Glutathione reductase (Gsr) regulates cell metabolism and signaling during neutrophil-pathogen interactions

Project Summary/Abstract During bacterial or fungal infections, neutrophils engulf the pathogens by phagocytosis and initiate a signaling process leading to the activation of NADPH oxidase. NADPH oxidase utilizes NADPH as an electron donor to produce superoxide (O2●-), which is subsequently converted to H2O2 and highly microbicidal hypochlorous acid. NADPH is produced from glucose metabolism primarily through the pentose phosphate pathway. Defects in the subunits of the NADPH oxidase and the pentose phosphate pathway are associated with chronic granulomatous disease. Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione (GSH) utilizing NAPDH as an electron donor. GSH protects cytoplasmic components from oxidative damage by reactive oxygen species (ROS). Rare patients deficient in GSR activity have been reported to have slower recovery after infections, hemolysis after consuming fava beans, and cataracts at a young age. Gsr-deficient (Gsr-/-) mice with a deletion from intron 1 to intron 5 in the Gsr gene failed to contain bacterial and fungal infections, despite the recruitment of larger number of leukocytes to the sites of infections and production of greater amounts of cytokine and chemokines than wildtype mice. Gsr-/- neutrophils phagocytosed fewer pathogen particles upon encountering either E. coli or C. albicans than did wildtype neutrophils. Gsr-/- neutrophils also produced substantially less ROS and had attenuated pentose phosphate pathway, and exhibited lower microbicidal activity and markedly greater protein tyrosine phosphorylation than did wildtype neutrophils. We hypothesize that during neutrophil-pathogen interaction, Gsr protects the enzymes in the glucose metabolic pathways from oxidative damage thus facilitating the production of ATP for phagocytosis and NADPH for respiratory burst. We postulate that Gsr also facilitates the microbicidal program by regulating protein phosphorylation. We will test these hypotheses through two Specific Aims. Aim 1 will assess contribution of hematopoietic, neutrophil, vs parenchymal/stromal cell Gsr to host defense against bacterial and fungal pathogens using bone marrow swaps between wildtype and Gsr-/- mice, adaptive transfer of wildtype neutrophils into Gsr-/- mice, and Gsr restauration in hematopoietic stem cells of Gsr-/- mice. Aim 2 will assess the effects of Gsr deficiency on: 1) glucose metabolism (glycolysis, Krebs cycle, and pentose phosphatase pathway) through metabolomics and Seahorse assays; 2) protein oxidation via OxiCAT and protein phosphorylation through proteomics. These studies will identify the proteins that are dependent on Gsr for protection against oxidative damage during neutrophil- pathogen interactions and reveal the critical function of Gsr in facilitating ROS production, cellular metabolism, and cell signaling during the execution of the microbicidal program. Successful completion of these specific aims will uncover the underlining mechanisms by which Gsr regulates phagocytic functions.