Microscopic Bioimaging Divulges 7‐Ketocholesterol‐ Mediated Morphological Alterations, Inflammatory Responses and Dyshomeostasis in the Immune Reactions of Macrophages

ABSTRACT 7‐ketocholesterol (7KCh), a cytotoxic oxysterol derived from cholesterol oxidation, plays a pivotal role in the progression of lipid‐driven inflammatory diseases such as atherosclerosis. Among its pathogenic mechanisms, 7KCh is known to trigger oxiapoptophagy, a unique convergence of oxidative stress, apoptosis and autophagy. While macrophages typically serve as key phagocytic cells for clearing lipid debris, exposure to 7KCh disrupts this homeostatic role, contributing instead to dysfunctional foam cell formation. Despite its clinical relevance, the precise morphological and mechanistic effects of 7KCh on macrophages remain insufficiently characterized. In this study, we investigated the impact of 7KCh on IC‐21 macrophages using a comprehensive, multimodal imaging strategy that integrates phase contrast, differential interference contrast (DIC), fluorescence microscopy and quantitative confocal imaging. Exposure to increasing concentrations (2–10 μg/ml) of 7KCh for 24 h revealed distinct morphological alterations, including cytoplasmic vacuolization, disrupted cellular architecture and pronounced lipid accumulation. Oil red O staining confirmed the accumulation of neutral lipids, while functional assays demonstrated impaired pinocytosis and phagocytosis, hallmark features of foam cell transformation. These findings were further supported by molecular analysis indicating induction of pro‐inflammatory markers and oxiapoptophagy signatures. Altogether, this work provides novel insight into the cytotoxic and inflammatory transformation of macrophages by 7KCh and highlights the power of advanced microscopy in delineating lipid‐induced immune dysfunction. Our imaging‐based approach offers a valuable platform for future investigations into oxysterol‐mediated pathologies and therapeutic targeting of foam cell dynamics.