Lipid droplet profiling during neutrophil differentiation by stimulated Raman scattering microscopy

Abstract

Lipid droplets (LDs) are dynamic organelles that serve as metabolic hubs and emerging regulators of immune cell fate. Although LDs have been implicated in immune regulation, how LD metabolism is remodeled during neutrophil differentiation and how stage‐specific LD dynamics shape mature neutrophil function remain poorly defined. Here, we profiled LD dynamics during neutrophil development and evaluated their role in innate immune function. Using stimulated Raman scattering (SRS) microscopy, we performed label‐free, quantitative mapping of LD accumulation in two complementary differentiation systems: murine Hoxb8 myeloid progenitors and human pluripotent stem cell (hPSC)‐derived neutrophils. To define metabolic requirements, we pharmacologically modulated LD biosynthesis and catabolism throughout neutrophil differentiation. Perturbing LD metabolism did not impair differentiation efficiency or lineage commitment, but significantly altered the functional output of mature neutrophils. In the murine system, inhibition of adipose triglyceride lipase (ATGL)‐mediated LD breakdown enhanced reactive oxygen species (ROS) production and increased anti‐tumor cytotoxicity against GL261 glioma cells. In the hPSC model, ATGL inhibition during the myeloid progenitor‐to‐neutrophil transition selectively increased intracellular LD accumulation without compromising neutrophil yield or purity. This metabolic rewiring also elevated ROS production in hPSC‐derived neutrophils, although cytotoxic enhancement against U87MG glioblastoma cells was less pronounced than in the murine system. Collectively, these findings define a stage‐specific LD metabolic landscape during neutrophil development and highlight targeted LD modulation as a potential strategy to enhance the functional potency of therapeutic neutrophils.