The bladder is a frequent site of infection, as ~50% of women aged 18-50 years will have at least one urinary tract infection (UTI) and ~40% of hospital-acquired nosocomial infections are UTIs after urinary catheter introduction. Most UTIs are caused by uropathogeneic E. coli (UPEC), starting with bladder colonization and then ascension to the kidneys. In severe cases of pyelonephritis, the bacteria can enter the bloodstream – causing bacteremia, sepsis (urosepsis), and death. Importantly, the immune system is involved in the development of these pathologies and response to treatment. The dominant in vivo mammalian model used in biomedical research is the mouse. However, environmental microbial exposure is an important difference between basic human and laboratory mouse biology that must be considered when using mouse models to evaluate the fitness of the immune system. Humans are naturally exposed to commensal and pathogenic microbes from birth, and the immune system of adult humans consequently becomes trained by each encounter. In contrast, laboratory mice are often housed under specific pathogen-free (SPF) conditions. While SPF housing has been key in increasing experimental reproducibility, it has simultaneously further distanced the mouse model from humans, largely because SPF mice live their lives with limited microbial exposure. This proposal leverages a novel mouse model that mimics a critical aspect of human biology – exposure to multiple ongoing and resolved infections trains the immune system for robust responses to new pathogens. Our central hypothesis holds that the matured immune system in microbially-experienced dirty mice will respond with a more robust immune response in the bladder after local UPEC infection and accelerated UTI clearance, but an exaggerated inflammatory response and increased mortality during systemic urosepsis. The overall goal of this project is to study how changing the “starting point” of the immune system (i.e., naïve, neonate-like immune system of SPF mice vs. mature, adult-like immune system in ‘dirty’ mice after physiological microbial exposure) affects the immune response local and systemic UPEC infections. Our rationale for the studies with ‘dirty’ mice is that we will amass a valuable new foundation of information regarding UTI and urosepsis-induced immune responses and pathophysiology. Ultimately, the data obtained from the proposed studies will be of great relevance to the understanding UTIs. It is important to emphasize our ‘dirty’ mouse model is meant to be a novel complement to, rather than a replacement of, the SPF mice typically used in research, and serve as a valuable tool to discover new efficacious therapies that may be sensitive to unique environmental perturbations resulting from physiological microbial exposure.