Nutrient Sensing of Apoptotic Cells by Macrophages Resolves Inflammation, Drives Atherosclerosis Regression, and Promotes Features of Plaque Instability

PROJECT SUMMARY/ABSTRACT Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of morbidity and mortality worldwide. While most atherosclerotic plaques remain clinically asymptomatic, a subset lead to myocardial infarction, stroke, or sudden death. These unstable atheromas feature a large necrotic core, primarily resulting from defective efferocytosis – a failure in the receptor-mediated process by which macrophages recognize, engulf, and degrade apoptotic cells (ACs). When efferocytosis fails, ACs become necrotic and generate a robust inflammatory response that drives atherosclerosis progression. Notably, the successive uptake of multiple ACs by individual macrophages, termed “continual efferocytosis”, is essential for inflammation resolution. However, this becomes increasingly compromised as atherosclerosis advances. Because the mechanisms underlying continual efferocytosis remain poorly understood, we aim to define the underpinnings driving the successive clearance of ACs by macrophages, revealing new therapeutic approaches to stabilize rupture-prone atheromas. Our supporting data provided herein indicate that nutrient-sensing of AC-derived cargo is a critical checkpoint that enables macrophages to successively clear multiple ACs rapidly. We show that the mammalian target of rapamycin complex 1 (mTORC1) is robustly activated in response to nutrient availability during efferocytosis, and inhibition of mTORC1 significantly reduces continual efferocytosis. Additionally, we provide evidence that the nutrient sensor SLC38A9 activates mTORC1 after AC uptake, drives phagolysosomal fission, and promotes continual efferocytosis. Mice lacking myeloid SLC38A9 (Slc38a9fl/fl Lyz2Cre+/-) exhibit defects in continual efferocytosis and increase necrosis in the dexamethasone-induced thymus injury model compared to controls (Slc38a9fl/fl). Notably, SLC38A9 expression is significantly reduced in lesional macrophages within unstable human atheromas and advanced plaques in mice, driven by TNFα- and IL-1β-mediated repression. Furthermore, unbiased transcriptomics comparing control and SLC38A9-deficient macrophages reveal significant differences in pathways associated with membrane trafficking, efferocytosis, and atherosclerosis. These data support our central hypothesis that SLC38A9-mediated nutrient sensing of AC-derived cargo activates the phagocytic machinery required for continual efferocytosis and drives inflammation resolution. We will address this hypothesis by pursuing the following aims: (1) to define the mechanisms by which SLC38A9-mediated mTORC1 activation promotes phagolysosomal fission, membrane trafficking, and continual efferocytosis; and (2) to elucidate the mechanisms through which SLC38A9 drives inflammation resolution, stimulates atherosclerosis regression, and promotes features of plaque stability. Successful completion of this proposal will provide new mechanistic insights into the role of nutrient sensing in efferocytosis and identify novel therapeutic strategies to stabilize rupture-prone atheromas.