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Intraerythrocytic polymerization of hemoglobin S promotes hemolysis and vaso-occlusive events in the microvasculature of sickle cell disease (SCD) patients. Although platelet-neutrophil aggregates-dependent vaso-occlusion is known to occur in the lung and contribute to lung injury, the etiological mechanisms that trigger acute chest syndrome are largely unknown. Here, we use intravital imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system to identify the innate-immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vaso-occlusion and injury in SCD. Platelet-inflammasome activation led to generation of IL-1β and caspase-1 carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation and platelet-neutrophil aggregation were enhanced by the presence of lipopolysaccharide at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1β pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. These results are the first to identify that platelet-inflammasome dependent shedding of IL-1β and caspase-1 carrying platelet EVs promote lung vaso-occlusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome dependent innate immune pathway to prevent acute chest syndrome in SCD.

Sickle cell disease (SCD) is a hematological disorder that leads to sickling of red blood cells within the microcirculation. The sickle red blood cells (sRBCs) are not only less deformable, but also express α4β1, ICAM-4, Sialyl Lewis X and glycolipids not expressed on non SCD-RBCs. These rigid sRBCs can get sequestered in the microcirculation to cause painful vaso-occlusive crisis (VOC) in SCD patients. In the lung, VOC can contribute to acute chest syndrome, which is a form of acute lung injury. Several factors including the pro-adhesive sRBCs, the large marginated pool of neutrophils, and the hyper-inflammatory state present in SCD can contribute to pulmonary VOC. Although neutrophil-RBC-endothelium interactions are known to play a role in systemic VOC, the cellular mechanism that drives pulmonary VOC remains unknown. Intravital study of the lung microcirculation of live BERK or Townes SCD mice is conducted using two-photon microscopy. Prior to observation, mice are challenged with lipopolysaccharide or PBS to cause lung inflammation and administered (iv) with fluorescent anti-mouse Gr-1 and Ter-119 antibodies to label circulating neutrophils and RBCs, respectively. Vaso-occlusion is defined as cellular aggregation and stasis of blood flow within the lumen of pulmonary capillaries. Preliminary results reveal that circulating macro-aggregates of neutrophils and RBCs may play a role in pulmonary VOC.

Cardiopulmonary disease remains a leading cause of morbidity and mortality in HIV infection, despite widespread use of anti-retroviral therapies. Chronic inflammation due to ongoing viral replication may lead to immune exhaustion, a potential mechanism for the development of chronic obstructive pulmonary disease (COPD) and/or pulmonary arterial hypertension (PAH). Previously, we have shown that simian immunodeficiency virus (SIV) infection of macaques results in PAH and COPD. In the current study, we examined whether lymphocyte exhaustion and/or activation is associated with cardiopulmonary disease in SIV infection. Macaques were intravenously infected with SIVΔB670 (n=6, PAH study) or SHIV89.6P (n=11, COPD study). Blood and bronchoalveolar lavage (BAL) fluid cells from S(H)IV-infected animals were analyzed by flow cytometry. Serial pulmonary function testing and right heart catheterization were performed to assess pulmonary obstruction and mean pulmonary arterial pressure (mPAP), respectively. Increased effector memory CD8+ T (Tem) cells (CD95+CD28-) (p=0.008) and expression of programmed death-1 (PD-1), a marker of exhaustion (p