Abstract
Introduction: Enhancing macrophage efferocytosis represents a potential therapeutic strategy for residual risk reduction in atherosclerotic cardiovascular disease. Our genome-wide CRISPR screening discovered Pdcd6ip as a top-ranked negative regulator of efferocytosis. PDCD6IP, also known as ALIX, is an accessory protein of ESCRT machinery, regulating multivesicular body formation and cytokinesis. Yet, how PDCD6IP may act as a molecular break on macrophage efferocytosis has not been characterized. Goals: Determine if knockout of Pdcd6ip enhances macrophage efferocytosis and protects against atherosclerosis pathogenesis. Methods: In vitro efferocytosis assays were performed in bone marrow-derived macrophages (BMDMs) and human monocyte-derived macrophages (HMDM). In vivo studies involved Ldlr -/- recipient mice with bone marrow transplantation (BMT) of WT and Pdcd6ip -/- BMs. Results: Pdcd6ip -/- induces cytokinesis arrest in proliferating BMDMs, resulting in an increased formation of binuclear polyploid BMDMs. Binuclear BMDMs have a remarkable capacity to engulf multiple apoptotic cells (ACs), termed continuing efferocytosis. While both mononuclear and binuclear Pdcd6ip -/- BMDMs demonstrate enhanced efferocytosis in primary efferocytosis, binuclear Pdcd6ip -/- BMDMs show enhanced continuing efferocytosis compared to binuclear WT BMDMs. Pdcd6ip -/- BMDMs have increased phagolysosomal acidification of the engulfed ACs, implicating proper degradation. Pdcd6ip -/- did not alter the engulfment of live cells, beads, or zymosan, supporting specific regulation of AC clearance. We hypothesize that Pdcd6ip -/- will be protective in atherosclerosis. Indeed, BMT of Pdcd6ip -/- BMs into Ldlr -/- mice led to increased atherosclerotic plaque stability after 22 weeks of Western diet feeding, characterized by decreased necrotic core area and increased fibrous cap thickness, without affecting the overall lesion area. Knockdown of PDCD6IP in HMDMs also led to an increased formation of binuclear HMDMs and enhanced continuing efferocytosis. Conclusion: We discovered a new role of PDCD6IP as a molecular break of macrophage efferocytosis. Inhibiting macrophage PDCD6IP may represent a therapeutic opportunity in atherosclerosis.
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