Figure 3.
NETs are shed in the liver and then embolize to the lung as cNETs in SCD. (A) Experimental scheme: control mice or mice with SCD were IV administered 10 µmol/kg oxy-Hb or saline. Intravital fluorescence microscopy was used to assess the absence or presence of NETs within the liver (B,C,E) and lung (H) microcirculation. Microcirculation (pseudo-colored purple), neutrophils (pseudo-colored red), and extracellular DNA (pseudo-colored green) were labeled in vivo by IV administration of FITC or Texas-red dextran, Pacific blue–anti-Ly6G Ab and Sytox orange or green, respectively. Alternatively (D), freshly cut-unfixed slices of excised liver were stained in vitro for neutrophils (Pacific blue–anti-Ly6G Ab), extracellular DNA (Sytox green), and NE (AF546–anti-NE Ab), and confocal microscopy was used to identify NETs based on colocalization of DNA (green) with neutrophils (pseudo-colored white) and NE (red). Refer to supplemental methods for details. (B) Three representative liver intravital microscopy images (FOVs #1, #2, and #3) and the corresponding videos (supplemental Videos 11-13) reveal numerous NETs (marked with white dotted ellipses) and areas with impaired blood flow evident by slow transit of erythrocytes (dark cells) in the liver microcirculation of mice with SCD administered IV oxy-Hb. NETs were identified based on colocalization of neutrophil (red) and extracellular DNA (green). A vaso-occlusion evident by lack of vascular dye (purple) is marked with white dotted polygon in FOV#1. (C) Magnified intravital images of 4 different NETs in the liver microcirculation of mice with SCD administered IV Oxy-Hb. (D) Representative confocal micrographs reveal abundance of NETs (neutrophil-associated DNA strands positive for NE) in the liver of an SCD but rare in the liver of a control mouse administered IV Oxy-Hb. Neutrophils (pseudo-colored white), NE (red), and extracellular DNA (green). Colocalization of red and white appears pink. Individual channels shown in supplemental Figure 9. (E) Two separate time series of liver intravital images (#1, top row, and #2, bottom row) showing shedding of NETs in the liver microcirculation of mice with SCD administered IV oxy-Hb. (#1) A fragment of ex-DNA (green; marked with dotted ellipse) starts to detach from the neutrophil (red) at t = 0.1 seconds and disappears into the microcirculation (purple) by t = 0.3 seconds. (#2) Several fragments of ex-DNA (green; marked with dotted ellipse) detach from the neutrophil (red) at t = 0.07 seconds and disappear into the microcirculation (purple) by t = 0.3 seconds. Time points are relative to the first frame shown at t = 0 seconds. Complete times series #1 and #2 shown in supplemental Videos 14 and , respectively. (F) Confocal micrographs (representative example shown in panel D) were quantified to reveal significantly more #NETs/FOV in the liver of SCD than control mice administered IV oxy-Hb. n = 6 FOVs in each group. FOV size ∼144 400 µm2. (G) Liver and kidney intravital microscopy images were quantified to estimate number of NETs per FOV (#NETs/FOV). #NETs/FOV was significantly higher in the liver than kidney of mice with SCD administered IV Oxy-Hb. SCD IV Oxy-Hb kidney (n = 3 mice; 35 FOVs), SCD IV Oxy-Hb liver (n = 4 mice; 44 FOVs). (H-I) Intravital lung microcopy was used to assess the effect of simultaneously ligating the hepatic artery and portal vein (liver clamping), on the arrival of cNETs in the lung microcirculation of mice with SCD administered IV oxy-Hb. In qFILM FOVs from same mouse (panel H and supplemental Video 18), cNETs (green; marked with dotted white circle) are seen entering the pulmonary arteriole pre- but not postclamping of liver blood flow. More FOVs are shown in supplemental Figure 13; supplemental Videos 25 and . (I) Number of cNETs arriving in the lung per FOV over a 1-minute duration (#cNETs/FOV/min) were significantly reduced (threefold) following clamping of liver blood flow in mice with SCD administered IV oxy-Hb (n = 5 mice; 45 FOVs preclamp; 33 FOVs postclamp). Intravital microscopy FOV size ∼65 536 µm2. Scale bars, 20 µm. Data in panels F, G, and I represent mean ± SE and were compared using Student t test. *P < .05. Arrow denotes the direction of blood flow.

NETs are shed in the liver and then embolize to the lung as cNETs in SCD. (A) Experimental scheme: control mice or mice with SCD were IV administered 10 µmol/kg oxy-Hb or saline. Intravital fluorescence microscopy was used to assess the absence or presence of NETs within the liver (B,C,E) and lung (H) microcirculation. Microcirculation (pseudo-colored purple), neutrophils (pseudo-colored red), and extracellular DNA (pseudo-colored green) were labeled in vivo by IV administration of FITC or Texas-red dextran, Pacific blue–anti-Ly6G Ab and Sytox orange or green, respectively. Alternatively (D), freshly cut-unfixed slices of excised liver were stained in vitro for neutrophils (Pacific blue–anti-Ly6G Ab), extracellular DNA (Sytox green), and NE (AF546–anti-NE Ab), and confocal microscopy was used to identify NETs based on colocalization of DNA (green) with neutrophils (pseudo-colored white) and NE (red). Refer to supplemental methods for details. (B) Three representative liver intravital microscopy images (FOVs #1, #2, and #3) and the corresponding videos (supplemental Videos 11-13) reveal numerous NETs (marked with white dotted ellipses) and areas with impaired blood flow evident by slow transit of erythrocytes (dark cells) in the liver microcirculation of mice with SCD administered IV oxy-Hb. NETs were identified based on colocalization of neutrophil (red) and extracellular DNA (green). A vaso-occlusion evident by lack of vascular dye (purple) is marked with white dotted polygon in FOV#1. (C) Magnified intravital images of 4 different NETs in the liver microcirculation of mice with SCD administered IV Oxy-Hb. (D) Representative confocal micrographs reveal abundance of NETs (neutrophil-associated DNA strands positive for NE) in the liver of an SCD but rare in the liver of a control mouse administered IV Oxy-Hb. Neutrophils (pseudo-colored white), NE (red), and extracellular DNA (green). Colocalization of red and white appears pink. Individual channels shown in supplemental Figure 9. (E) Two separate time series of liver intravital images (#1, top row, and #2, bottom row) showing shedding of NETs in the liver microcirculation of mice with SCD administered IV oxy-Hb. (#1) A fragment of ex-DNA (green; marked with dotted ellipse) starts to detach from the neutrophil (red) at t = 0.1 seconds and disappears into the microcirculation (purple) by t = 0.3 seconds. (#2) Several fragments of ex-DNA (green; marked with dotted ellipse) detach from the neutrophil (red) at t = 0.07 seconds and disappear into the microcirculation (purple) by t = 0.3 seconds. Time points are relative to the first frame shown at t = 0 seconds. Complete times series #1 and #2 shown in supplemental Videos 14 and , respectively. (F) Confocal micrographs (representative example shown in panel D) were quantified to reveal significantly more #NETs/FOV in the liver of SCD than control mice administered IV oxy-Hb. n = 6 FOVs in each group. FOV size ∼144 400 µm2. (G) Liver and kidney intravital microscopy images were quantified to estimate number of NETs per FOV (#NETs/FOV). #NETs/FOV was significantly higher in the liver than kidney of mice with SCD administered IV Oxy-Hb. SCD IV Oxy-Hb kidney (n = 3 mice; 35 FOVs), SCD IV Oxy-Hb liver (n = 4 mice; 44 FOVs). (H-I) Intravital lung microcopy was used to assess the effect of simultaneously ligating the hepatic artery and portal vein (liver clamping), on the arrival of cNETs in the lung microcirculation of mice with SCD administered IV oxy-Hb. In qFILM FOVs from same mouse (panel H and supplemental Video 18), cNETs (green; marked with dotted white circle) are seen entering the pulmonary arteriole pre- but not postclamping of liver blood flow. More FOVs are shown in supplemental Figure 13; supplemental Videos 25 and . (I) Number of cNETs arriving in the lung per FOV over a 1-minute duration (#cNETs/FOV/min) were significantly reduced (threefold) following clamping of liver blood flow in mice with SCD administered IV oxy-Hb (n = 5 mice; 45 FOVs preclamp; 33 FOVs postclamp). Intravital microscopy FOV size ∼65 536 µm2. Scale bars, 20 µm. Data in panels F, G, and I represent mean ± SE and were compared using Student t test. *P < .05. Arrow denotes the direction of blood flow.

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