NADPH oxidase isoform NOX-2 deficiency affects mitochondrial oxygen consumption and metabolic flexibility

Our recent study showed that the intracellular pattern recognition receptor Nlrp3 inflammasome, activated by reactive oxygen species (ROS), regulates metabolism in hematopoietic cells by maintaining proper "tonic activation" of the electron transport chain (ETC) in mitochondria. Therefore, we asked whether a deficiency in the expression of NADPH oxidase isoform NOX-2, a primary source of ROS in hematopoietic cells, would similarly affect mitochondrial function and metabolic adaptation to stress. In this study, we examined how NOX-2 influences ETC, redox balance, and glycolytic adaptation in lineage-negative Sca-1⁺c-Kit⁺ (SKL) bone marrow stem cells from wild-type (WT) and NOX-2 knockout (NOX2-KO) mice. Metabolic testing revealed that NOX2-KO cells have impaired mitochondrial respiration, lower ATP production, and reduced spare respiratory capacity. When exposed to hydrogen peroxide, NOX2-KO cells failed to activate a mitochondrial stress response and instead relied more on anaerobic glycolysis. Treatment with extracellular ATP (eATP), the most abundant signaling alarmin, increased ROS levels in WT cells but not in NOX2-KO cells, emphasizing eATP-NOX-2's role in redox regulation during stress conditions. Proteomic analysis identified proteins differentially expressed related to hypoxia, glycolysis, and oxidative stress response between WT and NOX2-KO SKL cells. These results demonstrate NOX-2 as a key regulator of mitochondrial function and metabolic flexibility in hematopoietic stem cells, highlighting its importance in maintaining redox balance during stress conditions.