Macrophage and Monocyte Metabolic Adaptation to Hemolysis and Sickle Cell Disease PROJECT SUMMARY Hemolysis is a unifying feature of a diverse set of pathologies including inherited disorders such as sickle cell disease (SCD), infectious diseases including malaria, systemic pathologies such as sepsis, as well as drug toxicity and autoimmune disease. The release of free heme causes oxidative damage, leading to immune cell activation, endothelial damage, ischemia, and end-organ toxicity. Innate immune cells such as macrophages are specialized in heme detoxification, and while it is now evident that macrophages alter their cellular metabolism in order to carry out specific effector functions, the metabolic adaptations that allow macrophages to survive the toxic stress of heme clearance remain unknown. Preliminary data from our group reveal that in response to heme loading macrophages shift glucose metabolism toward the pentose phosphate pathway (PPP), which allows for the rapid production of NADPH and maintenance of redox homeostasis. This metabolic adaptation is dependent on the activity of heme oxygenase, and that carbon monoxide (CO) released during heme breakdown is the mediator that induces the metabolic adaptation toward the PPP. We also find that activity of the PPP is required for effective heme detoxification, and that the PPP is upregulated at the enzymatic and transcriptional level in a mouse model of SCD, while, surprisingly, PBMCs from patients with SCD, PPP enzymes are downregulated during disease exacerbation when compared with the same patient at baseline. Taken together, these data suggest a critical role for macrophage/monocyte metabolic adaptation in the response to hemolysis, and point toward manipulation of metabolism as a potential therapeutic avenue for treatment of heme-driven pathologies. This proposal tests the hypothesis that carbon monoxide released by heme breakdown shifts glucose metabolism toward the PPP and that pharmacologic manipulation of this pathway to promote the PPP promotes heme clearance and ameliorates hemolysis-induced damage. This hypothesis will be tested through the completion of the following specific aims. Specific Aim 1 is to determine the intracellular mechanism by which CO drives metabolism toward the PPP: Subaim 1A tests the hypothesis that CO inhibition of cystathionine beta synthetase (CBS) results in loss of phosphofructokinase (PFK) FB3 activity, which shunts glucose metabolism away from glycolysis and toward the PPP. Subaim 1B then examines whether pharmacologic inhibition of PFKFB3 can promote heme clearance and reduce hemolysis-induced damage in hypoxia-induced vasoocclusion. Specific Aim 2 is to examine how the current SCD treatment glutamine alters immunometabolic profile of circulating monocytes in mouse and human SCD. Subaim 2A tests in vitro and in vivo whether glutamine alters cellular metabolism to promote the PPP and ameliorate hemolysis-induced pathology. Subaim 2B integrates transc...