Incorporation of Hydroxyeicosatetraenoic Acid Isomers into Macrophage Phospholipids Reveals Class-Specific Distribution.

Phospholipid fatty acid incorporation and remodeling are central processes through which immune cells adapt their membranes during activation. Macrophages are known to integrate oxidized fatty acids into phospholipids, yet the principles governing this distribution remain incompletely defined. Hydroxyeicosatetraenoic acids (HETEs) are abundant products generated during inflammation, and their integration into membrane phospholipids may influence signaling, trafficking, and membrane organization. Although individual HETE isomers differ in biosynthesis and function, it is not known whether macrophages handle them differently. Here, we address how 5-, 12-, and 15-HETE are incorporated into murine peritoneal macrophage phospholipids during inflammatory stimulation. We show that each isomer exhibits a distinctive phospholipid-class distribution, with 12-HETE preferentially entering choline phospholipids (PC), 15-HETE enriching phosphatidylinositol (PI), and 5-HETE distributing more broadly across PC, PI and ethanolamine phospholipids (PE). All three isomers are incorporated predominantly at the sn-2 position and showed similar molecular species distribution within each class, with diacyl PC, PE plasmalogens, and PI(18:0/HETE) serving as dominant acceptors. RAW264.7 cells reproduce these patterns. In ether phospholipid-deficient RAW.108 cells, incorporation into ether species is lost but compensated by increased routing into diacyl PC and PE, while PI incorporation remains unchanged. Collectively, these findings reveal that phospholipid class, not simple availability, determines where HETEs are incorporated. This distribution is preserved across macrophage cell types and remains intact even when ether phospholipids are absent, indicating that class specific pathways, rather than lipid subclass composition, primarily determine HETE incorporation.