Project Summary: As the most common inherited blood disorder in the United State, there are 70,000-100,000 Americans with sickle cell anemia. Sickle cell disease (SCD) is caused by a mutation in the β-globin gene, which leads to significant deformation red blood cell (RBC) membrane and promotes RBC adhesion to other cells to induce vaso-occlusive crises (VOC). Chronic sickle cell anemia is accompanied with progressive systemic multi- systemic organ dysfunction and cost over $475 million annually in hospital admission. Our laboratory has reported that sickle cell-induced hypoxic, oxidant, and inflammatory stress is perpetuated by aged neutrophils which positively correlates with VOC in humanized SCD mice. Our recent work has demonstrated that deletion of the microbiota in SCD mice by antibiotics restricted aged neutrophil expansion which consequently decreased VOC severity, and reduced organ damage as well as the iron overload. In addition, hydroxyurea, the only FDA- approved drug for SCD that promotes the anti-sickling fetal hemoglobin expression, also possesses anti- inflammatory, antiradical, and metal-chelating activities both in mammalian cells as well as in bacteria. In this application, we propose a 3-year experimental plan that will advance our understanding in the function of microbiota in SCD disease progression and will test whether manipulation of the microbiota will provide a potential novel SCD treatment. In Specific Aim 1, we will identify disease-modifying microbiota species that may contribute to neutrophil aging and SCD organ damage. 16S sequencing data has revealed microbiota differences between antibiotic treatment and untreated SCD mice, and the function of selected microbiota will be verified in germ-free SCD mice. In Specific Aim 2, we will examine if hydroxyurea reduces VOC and organ damage through microbiota manipulations in SCD mice in which fecal samples from hydroxyurea-treated and control SCD mice will be transplant to germ-free SCD to evaluate whether hydroxyurea-induced changes in the microbiota contribute to its therapeutic activity. In Specific Aim 3, we will study if iron restriction (by DFO or low-iron diet) or probiotics-induced changes in iron metabolism can ameliorate chronic organ damage in SCD mice. Iron-related changes in microbiota will be verified by germ-free SCD mice. These proposed studies, focused on strategies of microbiota manipulation in SCD, will allow us to identify the key microbial species that contribute to SCD pathophysiology, and provide potential novel cost-effective approaches for management of SCD’s life-long complications.