Watanabe_2026_Cell.Commun.Signal__

Background Lipid droplets (LDs) are vital organelles in cellular energy regulation, controlling lipid storage and mobilization. However, their specific roles in macrophage function, particularly in the context of immune regulation, remain elusive. This study aimed to elucidate how LD manipulation affects macrophage phenotypes and functions, with a focus on their role in efferocytosis and inflammatory modulation. We also explored the novel function of hormone-sensitive lipase (HSL) in regulating LD metabolism and macrophage polarization. Methods Human monocytes were differentiated into M1 (IFN-/LPS) or M2 (IL-4) macrophages. To determine the functional role of HSL, we employed pharmacological inhibition, siRNA-mediated knockdown, and plasmid-driven overexpression. Phenotypic and functional effects on efferocytosis, gene expression, and cytokine secretion were evaluated using flow cytometry, qRT-PCR, and ELISA. The underlying mechanisms were explored through unbiased quantitative lipidomics, real-time metabolic flux analysis to assess mitochondrial respiration, and high-resolution imaging via confocal and electron microscopy, which confirmed alterations in the LD dynamics. Results M2 macrophages exhibited increased efferocytosis and contained smaller, more numerous lipid droplets with greater lipase activity compared to M1 macrophages. Inhibition or siRNA-mediated knockdown of HSL reduced efferocytosis and downregulated the expression of M2 markers, including CD206, CD163, CD200R1, and Annexin A1, while promoting a pro-inflammatory state. Conversely, HSL overexpression in naive macrophages induced a strong M2-like phenotype, characterized by increased efferocytosis and elevated mitochondrial respiration. We highlighted the key role of the cAMP-protein kinase A pathway in promoting lipolysis of LD via HSL activation. Lipidomic analysis revealed distinct lipid profiles for M1 and M2 macrophages, as well as for M2 macrophages with HSL inhibition. Lipidomic results indicated that M2 macrophages contain specific monounsaturated fatty acids, such as Nervonic Acid (FA 24:1), which can enhance efferocytosis by M1 macrophages. Conclusions This study identifies a novel mechanism by which LD lipolysis, mediated by HSL, promotes M2 macrophage polarization and enhances efferocytosis. These findings highlight the importance of LD metabolism in macrophage function and suggest HSL as a potential therapeutic target for immune-mediated conditions.