Inflammatory processes are central to the progression of numerous chronic conditions, including cardiovascular and metabolic disorders, with macrophages playing a pivotal role in these responses. Monounsaturated fatty acids, including palmitoleic acid (16:1 n − 7), have been implicated in modulating inflammation, yet their precise molecular mechanisms of action remain incompletely understood. Notably, in macrophages, 16:1 n − 7 is preferentially esterified into a specific phosphatidylcholine (PC) species, PC(16:0/16:1 n − 7), raising the possibility that its biological activity is governed by this lipid-bound form. Here, we demonstrate that the anti-inflammatory effects of 16:1 n − 7 in macrophages are mediated through its incorporation into this PC species. Using synthetic phospholipids and multiple activation stimuli, we show that PC(16:0/16:1 n − 7) directly regulates macrophage activation. It suppresses NF-κB signaling, reprograms gene expression, and promotes a shift toward an anti-inflammatory, M2-like phenotype that enhances phagocytic capacity. These effects are preserved in ether analogs resistant to phospholipase-mediated hydrolysis, confirming that the release of free 16:1 n − 7 is not required. These findings reveal a previously unrecognized lipid-driven mechanism of immunomodulation, in which specific structural features of PC(16:0/16:1 n − 7) confer intrinsic bioactivity. Our study broadens understanding of immunometabolic regulation by membrane phospholipids, and provides a mechanistic basis for the pharmacotherapeutic potential of defined lipid species in reprogramming macrophage function in inflammatory diseases.
