Abstract
INTRODUCTION: Monophosphoryl lipid A (MPLA), a toll-like receptor (TLR) 4 agonist and licensed vaccine adjuvant, reprograms innate immune cells to confer protection against diverse pathogens. However, the metabolic and molecular adaptations supporting this response remain poorly defined. METHODS: The contributions of discrete reactive oxygen species (ROS) sources-including NADPH oxidase 2 (NOX2), xanthine oxidase (XO), mitochondria, and inducible nitric oxide synthase (iNOS)-to MPLA-induced macrophage antimicrobial activity were examined using genetic deletion or pharmacologic inhibition. Metabolic and redox adaptations supporting this response were assessed by analyzing oxidative pentose phosphate pathway (oxPPP) activity, glutathione-dependent antioxidant systems, and mitochondrial oxidative phosphorylation in MPLA-primed macrophages. RESULTS: MPLA enhanced macrophage clearance of Pseudomonas aeruginosa by coordinating source-specific ROS generation. NOX2 was essential for this response, as its pharmacologic inhibition or genetic deletion markedly diminished MPLA-induced microbicidal responses. MPLA also induced XO, providing auxiliary ROS that acted additively with NOX2-derived ROS to support bacterial clearance. MPLA activated the oxPPP to generate NADPH, which was essential for supporting phagocytosis and maintaining glutathione-dependent redox homeostasis. Additionally, MPLA promoted mitochondrial oxidative phosphorylation to sustain phagocytic capacity. Mitochondrial ROS (mROS) were tightly constrained by induction of antioxidant systems, including superoxide dismutase 2 (SOD2), heme oxygenase-1 (HO-1) and glutathione, and were dispensable for antimicrobial protection. iNOS-derived nitric oxide did not contribute to the MPLA-induced antimicrobial phenotype. CONCLUSION: These findings define the metabolic and redox circuits driving MPLA-induced antimicrobial immunity and establish its potential as a host-directed antimicrobial therapy beyond vaccine adjuvancy.