Formation of cholesterol crystals (CCs) is implicated in atherogenesis, but the cellular mechanisms that degrade CCs inside macrophages have been incompletely understood. Using labeled crystals and advanced imaging, the authors identify the lysosomal pathway—specifically late endosomes and lysosomes (LE/Lys)—as the central compartment where CCs are processed and their cholesterol content mobilized.
The study reports that macrophages internalize CCs and route sterol from crystals into intracellular membranes and storage compartments. This processing culminates in the generation of lipid droplets (LDs) that contain cholesterol derived from dissolved crystals. These observations support a model in which LE/Lys act as the primary site for CC dissolution and subsequent cholesterol trafficking within macrophages.
To visualize CC handling, the investigators incorporated the fluorescent cholesterol analogue TopFluor-Cholesterol (TF-Chol) into crystals. This labeling enabled correlative imaging using fluorescence microscopy together with soft X-ray microscopy, providing complementary contrast and localization. In parallel, quantitative 3D live-cell imaging was used to follow crystal uptake and intracellular fate over time.
These multimodal approaches allowed direct visualization of crystal uptake, localization to LE/Lys, and the appearance of TF-Chol signals in newly formed LDs. The combination of fluorescence labeling and soft X-ray imaging provided structural and chemical context for the intracellular processing events described.
Time-resolved 3D imaging showed that after CC uptake, TF-Chol is transferred into intracellular compartments identified as late endosomes and lysosomes and subsequently appears in lipid droplets. The data indicate a sequence of events in which crystal-derived sterol becomes bioavailable in LE/Lys and is then trafficked into neutral lipid stores.
The appearance of TF-Chol–enriched LDs over time provides a readout for successful intracellular dissolution and redistribution of cholesterol originally present in crystals.
The authors tested pharmacologic interventions that perturb lysosomal cholesterol handling. Treatment with U18666A, an inhibitor of lysosomal sterol export, caused accumulation of TF-Chol in LE/Lys, consistent with impaired egress of cholesterol from these compartments.
Inhibition of lysosomal acidification with bafilomycin A1 produced reduced dissolution of CCs, indicating that lumenal acidic conditions in lysosomes contribute to crystal breakdown. Together, these manipulations support a functional requirement for intact lysosomal activity—both export mechanisms and acidification—in processing and dissolving CCs.
Using a novel assay combined with 3D image processing, the study examined how larger CCs that contact macrophages are handled. The data indicate that such crystals are processed via lysosomal exocytosis, after which both extracellular and intracellular degradation of CCs occurred. This pathway suggests a mechanism whereby macrophages can act at the cell surface through lysosome-mediated secretion, followed by crystal dissolution that involves extracellular and intracellular steps.
The assay and image analysis highlighted the dynamic interactions between the plasma membrane, exocytosed lysosomal content, and crystal material during the degradation process.
Treatment of macrophages with a fluorescently labeled cyclodextrin (CD) enhanced dissolution of CCs and increased formation of TF-Chol–containing LDs. Fluorescent CD largely co-localized with a marker for LE/Lys during the process, suggesting that intracellular delivery of CD to LE/Lys may support the dissolution of crystals within these compartments.
These findings indicate that CD can act at least in part via intracellular routes to promote CC solubilization and redistribution of sterol into lipid storage compartments.
To complement TF-Chol and cellular imaging experiments, the authors used dehydroergosterol (DHE), an intrinsically fluorescent sterol that self-associates into aggregates and crystals. Using fluorescence spectroscopy and specialized ultraviolet (UV) microscopy, they observed that CD enhances dissolution of DHE crystals in vitro and in cells. These orthogonal assays support the conclusion that CD can facilitate solubilization of sterol crystals and that this effect is observable across model sterols and experimental systems.
The combined imaging, pharmacologic perturbation, and biochemical assays in this preprint identify the lysosomal pathway—including late endosomes and lysosomes and processes such as lysosomal acidification, sterol export, and exocytosis—as central to the dissolution of cholesterol crystals taken up by macrophages. Cyclodextrin promotes crystal dissolution and appears to act, at least partially, via delivery to LE/Lys.
These results position lysosomal function as a key determinant of how macrophages clear crystalline cholesterol and redistribute sterol into lipid droplets. The authors note that the work is presented as a preprint and has not undergone peer review. Details about experimental conditions, quantitative metrics, and additional methodological specifics are reported in the source article and its supplementary materials.