Fluorescent high-content imaging allows the discrimination and quantitation of E-LDL-induced lipid droplets and Ox-LDL-generated phospholipidosis in human macrophages.
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-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-LDL-loaded macrophages show increased intensity of NBD-PE and LipidTOX-Red detectable phospholipidosis in the endolysosomal compartment compared to E-LDL-loaded and M-CSF-differentiated macrophages. Treatment with the peroxisome proliferator-activated receptor-gamma 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.
Grandl
Margot
M
Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany.
Schmitz
Gerd
G
D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D005487:Foam Cells / Q000166:cytology; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008077:Lipoproteins, LDL / Q000737:chemistry*; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D009000:Monocytes / Q000166:cytology; D010100:Oxygen / Q000737:chemistry; D047495:PPAR gamma / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry*
D005456:Fluorescent Dyes; D008055:Lipids; D008077:Lipoproteins, LDL; D047495:PPAR gamma; D010743:Phospholipids; C099252:oxidized low density lipoprotein; D010100:Oxygen