Comprehensive Lipidomic Profiling Reveals Distinct Metabolic Remodeling during Differentiation of Human Monocyte-Derived Macrophages.

Macrophages are central players of innate immunity with diverse functions and involvement in numerous diseases, making it essential to understand their metabolic regulation. Here, we provide a comprehensive analysis of lipid metabolic dynamics during human monocyte-to-macrophage differentiation. Primary blood monocytes were differentiated for 5 days with either M-CSF or GM-CSF. Lipidomic profiling using the differential mobility spectrometry-shotgun lipidomics assistant (DMS-SLA) platform reliably quantified ∼400 lipids across 16 classes, while spectral flow cytometry was used to assess metabolic enzyme expression. Differentiation was marked by remodeling toward membrane lipids, accompanied by shorter acyl chains and reduced unsaturation in triglyceride (TG) and phosphatidylcholine (PC) species. PC species incorporated a more diverse range of pro- and anti-inflammatory precursors, whereas TG species mainly incorporated fatty acid FA 22:6. Metabolic enzyme expression showed dynamic, marker-specific changes over time, with G6PD and SDHA upregulation indicating enhanced pentose phosphate and oxidative phosphorylation pathways. M-CSF MDMs exhibited higher GLUT1 and CD36 expression and greater FA 22:6 incorporation within TGs. Together, these findings reveal extensive lipidome remodeling that underlies macrophage differentiation and distinct functional states.