Proteomics analysis reveals headgroup-dependent modulation of lipopolysaccharide-induced inflammation by arachidonoyl...

Aminophospholipids, including phosphatidylserines (PS) and phosphatidylethanolamines (PE), are major components of the inner leaflet of cellular membranes with emerging immunomodulatory functions, yet the contribution of their polar headgroups remains poorly defined. To address this, we compared PS16:0/20:4 and PE16:0/20:4, two phospholipids with identical fatty-acyl composition, in resting and lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages using LC-MS/MS proteomics, gene set enrichment analysis, and targeted qPCR. Among the 1611 proteins retained for statistical analysis, both phospholipids produced distinct basal proteomic profiles and substantially modified the subsequent response to LPS. A core of 667 LPS-responsive proteins was preserved across control, PS-, and PE-supplemented macrophages, while interaction analyses revealed marked headgroup-dependent differences in response magnitude. These differences mapped onto distinct pathways. PS supplementation was associated with vesicle trafficking, lipid recognition, redox regulation, and ER proteostasis, whereas PE preferentially affected glutathione and redox-related proteins. Both phospholipids reduced selected inflammatory markers and LPS-induced nitric oxide production. Together, these findings demonstrate headgroup-dependent modulation of macrophage proteomic responses by matched arachidonoyl phospholipids, underscoring the functional relevance of phospholipid headgroup. Significance: Membrane phospholipids are increasingly recognized as active regulators of immune cell function, yet the contribution of the polar headgroup remains poorly defined. By directly comparing PS16:0/20:4 and PE16:0/20:4, this study shows that two native phospholipids carrying identical acyl chains generate distinct macrophage proteomic responses. Both lipids remodel the resting proteome and modify subsequent LPS responsiveness, but with different associations with redox regulation, membrane trafficking, ER proteostasis, and inflammatory signalling. These findings advance our understanding of the role of phospholipid headgroup as an important modulator of macrophage responses and support the development of phospholipid-based approaches to modulate inflammation and immunometabolism.