Project 2: Macrophage Lipid Homeostasis and Inflammatory Signaling ABSTRACT/SUMMARY The objective of Project 2 of this PPG is to understand how cellular lipid composition and lipid trafficking influence the inflammatory function of macrophages. Although perturbations in lipid homeostasis are recognized to be associated with inflammation in a number of human diseases, our understanding of “how” and “why” remains limited. Recent work has revealed that pro-inflammatory signals reprogram the lipid metabolic state of macrophages. It has also become clear that perturbations in lipid homeostasis can be sensed by the inflammatory machinery of macrophages so as to induce and to regulate inflammatory responses. Thus, lipid homeostasis and inflammation are interrelated, and perturbations in one affect the other. In this project, our PPG team will combine advanced analytical mass spectrometry–based approaches with genetic models of inflammation, with the goal of defining mechanisms by which inflammation drives reprogramming of the lipidome (and vice versa). We will assess the consequences of changing the subcellular levels of lipids on inflammatory signaling. Specific Aim 1 is to apply advanced analytic techniques to determine how pro- and anti-inflammatory signals change the subcellular lipidome in macrophages. We will use mass spectrometry approaches, including shotgun lipidomics, NanoSIMS imaging, and isotope labeling, to understanding how pro- and anti-inflammatory signals influence lipid localization and trafficking in macrophages. Specific Aim 2 is to determine the mechanisms by which alterations in cholesterol homeostasis potentiate the STING signaling pathway. We will pursue our discovery that perturbations in de novo cholesterol synthesis change type I IFN inflammatory responses via STING. Using a combination of biochemical approaches, confocal and NanoSIMS imaging, and chemoproteomics, we will test the hypothesis that cholesterol regulates STING function through direct binding. We will also test whether disease-associated mutations in STING abrogate the regulatory impact of cholesterol. Specific Aim 3 is to determine the importance of the STING signaling pathway on the development of dyslipidemia, inflammation, and atherogenesis in mice. Type I IFNs have been shown to influence the pathogenesis of atherosclerosis, but the molecular pathways underlying this sterile inflammatory response have not been elucidated. We will test the hypothesis that the cGAS/STING inflammatory axis is required to generate type I IFN in the setting of dyslipidemia and atherosclerosis. These studies will define the influence of the STING pathway on dyslipidemia, inflammation, immune cell ...