Extensive cholesterol accumulation in human macrophages inhibits lysosome function; a key attribute of many late-stage atherosclerosis foam cells. Lipophagy (a specialized form of autophagy) delivers lipids, like cholesteryl esters, to lysosomes where the esters are hydrolyzed to unesterified cholesterol which can be utilized in membranes or effluxed from the cell. Lipophagy has been shown to be an important pathway in foam cell sterol metabolism. We investigated what occurs to lipophagy when lysosome function is disrupted by excess cholesterol. Does autophagy continue? Does autophagy transport lipid to non-functioning lysosomes and thus contribute to overall lysosomal dysfunction and lysosomal sterol accumulation? In macrophages with dysfunctional lysosomes, how does inhibition of autophagy effect overall sterol balance and macrophage function?. We incubated human macrophages with aggregated LDL to produce macrophages with various degrees of lysosome dysfunction (confirmed by lysosome pH measurement and cholesteryl ester hydrolysis). To assess the role of lipophagy in changes in cellular sterol metabolism, we compared lipid metabolism as lysosomes became more dysfunctional and also with identical cells in which autophagy was inhibited with vinblastin. Increases in lysosome dysfunction did not alter the constitutive rate of autophagy as measured by proteins critical to autophagosome assembly (Atg7, Atg5, p62, LC3) and visualization of autophagosomes. Moreover, autophagy targeted lipid droplet material to lysosomes as indicated by the progressive accumulation of perilipin 2 in lysosomes and a significant decrease in lysosome cholesteryl ester content when autophagy was inhibited. In cells with normal functioning lysosomes, inhibition of autophagy increased total cell sterol levels. However, in cells where lysosomal dysfunction was prominent, lysosomal levels of sterol decreased and efflux of cholesterol was slightly enhanced when autophagy was inhibited. Our data indicates that lipophagy continues in the face of lysosome dysfunction and autophagy-delivered cholesteryl ester can exacerbate overall lysosome sterol accumulation and macrophage dysfunction when foam cell sterol is excessive.
Alveolar macrophage dysfunction in malignant lung tumours.
