (UTI). The proposed studies are relevant to the part of the NIH's mission to reduce both the psychosocial and economic burdens associated with UTIs in the female population, and reveal new targets for strategically focused diagnostic and therapeutic interventions for female urinary tract disorders. The acquired information can be further extrapolated to other areas of female urological diseases, as well as global epithelial inflammatory or infectious disease research involving neuroendocrine dysregulation.