Neutrophil NADPH oxidase regulation by oxidative phosphorylation and accessory glucose metabolic pathways

Abstract

Neutrophils produce reactive oxygen species (ROS) to extinguish pathogens and regulate intracellular signaling. The major producer of ROS in neutrophils is the NADPH oxidase, which is fueled by NADPH generated via the oxidative pentose phosphate pathway. It remains unclear how other accessory pathways of glucose metabolism and mitochondrial activity influence the oxidative burst in neutrophils. The respiratory burst in bone marrow-derived neutrophils was stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of an inhibitor of glycogen breakdown (glycogen phosphorylase inhibitor; GPi), inhibitors of mitochondrial respiration (antimycin A and rotenone), or an inhibitor of serine biosynthesis (NCT503). Respiratory burst was also measured under conditions of limited glucose and glutamine. ROS production was measured via extracellular flux analysis or via dihydrorhodamine fluorescence. In both male and female mouse neutrophils, inhibition of glycogen breakdown by GPi delayed initiation of the oxidative burst in response to PMA (n = 6, p<0.05, Student’s T-test and Repeated Measure’s ANOVA). Inhibition of mitochondrial oxidative phosphorylation or inhibition of the serine biosynthetic pathway sustained ROS production in PMA-stimulated neutrophils (n = 6, p<0.05, Student’s T-test). Removal of glucose and glutamine showed a truncated oxidative burst (n = 6, p<0.05, Student’s T-test). Ultimately, these findings suggest that glycogen breakdown fuels the initial activation of the oxidative burst, while mitochondrial activity and the serine biosynthesis pathway control the magnitude and duration of neutrophil ROS production. Furthermore, utilization of exogenous glucose and glutamine are necessary for the entire oxidative burst. Future studies will continue to illustrate the mechanisms of neutrophil metabolic plasticity as well as connect their significance in the context of wound healing, exercise, and chronic inflammatory diseases. NIH: R01HL141191, NIH: P01 HL078825. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.