Phase partitioning of the neutrophil oxidative burst is coordinated by accessory pathways of glucose metabolism and mitochondrial activity

Neutrophils are a part of the innate immune system and produce reactive oxygen species (ROS) to extinguish pathogens. The major source of ROS in neutrophils is NADPH oxidase, which is fueled by NADPH generated via the pentose phosphate pathway; however, it is unclear how other accessory glucose metabolism pathways and mitochondrial activity influence the respiratory burst. We examined the temporal dynamics of the respiratory burst and delineated how metabolism changes over time after neutrophil activation. Bone marrow-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate, and the respiratory burst was measured via extracellular flux analysis. Metabolomics experiments utilizing ¹³C₆-glucose highlighted the activation of glycolysis as well as ancillary pathways of glucose metabolism in activated neutrophils. Phorbol 12-myristate 13-acetate stimulation acutely increased ¹³C enrichment into glycerol 3-phosphate (G3P) and citrate, whereas increases in ¹³C enrichment in the glycogen intermediate, UDP-hexose, and end products of the hexosamine and serine biosynthetic pathways occurred only during the late phase of the oxidative burst. Targeted inhibition of the G3P shuttle, glycogenolysis, serine biosynthesis, and mitochondrial respiration demonstrated that the G3P shuttle contributes to the general magnitude of ROS production; that glycogen contributes solely to the early respiratory burst; and that the serine biosynthetic pathway activity and complex III-driven mitochondrial activity influence respiratory burst duration. Collectively, these results show that the neutrophil oxidative burst is highly dynamic, with coordinated changes in metabolism that control the initiation, magnitude, and duration of ROS production.