Lipid-Loaded Macrophages in Atherosclerotic Plaque Inflammation

Mayo Clinic Rochester, Rochester MN

Investigators

Abstract

Project Summary Cardiovascular disease (CVD) has remained the leading cause of death in the United States for over a century. Underlying CVD is atherosclerosis, a pathogenic lipid-driven and chronic inflammatory response. Lipid buildup leads to plaque formation and artery stenosis, presenting clinically as myocardial infarction, cerebrovascular disease, peripheral arterial disease, and aortic aneurysm. In preliminary studies, we show through an integrated atheroma-derived single-cell RNA sequencing (scRNA-seq) analysis that immune cells, most of which are T cells and macrophages (Mφ), dominate the atherosclerotic milieu. Further analysis revealed five distinct Mφ subpopulations, including one showing upregulation of lipid-associated genes. Examination of this lipid- associated Mφ (LAM) demonstrated marked upregulation of bioenergetic processes. However, specialization of metabolic processing and lipid clearance came at the expense of core Mφ functions such as antigen presentation and cytokine production. Comparing LAMs to in vitro lipid-loaded Mφ confirmed that lipid feeding polarized Mφ towards the LAM phenotype. Epidemiologic data indicated that high LAM scores in the carotid atheroma placed patients at higher risk for future ischemic events, suggesting that LAMs play a pro-inflammatory and tissue- destructive role. Assessment of in vitro lipid-loaded Mφ revealed that lipid challenge increased production of extracellular adenosine triphosphate (eATP), a pro-inflammatory metabolite. Based on these data, we hypothesize that LAMs, under lipid-dependent metabolic overload, release ATP into the tissue microenvironment and utilize this metabolite as a signaling molecule to regulate surrounding Mφ. In pursuit of this hypothesis, we will execute the following aims. In Aim 1, we will characterize the effects of lipid loading on the metabolic landscape and cell fate decisions of atheroma-derived macrophages. Employing an in vitro human Mφ model, we will identify CVD-specific Mφ population enrichment, lipid-induced trajectory shifts, and gene regulatory networks with paired single-nucleus RNA-seq and scATAC-seq multi-omic analysis. We will also assess for lipid-induced bioenergetic perturbations at the bulk and single-cell level. In parallel, we will execute comprehensive analyses (e.g. phagocytosis, antigen presentation) to create a LAM functional atlas. In Aim 2, we will investigate how LAMs exacerbate plaque inflammation by controlling the function of neighboring macrophage subpopulations. Here, we will lipid load Mφ, assess for steady-state eATP production, and determine efflux kinetics. We will also develop a comprehensive understanding of how eATP affects atheroma- derived Mφ subsets by assessing for differential eATP-induced responses (e.g. inflammasome activation) in faithfully reproduced in vitro Mφ. To identify disease-specific responses, we will perform assays with CVD patient- and healthy control-derived Mφ. Ultimately, results from this proposal will clarify the link between dyslipidemia and atherosclerotic inflammation, allowing for novel diagnostic approaches and targeted CVD therapies.