Inhibiting the GSDMD pathway ameliorates P-selectin–independent lung vaso-occlusion in SCD. (A) Experimental scheme: SCD or SCD-Selp^−/− mice and WT or Gsdmd^−/− mice were IV administered 10 µmol/kg oxy-Hb (10 oxy-Hb) and 20 µmol/kg oxy-Hb (20 oxy-Hb), respectively, without or with 10 mg/kg GSDMD inhibitor (LDC7559) or 0.004 µmol/kg pan-caspase inhibitor (Z-VAD-FMK) or 20 mg/kg GSDMD inhibitor NSA or 10 mg/kg GSDMD inhibitor (disulfiram). qFILM was used to assess the absence or presence of platelet-neutrophil aggregate-mediated PVO. Pulmonary microcirculation (pseudo-colored purple), neutrophils (red), and platelets (pseudo-colored green) were labeled in vivo by IV administration of FITC dextran, AF546–anti-Ly6G Ab and V450–anti-CD49b Ab, respectively. Representative qFILM images are shown in panels B to E. (B) Representative qFILM image and supplemental Video 21 reveal 5 neutrophil-platelet aggregates (marked with dotted white ellipses) occluding pulmonary arteriole-bottleneck in the lung of a mouse with SCD IV administered 10 µmol/kg oxy-Hb (10 oxy-Hb). Representative qFILM images and corresponding videos reveal absence of lung vaso-occlusion and significantly improved blood flow (evident by rapidly transiting erythrocytes [dark cells]) in the lung of a mouse with SCD IV administered 10 oxy-Hb + Z-VAD-FMK (C; supplemental Video 22), 10 oxy-Hb + LDC7559 (D; supplemental Video 23), and 10 oxy-Hb + NSA (E; supplemental Video 24). White arrows denote the direction of blood flow within the pulmonary arterioles. Alveoli are marked with asterisks. Scale bars, 20 µm. The diameter of pulmonary arteriole in panels B to E is ∼22 µm, 32 µm, 31.5 µm, and 31 µm, respectively. (F-K) PVOs were compared between treatment groups using following 2 parameters: #PVOs/FOV and number of large PVOs (with area >1000 µm²) per FOV. Both #PVOs/FOV and #PVOs (with area >1000 µm²) per FOV were significantly less in mice with SCD IV administered (F,G) 10 oxy-Hb + Z-VAD-FMK (n = 4 mice; 51 FOVs) or (H,I) 10 oxy-Hb + LDC7559 (n = 4 mice; 41 FOVs) or (J,K) 10 oxy-Hb + NSA (n = 3 mice; 38 FOVs) than mice with SCD IV administered 10 oxy-Hb alone (n = 5 mice; 75 FOVs). Both (L) #PVOs/FOV and (M) #PVOs (with area >1000 µm²) per FOV were significantly less in Gsdmd^−/− (n = 3 mice; 28 FOVs) than littermate WT mice (n = 3 mice; 31 FOVs) IV administered 20 oxy-Hb. (N) #PVOs/FOV were significantly reduced and (O) #PVOs (with area >1000 µm²) per FOV were absent in SCD-Selp^−/− mice IV administered 10 oxy-Hb + LDC7559 (n = 3 mice; 35 FOVs) compared with SCD-Selp^−/− mice IV administered 10 oxy-Hb alone (n = 5 mice; 69 FOVs). A similar effect of disulfiram on PVOs in SCD-Selp^−/− mice shown in supplemental Figure 25. The data for mice with SCD IV administered 10 oxy-Hb (n = 5 mice; 75 FOVs) are included in panels N and O for relative comparison. qFILM FOV size ∼65 536 µm². Data represent mean ± SE and are compared using Student t test. *P < .05. (P) Schematic showing the main findings of the study. The sterile inflammatory milieu (DAMPs, IFN-I signaling, and ROS) in SCD promotes caspase-4 (humans) or caspase-11 (mouse)-dependent cleavage of neutrophil GSDMD into the active form GSDMD-NT, leading to the shedding of NETs in the liver microcirculation. Once shed, these NETs are carried by the blood as cNETs to the lung, where they promote neutrophil-platelet aggregation in the pulmonary arterioles, leading to pulmonary vaso-occlusion and lung injury (acute chest syndrome).