PROJECT SUMMARY / ABSTRACT Macrophages play a pivotal role in maintaining homeostasis by tightly regulating the inflammatory response. In response to endotoxins like lipopolysaccharide, they undergo dynamic reprogramming, transitioning from a pro-inflammatory to suppressive to tolerant state, resulting in endotoxin tolerance. In this tolerant state, the inflammatory response to repeated endotoxin exposure is dampened. This can be protective, but insufficient or excessive tolerance can cause hyperinflammation or immunosuppression as seen in sepsis. The underlying mechanism of this process, particularly the contribution of long-term metabolic change, continues to be a key knowledge gap. We found there is a persistent and sustained long-term decrease in acetyl-CoA levels in stimulated macrophages based on preliminary data. However, the role of acetyl-CoA metabolism – a central metabolic pathway – in endotoxin tolerance remains unknown. This proposal aims to investigate the role of acetyl-CoA metabolism in mediating endotoxin tolerance induced by classical activation in macrophages. The specific aims are to (1) determine how acetyl-CoA availability affects histone acetylation in endotoxin tolerance, (2) profile the global effect of acetyl-CoA metabolic reprogramming on non-histone protein acetylation in endotoxin tolerance, and (3) characterize the role of acetyl-CoA metabolism in endotoxin tolerance in murine models. To achieve these aims, endotoxin tolerance will be recapitulated in cell lines, primary cell cultures, and murine models, while acetyl- CoA availability will be modulated using different genetic perturbation and treatment approaches. Identification of histone acetylation changes will be achieved through immunoblotting for selected acetylation sites and genome wide histone acetylation changes identified by chromatin immunoprecipitation sequencing with resulting gene expression changes determined by quantitative PCR. The global acetylated proteome will be profiled by mass spectrometry. Furthermore, the in vivo characterization of endotoxin tolerance will be accomplished by measuring serum cytokine levels and clinical phenotyping. This study focusing on the reprogramming of acetyl-CoA metabolism in macrophages aims to provide mechanistic insights into how the metabolic state of macrophages influences endotoxin tolerance. By understanding this relationship, we can gain valuable knowledge about how metabolic reprogramming impacts immune cell function and identify potential therapeutic targets within acetyl-CoA metabolism to regulate the inflammatory response. The Morgridge Institute for Research and the University of Wisconsin-Madison offer a strong foundation for conducting this project, given their extensive metabolism research. With the support and guidance from Dr. Jing Fan and collaborators, the project is expected to be completed efficiently. Through this training plan, the applicant will develop essential technical, scientific communication, and additional cli nical skills and knowledge, paving the way for a successful career as an academic physician-scientist.

PROJECT NARRATIVE Macrophages’ precise regulation of inflammation is vital in maintaining homeostasis and preventing excessive or insufficient inflammation observed in critical illnesses like sepsis, affecting 1.7 million US adults annually. One method of control is endotoxin tolerance, which is the ability for a secondary innate immune response to be diminished to prevent a hyperinflammatory or persistent response upon repeated exposure to endotoxins. This project aims to investigate the role of dynamic macrophage metabolism reprogramming in modulating the inflammatory response during endotoxin tolerance, potentially uncovering novel metabolic targets for managing critical illnesses such as sepsis and promoting human health.