Red blood cell (RBC) transfusions remain a cornerstone treatment in the management of sickle cell disease (SCD). However, patients may experience delayed hemolytic transfusion reaction (DHTR) which in this patient population has an unpredictable progression from mild to life-threatening severe reactions where both transfused and patient’s own RBCs are destroyed along with reticulocytopenia at the time of hemolytic crisis, exacerbating the anemia. The mechanisms underlying severe DHTR progression are poorly understood, posing challenges for prevention and effective treatments for this transfusion complication which disproportionately is encountered in patients with SCD. We recently found that acute hemolysis induces type I interferon (IFN-I) production in innate immune cells, leading to increased differentiation and activation of monocyte-derived macrophages (MoMΦ) in SCD, and exacerbating destruction of antibody (Ab)-coated transfused RBCs. Our preliminary data showed that Ab-sensitized RBC destruction alone also led to IFN-I production. Interestingly, only when the incompatible RBC destruction occurs under hemolytic conditions inducing highest levels of IFN-I, we detect bystander hemolysis of sickle RBCs, mimicking hyperhemolysis reaction in SCD. We also found that hemolysis-induced IFN-I impairs erythropoiesis along with inhibition of EPO/EPOR signaling. Based on these data, we hypothesize that Fc receptor crosslinking in a hemolytic backdrop of SCD leads to increase in IFN-I levels, causing heightened IFN- I signaling in phagocytes and erythroid cells which trigger increased RBC destruction and inhibits RBC production, respectively, in severe DHTR. In aim 1, we will focus on identifying mechanisms of bystander hemolysis by examining the role of key phagocytosis activation molecules, including thrombospondin (TSP-1) and its ligands which are upregulated in our hyperhemolytic models. We will compare the role of Ab-mediated erythrophagocytosis versus Ab-independent RBC engulfment in triggering bystander hemolysis and interrogate the relative contribution of murine/human FcγR/SYK pathway and heme activation pathways in bystander hemolysis. We will also examine the potential of IFN-I as a biomarker of DHTR severity by examining IFN- I/STAT1 driven changes in monocyte phagocytosis associated profiles in SCD patient samples, comparing patients experiencing severe and mild DHTR and after recovery to steady state. For aim 2, we will define the mechanisms by which IFN-I suppresses erythropoiesis using primary erythroid cell culture system and targeted deletion of key downstream pathways in human erythroblast cell lines and mouse models. We will also test the therapeutic effects of inhibiting IFN-I production/IFN-I signaling or/and increasing EPO/EPOR signaling on reversing impaired BM erythropoiesis in SCD mice and on human erythropoiesis in vitro and in cultures treated from SCD patient plasma. We believe that our proposed studies to examine the basis ...