Project Abstract A chronic inflammatory state is now considered a hallmark of sickle cell disease (SCD) resulting from ongoing hemolytic insult to the underlying vasculature and repeated cycles of vaso-occlusion crisis (VOC). Transfusions remain a cornerstone treatment for patients with SCD, but unlike other transfused patient populations, a higher proportion (20-50%) of patients with SCD develop alloantibodies with potentially life-threatening sequalae. These include poorly understood delayed transfusion reactions (DHTRs) whose occurrence, as well as clinical progression from mild to severe, is unpredictable. A long-standing interest of our research program has been to understand the underling immune mechanisms leading to SCD complications including VOC and transfusion complications. Our studies have uncovered an underappreciate role of circulating monocytes and the innate immune response to hemolysis, a hallmark of SCD, in transfusion complications including alloimmunization and DHTRs. Our projected studies as part of this R35 will be focused on innate immune cellular pathways that magnify the inflammatory SCD state versus the cells' heme protective pathways such as the heme detoxifying key enzyme heme oxygenase 1 in alloimmunization and in the development of DHTRs. Our laboratory has also identified a novel function for a subset of circulating monocytes, referred to as patrolling monocytes (PMo), in their ability to scavenge and remove damaged endothelium and endothelial-attached sickle RBCs. The discovery of a novel mechanism of action of PMo in SCD offers a paradigm shift in our understanding of VOC and opens up the possibility that PMos can be manipulated to prevent painful VOC. An objective of the proposed studies in this R35 is to understand the mechanisms by which PMo protect SCD vasculature and how they are affected both functionally and numerically during a vaso-occlusive event with the goal to help establish their role in VOC pathophysiology, and their potential as a therapeutic target for VOC. As we have done in the past, all the proposed studies for both objectives will combine the use of in vivo mouse models, in vitro human model systems, and patient samples to mechanistically dissect in a rigorous manner how specific immune cell types and molecular pathways contribute to SCD alloimmunization and VOC. Altogether, we believe that our laboratory's approach of integrating studies in humans and experimental models to mechanistically dissect immune pathways that regulate SCD complications is likely to be impactful and generate novel findings in areas critical to the NHLBI scientific mission.