Monophosphoryl lipid A boosts macrophage antimicrobial immunity by metabolically regulating source-specific ROS generation

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.