CD36-Pyruvate Kinase M2 Signaling Promotes Macrophage Phagocytosis Through Mitochondrial Reactive Oxygen Species

OBJECTIVE Macrophage phagocytosis function is critical for tissue homeostasis, but excessive phagocytosis leads to lipid overloading and foam cell formation, a hallmark of atherosclerosis. However, regulatory mechanisms of phagocytosis under atherogenic conditions remain poorly understood. We previously reported that macrophage oxLDL/CD36 signaling drives mitochondrial reactive oxygen species (mtROS) production during atherosclerosis initiation. Here we aim to determine the role of CD36-mtROS axis in regulation of phagocytosis and explore the underlying molecular mechanism, which could provide new potential therapeutic targets. METHODS AND RESULTS Feeding macrophages with a variety of large particles including pHrodo-conjugated E.coli, green fluorescence beads, and fluorescence-labeled apoptotic cells, we showed that oxLDL upregulated phagocytosis efficiency in macrophages. This effect was dependent on CD36 as it was attenuated in Cd36 -null macrophages. Through use of mtROS scavenger MitoTEMPO, MCAT transgenic strategy and antioxidant signaling stimulation, we observed that mtROS were required for oxLDL-upregulated phagocytosis. Using the atherosclerosis-prone Apoe -null mouse model, we found that aortic foamy macrophages displayed both elevated levels of mtROS and phagocytosis functions during the initiation of atherosclerosis. Employing a combination of co-immunoprecipitation, mass spectrometry, genetic knockdown by siRNA, and small chemical inhibitors, we identified a cytosolic enzyme PKM2 downstream of oxLDL/CD36 signaling, which translocated to the mitochondria with the assistance of a chaperone GRP75. Subsequently, mitochondrial PKM2 bound to the electron transport chain Complex III, potentially facilitating mtROS production. CONCLUSIONS These findings reveal a novel CD36-PKM2-mtROS pathway in macrophages that could lead to excess phagocytosis during the initiation of atherosclerosis. ### Competing Interest Statement The authors have declared no competing interest. * AC : apoptotic cell bodies 2-DG : 2-deoxy-D-glucose ETC : electron transport chain FSC : forward scatter HFD : high fat diet HMDM : human monocyte-derived macrophages IP : immunoprecipitation MCAT : mitochondrial-targeted human catalase expression MFI : mean fluorescence intensity mtROS : mitochondrial reactive oxygen species oxLDL : oxidized low-density lipoprotein PKM2 : pyruvate kinase muscle 2 PLA : proximity ligation assay RT : room temperature scRNA-seq : single-cell RNA sequencing Trem2 : triggering receptor expressed on myeloid cells 2 TUNEL : terminal deoxynucleotidyl transferase dUTP nick end labeling UMAP : uniform manifold approximation and projection WT : wild type