Wdfy3 promotes macrophage efferocytosis via accelerating the uptake and degradation of dying cells

Efferocytosis is the phagocytic clearance of apoptotic cells (ACs) by phagocytes, mainly macrophages. Defective efferocytosis leads to unresolved inflammation, driving important diseases including autoimmune diseases and atherosclerosis, while enhancing efferocytosis has therapeutic potential. Therefore, discovering novel regulators and mechanisms of macrophage efferocytosis as a therapeutically targetable pathway has broad impact on many diseases. Our lab has recently performed a genome-wide CRISPR screening for novel genes essential for the uptake of ACs using primary macrophages. WDFY3 was identified and validated as a top hit that has never been shown to regulate efferocytosis. The goal of the proposal is to address how WDFY3 regulates macrophages efferocytosis and whether enhancing WDFY3 can be beneficial. My preliminary data suggest that both the uptake and degradation of the engulfed ACs were impaired in Wdfy3-deficient macrophages. I hypothesize that WDFY3 regulates multiple stages of macrophage efferocytosis likely through distinct mechanisms. Using bone-marrow derived macrophages (BMDM) and mice with myeloid-specific Wdfy3 knockout (LysMCre+/-Wdfy3fl/fl) and transgenic overexpression of human WDFY3 (LysMCre+/-hWDFY3Tg), as well as human THP-1 macrophages and monocyte-derived macrophages (HMDM) with siRNA knockdown or lentiviral overexpression. Aim 1 will determine how WDFY3 regulates the uptake of ACs by macrophages. I will test whether Wdfy3 knockout leads to delayed or impaired phagocytic cup formation and sealing; whether Wdfy3 knockout decreases, yet WDFY3 overexpression enhances AC uptake in vivo by both thymus and peritoneal macrophages. Aim 2 will determine how WDFY3 regulates the degradation of ACs by macrophages. I will test how WDFY3 translocates to the engulfed ACs to form phagolysosome and regulate lysosomal acidification and degradation; and effects of impaired WDFY3-mediated AC degradation on inflammation in vivo; whether there is direct interaction between WDFY3 and LC3/GABARAP and other proteins to promote LC3 lipidation through specific molecular domains of WDFY3. This study will (1) reveal novel fundamental mechanisms of efferocytosis regulated by Wdfy3 discovered through unbiased screen; (2) provide deep mechanistic insights into the role of Wdfy3-mediated efferocytosis in vivo and its potential as a target for pro-efferocytotic therapy.