The sleep-immune axis is highly complex, and the negative impact of inadequate sleep on immune dysfunction is well recognized. Unfortunately, sleep disorders are highly prevalent among Veterans; as a result, understanding how sleep influences immune regulation offers novel therapeutic strategies for inflammatory diseases associated with poor sleep. The transcription factors (TFs) and gene regulatory programs involved in the sleep-immune axis are not fully understood. Analysis of genome-wide changes in the activity of cis-regulatory elements, or cistrome, offers an unbiased assessment of TF activity to identify key pathways regulating the sleep- immune axis. The studies I propose in this CDA-2 application will identify and test TF network pathways contributing to alterations in neutrophil function during sleep disruption. Because of my clinical practice in critical care medicine, I am focusing on understanding the mechanistic impact of sleep on sepsis. Using an established murine model of sleep fragmentation, I have identified phenotypes of immune dysfunction related to sleep disruption. Previous studies showed sleep disrupted mice had worse outcomes in pathogen-based sepsis models. Whether the mortality is a consequence of an early hyperinflammatory response or a sequala of poor pathogen clearance is uncertain. In this proposal, I specifically tested for the impact of sleep on the early inflammatory response to sepsis using the pathogen-free, endotoxin-induced peritonitis model. Mice with sleep disruption had less mortality than matched controls upon LPS-induced peritonitis, suggesting a dampened acute inflammatory response. Consistent with this notion, peritoneal neutrophils from sleep-disrupted septic mice have lower expression of Myeloperoxidase (Mpo), a critical enzyme in reactive oxygen species (ROS) production. Single-cell RNA sequencing from the bone marrow of sleep-disrupted mice showed global transcriptomic changes in mature neutrophils with activity signature of Stat3, a TF that mediates immunosuppressive response. These findings support my hypothesis that neutrophil phenotype and function are sensitive to sleep perturbation. Using the innovative approach of cistrome analysis, I aim to identify the mechanistic TF pathways by which sleep disruption impacts neutrophil function. The overarching goal is to identify pathways that could be targeted to modulate neutrophil activation to treat Veterans suffering from the adverse consequences of sleep disruption. This proposal addresses the impact of sleep on neutrophil function through transcriptional regulation. First, I will investigate whether sleep disruption reduces the fundamental capabilities of neutrophils, including ROS production, phagocytosis, and NETosis (AIM1). I will then use cistromic tools to test whether the sleep disruption affects the transcriptional activity of Stat3 (AIM2). Using ChIP-seq, I expect a global change in Stat3 binding in cis-regulatory elements at a genome-wid...