Project Summary Acute chest syndrome (ACS) is a type of acute lung injury and one of the leading causes of mortality in Sickle Cell Disease (SCD). The etiological mechanism that triggers ACS remains poorly understood. 10-20% of SCD patients hospitalized with acute systemic painful vaso-occlusive episodes develop ACS within next few days, suggesting that molecular events surrounding vaso-occlusion contribute to lung injury. This epidemiology also offers a therapeutic window to halt the development of ACS, provided that targeted therapies are identified. In the first cycle of R01, we used real time in vivo multi-photon-excitation microscopy to make a novel finding that ACS in SCD mice is secondary to micro-embolism of precapillary pulmonary arterioles by neutrophil-platelet aggregates. These findings have been published in AJRCCM 2019, JCI-Insight 2017 & 2020, Blood Advances 2017 & 2020, Experimental Hematology 2020, Blood 2020 and Haematologica 2015. We found that platelet P- selectin contributed to formation of these micro-embolic cellular aggregates and P-selectin inhibition reduced lung vaso-occlusion by ~50%. Supporting the relevance of our mouse model, recent clinical trial reported ~50% reduction in pain episodes in SCD patients given P-selectin Ab therapy. In the present renewal of our 5-year R01, we identify additional P-selectin-independent pathological inflammatory signaling events that can be targeted to further inhibit lung vaso-occlusion and ACS in SCD. Based on our new preliminary findings, we hypothesize that liver-derived circulating-fragments of neutrophil extracellular traps (cNETs) arrive in the lung to promote neutrophil-platelet aggregate-enabled pulmonary arteriole micro-embolism in SCD. We also propose that inhibiting pore forming protein gasdermin-D (GSDMD)-dependent signaling in neutrophils prevents cNETs generation and development of ACS. We will test this hypothesis using our newly developed model of intravenous hemoglobin induced ACS in SCD mice, in vivo imaging of lung in live mice, in vitro microfluidic studies with patient blood, and SCD mice genetically deficient in GSDMD or platelet-P-selectin. In Aim 1, we will determine whether SCD mice genetically deficient in platelet-P-selectin are only partially protected from pulmonary arteriole micro-embolism and ACS. In Aim 2, we will determine whether cNETs shed in the liver, travel to the lung to promote P-selectin-independent pulmonary arteriole micro-embolism in SCD. In Aim 3, we will determine whether caspase-4/11-dependent activation of neutrophil-GSDMD promotes shedding of cNETs and development of ACS in SCD. These studies will introduce a novel paradigm that translocation of DAMPs from liver to lung promotes lung injury in SCD, and also identify a new GSDMD-mediated, P-selectin-independent mechanism of ACS in SCD.