Phospholipase D1 activates NAD kinase fuelling neutrophil extracellular trap formation

Abstract Neutrophil extracellular trap formation is a central innate immune defence mechanism that depends on NADPH oxidase–driven reactive oxygen species production. How NADP⁺ availability is regulated during neutrophil activation remains unclear. Using integrated lipidomic, biochemical and functional analyses in human neutrophils, we identify NAD kinase, the sole enzyme responsible for NADP⁺ synthesis, as a direct lipid-regulated effector downstream of phospholipase D1. Neutrophil activation induces rapid, phospholipase D1-dependent production of phosphatidic acid and phosphatidylinositol 4,5-bisphosphate, which bind and activate NAD kinase. The resulting cytosolic NADP⁺ is reduced to NADPH by the oxidative pentose phosphate pathway and consumed by NADPH oxidase during neutrophil extracellular trap formation. Pharmacological or genetic disruption of phospholipase D1 suppresses NAD kinase activity, depletes NADP⁺ and impairs neutrophil extracellular trap formation independently of direct NADPH oxidase inhibition. These findings define a lipid–metabolic axis coupling phospholipase D1-driven membrane remodelling to NADP⁺ availability and innate immune function.