PROJECT SUMMARY: We have developed methods to profile the metabolome of hematopoietic stem cells (HSCs) and other rare cell types purified from tissues. Each hematopoietic cell type had a distinct metabolite identity. Most metabolites were enriched or depleted in specific cell types, suggesting they may have novel cell-type specific roles. HSCs and multipotent progenitors (MPPs) in mouse and human bone marrow had high levels of ascorbate (Vitamin C), which promoted the activity of the enzyme TET2, a suppressor of HSC function. Hematopoietic-specific ascorbate deficiency promoted HSC function, myelopoiesis and the generation of inflammatory myeloid cells, and caused early lethality. Ascorbate deficiency is common in the human population because in early primate evolution we lost the ability to synthesize ascorbate. Ascorbate deficiency in healthy people is associated with increased risk of mortality for unknown reasons. Hematopoietic TET2 loss of function mutations are also common in humans, and drive a clonal expansion of mutant blood cells termed clonal hematopoiesis. TET2- deficient blood cells may contribute to an increased risk of mortality. This application’s objective is to understand the role of ascorbate in the regulation of myelopoiesis. Our central hypothesis is that ascorbate suppresses myelopoiesis, and that ascorbate deficiency increases myelopoiesis and inflammation after plasmodium infection. To test this hypothesis, we will use genetically engineered ascorbate deficient mice, to mimic the human condition, and Tet2-deficient mice. In Aim 1 we will test if ascorbate suppresses the generation of inflammatory myeloid cells by acting on HSCs or restricted myeloid progenitors, and if this is mediated by Tet2. In Aim 2 we will determine the effects of ascorbate deficiency or Tet2 deficiency on the myelopoietic response to Plasmodium infection in a mouse model of malaria. In Aim 3 we will investigate the mechanisms by which ascorbate deficiency and Tet2 deficiency promote morbidity and mortality in Plasmodium infection. These experiments may have significant public health implications. They could identify physiological situations, such as infection, in which the presence of ascorbate deficiency and Tet2-deficient clonal hematopoiesis are deleterious to the organism. They may also identify mechanisms by which aberrant myelopoiesis contributes to the pathogenesis of malaria which afflicts more than 200 million people worldwide.