Project 2 Summary Atherosclerosis is the primary cause of cardiovascular disease (CVD), which manifests in myocardial infarction and stroke, a major source of mortality and morbidity in the US. Hypercholesterolemia and chronic inflammation are leading causative factors in the development of atherosclerosis. Many inflammatory receptors and other proteins involved in the inflammatory response localize and become activated in cholesterol-rich membrane microdomains, often designated as lipid rafts, which provide a solid platform for protein assembly within a liquid membrane. Here, we propose the concept of inflammarafts, enlarged lipid rafts hosting assembled inflammatory signaling complexes, to help formalize the lipid raft-centric view of inflammation and its control by cholesterol metabolism. Cholesterol depletion from inflammarafts in activated cells disrupts inflammatory signaling, and the approaches that allow for targeting cholesterol removal selectively to inflammatory cells may be used for atheroprotection. In the context of atherosclerosis, monocytes and macrophages develop long-term adaptation, or trained immunity, which results in sustained inflammatory phenotypes and includes epigenetic memory, as well as transcriptional and metabolic alterations leading to a chronic inflammatory state. In this application, we will test the hypotheses that inflammarafts serve as a signaling platform that mediates cell reprogramming and that trained immune cells sustain lipid rafts to maintain the inflammatory response. We propose that this positive feedback mechanism between inflammarafts and metabolic, gene expression and epigenetic alterations plays a major role in the development of atherosclerosis. Specifically, to test the hypothesis that inflammarafts serve as gatekeepers and effectors of trained immunity in atherosclerosis, we will use inflammaraft assays to image and quantify lipid rafts, accessible cholesterol, membrane order, TLR4 dimerization and the assembly of other receptor complexes. RNA-seq, ATAC-seq, ChIPseq and metabolomics will be used to characterize trained immunity. To modulate ligand-dependent inflammaraft assembly, we will neutralize oxidized lipid DAMPs, abundant in plaques, in transgenic mice that overexpress oxidation-specific antibodies. APOA1, AAV-AIBP (apoA-I binding protein) and cyclodextrin interventions will be used for systemic and targeted depletion of cholesterol. Conversely, cholesterol and inflammarafts will be increased in inducible, macrophage-specific ABCA1/ABCG1 knockout mice. Statins/mevalonate and desmosterol mimetics will be used to modulate macrophage reprogramming. Importantly, we will evaluate the diagnostic and prognostic power of inflammaraft phenotyping of blood leukocytes in CVD. By using the resources of PPG’s clinical and single cell protein and RNA sequencing cores, we will test the hypotheses that the abundance of inflammarafts and raft-dependent receptor assemblies in peripheral blood monocytes and...