A Rosella-PLIN2 knock-in mouse reveals lipophagy and immunometabolic interplay in atherosclerosis

Cytosolic lipid droplets (LDs) regulate lipid homeostasis, with abnormal LD dynamics linked to metabolic diseases like atherosclerosis. In macrophage foam cells, LDs undergo autophagic degradation via lipophagy, but the extent of this process in vascular smooth muscle cell (VSMC) foam cells remains unclear. To track lipophagy in real time, we developed a Rosella-PLIN2 (perilipin 2) biosensor by tagging PLIN2 with the fluorescent pH-biosensor Rosella. We show that proatherogenic lipoproteins and autophagy activators stimulate lipophagy in human macrophages. Targeting LDs with an LC3 fusion protein or LD-autophagy tethering compounds (LD-ATTECs) selectively enhanced lipophagy, promoting foam cell LD clearance. In an atherosclerosis model, Rosella-PLIN2 accurately tracked lipophagy in arterial foam cells, revealing distinct PLIN2 expression patterns in macrophage and non-leukocyte foam cells. We identified a lipophagy deficiency in VSMC foam cells and demonstrate that enhancing lipophagy promotes LD catabolism in primary VSMC foam cells. TREM2⁺ macrophages exhibited high lipid content and low lipophagy flux, whereas TREM2^- macrophages had low lipid content and high lipophagy flux. Our findings highlight a cell-specific interplay between lipophagy and immunometabolism in arterial foam cells, unveiling novel therapeutic avenues for atherosclerosis. Additionally, the Rosella-PLIN2 model provides a powerful tool for studying LD metabolism, offering new insights into lipid homeostasis and disease mechanisms.Abbreviations: AgLDL: aggregated LDL; Baf-A1: bafilomycin A₁; BMDMs: bone marrow-derived macrophages; HDL: high-density lipoproteins; LD: lipid droplets; LD-ATTECs: LD-autophagy tethering compounds; LDL: low-density lipoproteins; LIPA/LAL: lysosomal acid lipase A; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LTA: lipoteichoic acid; MFI: mean fluorescence intensity; OA: oleic acid; p-ATG16L1: phospho-ATG16L1; PLIN2: perilipin 2; PLINs: perilipins; RCT: reverse cholesterol transport; TLR: toll-like receptor; VSMCs: vascular smooth muscle cells; WSC: water-soluble cholesterol.