Fluorescent high‐content imaging allows the discrimination and quantitation of E‐LDL‐induced lipid droplets and Ox‐LDL‐generated phospholipidosis in human macrophages

Abstract

Macrophage foam cells formed during uptake of atherogenic lipoproteins are a hallmark of atherosclerotic lesion development. In this study, human macrophages were incubated with two prototypic atherogenic LDL modifications enzymatically degraded LDL (E-LDL) and oxidized LDL (Ox-LDL) prepared from the same donor LDL. To detect differences in macrophage lipid storage, fluorescent high-content imaging was used. Lipid droplets were stained using Bodipy 493/503, and the fluorescent phospholipid probe NBD-PE was used to detect endolysosomal phospholipidosis in high-content imaging assays. The phospholipidosis assay was validated using phospholipidosis-inducing cationic amphiphilic drugs. In addition, neutral lipids and phospholipidosis were determined using LipidTOX. Images of 96-well cell culture microtiter plates were captured with multichannel laser-based high-content confocal microscopy, and subsequently cell-and well-based data were analyzed. E-LDL-loaded macrophages show increased intensity of Bodipy 493/503 and LipidTOX TM -Green neutral lipid droplet staining and a greater mean area and number of lipid droplets per cell compared to Ox-LDL-loaded and M-CSF-differentiated control macrophages. In contrast, Ox-LDLloaded macrophages show increased intensity of NBD-PE and LipidTOX TM -Red detectable phospholipidosis in the endolysosomal compartment compared to E-LDL-loaded and M-CSF-differentiated macrophages. Treatment with the peroxisome proliferatoractivated receptor-c agonist pioglitazone leads to lipid droplet induction depending on the lipid loading state of the macrophages. These results indicate that E-LDL preferentially induces lipid droplets, while Ox-LDL provokes endolysosomal phospholipidosis in human macrophages representing two different lipid storage principles. Therefore, fluorescent high-content imaging is a useful tool to discriminate between and quantify lipid storage compartments in macrophages also in response to drugs affecting cellular lipid metabolism. ' International Society for Advancement of CytometryKey terms E-LDL; Ox-LDL; human macrophages; lipid droplets; phospholipidosis MONOCYTE derived macrophages and lipid-loaded macrophage foam cells play a central role in the initiation and progression of atherosclerotic lesions. Human blood monocytes are a disease relevant tool to unravel lipid storage and release mechanisms, and differential processing of atherogenic lipoproteins like enzymatically modified LDL (E-LDL) or oxidized LDL (Ox-LDL). In macrophages treated in vitro with Ac-LDL or E-LDL, much of the internalized cholesterol accumulates in cytoplasmic cholesterylester containing lipid droplets (1,2) as is the case also in atherosclerotic lesions (3,4). Lipid droplets form the major lipid store in mammalian cells and serve as a reservoir of cholesterol and acyl-glycerols for the synthesis and maintenance of membranes. Lipid droplets consist of a core of neutral lipids, predominantly triacylglycerols or cholesterylesters, that are surrounded by a monolayer of phospholipids

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