Understanding how appropriate apoptotic cell degradation ensures lung homeostasis

Efferocytosis, the phagocytic uptake of apoptotic cells (ACs), is a critical homeostatic mechanism that regulates all metazoan tissues. These ACs, produced daily during tissue turnover, are cleared by professional phagocytes (typically tissue-resident macrophages) in a sequence that first involves the sensing of an AC, followed by the engulfment and digestion of that AC. Digestion of ACs is potentially hazardous, as the AC contains dangerous biomass that a phagocyte must handle in order to maintain cellular and tissue homeostasis. For example, in atherosclerosis, aberrant macrophage lipid digestion causes “foamy cell” build-up which prevents subsequent efferocytosis, causing plaque formation and inflammatory necrosis. In lung granulomatous and lysosomal storage diseases, macrophages feature undigested ACs. Critically, these diseases often implicate lysosome-associated proteins that are often uniquely expressed in tissue-resident macrophages. As a result, our lab has proposed that the failure to appropriately digest ACs (FAD) during efferocytosis is a main driver of pathogenesis. However, the cellular mechanisms behind FAD have not been fully characterized. To understand how FAD impacts lung homeostasis, we will target lysosome-associated proteins in alveolar macrophages implicated in pulmonary diseases (Aim 1). We will utilize our novel, highly resolved ex vivo efferocytosis assay that better-represents physiological tissue turnover to identify candidate genes that disrupt AC digestion. We will then investigate putative AC digestion genes using a medium throughput assay that allows us to rapidly generate and study the functional and physiological consequences of gene deletions in alveolar macrophages in vivo. In parallel, we will use novel fluorescence reporters to visualize organelle dynamics during efferocytosis in order to understand how FAD impacts cellular homeostasis (Aim 2). As AC- derived biomass must be shuttled through solute transport carriers to organelles such as the ER and mitochondria, we hypothesize that FAD impacts cellular homeostasis by impairing metabolite transport between organelles. As part of this aim, we will visualize contact site formation and duration between the phagolysosome (site of AC digestion) and the ER/mitochondria. To test our hypothesis, we will quantify these contact sites during healthy efferocytosis and in macrophages with digestion deficiency to assess whether FAD disrupts cellular organelle homeostasis. Collectively, these studies will probe FAD in both cellular and tissue (lung) settings. These studies will demonstrate a direct link between lysosome-associated proteins and FAD in promoting lung pathogenesis. Additionally, this work will explore the mechanisms of cellular dysfunction during aberrant AC digestion in macrophages and provide novel visualizations of subcellular organelle dynamics during real-time efferocytosis. The completion of these studies will provide tools to dissect FAD during efferocytosis and highlight the importance of AC digestion in maintaining tissue, especially lung, homeostasis. Project Number: 1F31HL182129-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Achuth Nair | Institution: SLOAN-KETTERING INST CAN RESEARCH, NEW YORK, NY | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F05-A (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F31HL18212901