PROJECT ABSTRACT The interplay between metabolic and inflammatory processes is critical to maintaining overall health where a delicate balance exists between pathogen clearance and limiting host tissue damage. Severe bacterial infections upset this equilibrium and lead to a critical condition known as septicemia, where metabolic and immunologic dysregulation rapidly overwhelms the body’s defenses and poses a life-threatening challenge. Neutrophils are crucial in establishing innate immunity and undergo an enigmatic cell death process termed neutrophil extracellular trap (NET) formation (NETosis) that elicits antimicrobial activities but also induces collateral damage to host tissues during sepsis. We aim to investigate the intricate mechanisms underlying NETosis and how shifts in metabolic homeostasis during septicemia antagonize NET formation thereby causing aberrant inflammation. We previously demonstrated that mitochondrial superoxide production promotes downstream NETosis, and preliminary data indicate a critical role for lactate in this cascade. My research program aims to determine (i) how excess lactate during bacterial septicemia antagonizes NETosis and the impact this has on disease outcome. This study reframes mitochondria as ‘sensory organelles’ within neutrophils that respond to perturbations in the metabolic environment and dictate downstream inflammatory responses rather than acting as the ‘powerhouse’ of the cell. These metabolic shifts during sepsis may play a direct role in modulating innate immunity and (ii) we aim to understand how hyperglycemia and increased lactate availability during septicemia influence lactylation of histones and downstream gene expression in neutrophils. These studies are aided by our discovery of a biomarker that transiently accumulates on the surface of neutrophils that accurately predicts neutrophils that will undergo NETosis 2-3 hr later, which will allow us to decipher transcriptional changes related to NETosis and contrast transcriptomes across different biologically and clinically relevant bacterial inducers of sepsis in various tissues during septicemia. While much of this proposal focuses on the impacts of glucose and lactate and inflammation, the complex metabolic environment during sepsis likely has diverse effects on innate immunity in differing tissues. Therefore, (iii) we will employ chimeric immune cell editing (CHIME) using small CRISPR-based libraries to target 40-50 metabolic genes so that each neutrophil in a mouse has a single gene disrupted. This technology will allow us to decipher the metabolic dependencies driving neutrophil recruitment and inflammatory processes across differing tissues or in response to common bacterial pathogens associated with sepsis. These studies are significant as they begin to unravel the mechanistic links between metabolic and transcriptional dysregulation during sepsis and the impact this has in skewing innate immunity of neutrophils to combat the invadin...