Figure 6.
Neutrophil-GSDMD promotes liver-to-lung embolization of cNETs in SCD. (A) Experimental scheme used in panels B-D: SCD patient blood preincubated with 20 μM hemin with or without GSDMD inhibitor (LDC7559) or caspase-4 inhibitor (LEVD-CHO) or NAC, processed to generate PPP, PPP incubated with Sytox green and fluorescent Abs against NE and citrullinated histones (H3-Cit), and cNETs detected in PPP by imaging flow cytometry as described in Figure 2. Concentration of cNETs (#cNETs/μL of plasma) was significantly reduced in hemin-treated SCD patient blood following incubation with (B) 20 µM LDC7559 (n = 4 patients), (C) 20 µM LEVD-CHO (n = 5 patients), and (D) 20 µM NAC (n = 5 patients). Straight line connects cNETs concentrations in the same SCD patient blood pre- (red circle) and post- (blue circle) inhibitor treatment. (E) Experimental scheme used in panels F to O: 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 necrosulfonamide (NSA) or 10 mg/kg GSDMD inhibitor (disulfiram). In panels F-G, venous blood was processed to generate PPP and cNETs detected in PPP using imaging flow cytometry as in panel A. In panels H to O, microcirculation, neutrophils, and extracellular DNA were labeled in vivo by IV administration of FITC or Texas-red dextran, Pacific blue–anti-Ly6G Ab, and Sytox orange or green, respectively, and intravital fluorescence microscopy was used to assess the absence or presence of NETs within the liver (H-J) and lung (K-O) microcirculation. (F) Plasma concentration of cNETs was significantly less in mice with SCD IV administered 10 oxy-Hb + LDC7559 (n = 4 mice) than 10 oxy-Hb alone (n = 4 mice). (G) Plasma concentration of cNETs was significantly less in SCD-Selp−/− mice IV administered 10 oxy-Hb + LDC7559 (n = 4 mice) than 10 oxy-Hb alone (n = 4 mice). (H) Left, representative liver intravital microscopy image and supplemental Video 19 reveal numerous large NETs (marked with white ellipses) and areas with impaired blood flow evident by slow transit of erythrocytes (dark cells) in the liver microcirculation of a mouse with SCD administered IV 10 oxy-Hb. Right, representative liver intravital microscopy image and supplemental Video 20 reveal only a single small NET (marked with white ellipse) and significantly improved blood flow evident by rapidly transiting neutrophils (red) and erythrocytes (dark cells) in the liver microcirculation of a mouse with SCD administered IV 10 oxy-Hb + 10 mg/kg LDC7559. NETs were identified based on colocalization of neutrophil (pseudo-colored red) and extracellular DNA (green) in microcirculation (pseudo-colored purple). Scale bars, 20 µm. Arrow denotes the direction of blood flow. Liver intravital microscopy images were analyzed as in Figure 3 to estimate number of NETs per FOV (#NETs/FOV). (I) #NETs/FOV were significantly less (threefold) in the liver microcirculation of mice with SCD administered IV 10 oxy-Hb + LDC7559 (n = 4 mice; 42 FOVs) than 10 oxy-Hb alone (n = 4 mice; 44 FOVs). (J) #NETs/FOV were significantly less (fourfold) in the liver microcirculation of Gsdmd−/− (n = 4 mice; 35 FOVs) than littermate WT mice (n = 4 mice; 38 FOVs) administered IV 20 oxy-Hb. (K-O) Lung intravital microscopy images were analyzed as in Figure 2 to estimate number of cNETs entering per FOV in the lung over a 1-minute duration (#cNETs/FOV/min). #cNETs/FOV/min in the lung were significantly less in mice with SCD IV administered (K) 10 oxy-Hb + 0.004 µmol/kg Z-VAD-FMK (n = 4 mice; 51 FOVs), (L) 10 oxy-Hb + 10 mg/kg LDC7559 (n = 4 mice; 41 FOVs), and (M) 10 oxy-Hb + 20 mg/kg NSA (n = 3 mice; 38 FOVs) than 10 oxy-Hb alone (n = 4 mice; 44 FOVs). (N) #cNETs/FOV/min in the lung were significantly less in Gsdmd−/− (n = 3 mice; 29 FOVs) than littermate WT mice (n = 3 mice; 33 FOVs) IV administered 20 oxy-Hb. (O) #cNETs/FOV/min in the lung were significantly less (fourfold) in SCD-Selp−/− mice IV administered 10 oxy-Hb + LDC7559 (n = 3 mice; 35 FOVs) than 10 oxy-Hb alone (n = 5 mice; 71 FOVs). A similar effect of disulfiram on #cNETs/FOV/min in the lung of SCD-Selp−/− mice shown in supplemental Figure 22. Data in panels B to D compared using a paired Student t test. Data in panels F, G, and I to O represent mean ± SE and compared using Student t test. *P < .05. FOV size ∼65 536 µm2.

Neutrophil-GSDMD promotes liver-to-lung embolization of cNETs in SCD. (A) Experimental scheme used in panels B-D: SCD patient blood preincubated with 20 μM hemin with or without GSDMD inhibitor (LDC7559) or caspase-4 inhibitor (LEVD-CHO) or NAC, processed to generate PPP, PPP incubated with Sytox green and fluorescent Abs against NE and citrullinated histones (H3-Cit), and cNETs detected in PPP by imaging flow cytometry as described in Figure 2. Concentration of cNETs (#cNETs/μL of plasma) was significantly reduced in hemin-treated SCD patient blood following incubation with (B) 20 µM LDC7559 (n = 4 patients), (C) 20 µM LEVD-CHO (n = 5 patients), and (D) 20 µM NAC (n = 5 patients). Straight line connects cNETs concentrations in the same SCD patient blood pre- (red circle) and post- (blue circle) inhibitor treatment. (E) Experimental scheme used in panels F to O: 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 necrosulfonamide (NSA) or 10 mg/kg GSDMD inhibitor (disulfiram). In panels F-G, venous blood was processed to generate PPP and cNETs detected in PPP using imaging flow cytometry as in panel A. In panels H to O, microcirculation, neutrophils, and extracellular DNA were labeled in vivo by IV administration of FITC or Texas-red dextran, Pacific blue–anti-Ly6G Ab, and Sytox orange or green, respectively, and intravital fluorescence microscopy was used to assess the absence or presence of NETs within the liver (H-J) and lung (K-O) microcirculation. (F) Plasma concentration of cNETs was significantly less in mice with SCD IV administered 10 oxy-Hb + LDC7559 (n = 4 mice) than 10 oxy-Hb alone (n = 4 mice). (G) Plasma concentration of cNETs was significantly less in SCD-Selp−/− mice IV administered 10 oxy-Hb + LDC7559 (n = 4 mice) than 10 oxy-Hb alone (n = 4 mice). (H) Left, representative liver intravital microscopy image and supplemental Video 19 reveal numerous large NETs (marked with white ellipses) and areas with impaired blood flow evident by slow transit of erythrocytes (dark cells) in the liver microcirculation of a mouse with SCD administered IV 10 oxy-Hb. Right, representative liver intravital microscopy image and supplemental Video 20 reveal only a single small NET (marked with white ellipse) and significantly improved blood flow evident by rapidly transiting neutrophils (red) and erythrocytes (dark cells) in the liver microcirculation of a mouse with SCD administered IV 10 oxy-Hb + 10 mg/kg LDC7559. NETs were identified based on colocalization of neutrophil (pseudo-colored red) and extracellular DNA (green) in microcirculation (pseudo-colored purple). Scale bars, 20 µm. Arrow denotes the direction of blood flow. Liver intravital microscopy images were analyzed as in Figure 3 to estimate number of NETs per FOV (#NETs/FOV). (I) #NETs/FOV were significantly less (threefold) in the liver microcirculation of mice with SCD administered IV 10 oxy-Hb + LDC7559 (n = 4 mice; 42 FOVs) than 10 oxy-Hb alone (n = 4 mice; 44 FOVs). (J) #NETs/FOV were significantly less (fourfold) in the liver microcirculation of Gsdmd−/− (n = 4 mice; 35 FOVs) than littermate WT mice (n = 4 mice; 38 FOVs) administered IV 20 oxy-Hb. (K-O) Lung intravital microscopy images were analyzed as in Figure 2 to estimate number of cNETs entering per FOV in the lung over a 1-minute duration (#cNETs/FOV/min). #cNETs/FOV/min in the lung were significantly less in mice with SCD IV administered (K) 10 oxy-Hb + 0.004 µmol/kg Z-VAD-FMK (n = 4 mice; 51 FOVs), (L) 10 oxy-Hb + 10 mg/kg LDC7559 (n = 4 mice; 41 FOVs), and (M) 10 oxy-Hb + 20 mg/kg NSA (n = 3 mice; 38 FOVs) than 10 oxy-Hb alone (n = 4 mice; 44 FOVs). (N) #cNETs/FOV/min in the lung were significantly less in Gsdmd−/− (n = 3 mice; 29 FOVs) than littermate WT mice (n = 3 mice; 33 FOVs) IV administered 20 oxy-Hb. (O) #cNETs/FOV/min in the lung were significantly less (fourfold) in SCD-Selp−/− mice IV administered 10 oxy-Hb + LDC7559 (n = 3 mice; 35 FOVs) than 10 oxy-Hb alone (n = 5 mice; 71 FOVs). A similar effect of disulfiram on #cNETs/FOV/min in the lung of SCD-Selp−/− mice shown in supplemental Figure 22. Data in panels B to D compared using a paired Student t test. Data in panels F, G, and I to O represent mean ± SE and compared using Student t test. *P < .05. FOV size ∼65 536 µm2.

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