Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury

Abstract

There is emerging evidence that platelets are major contributors to inflammatory processes through intimate associations with innate immune cells. Here, we report that activated platelets induce the formation of neutrophil extracellular traps (NETs) in transfusion-related acute lung injury (TRALI), which is the leading cause of death after transfusion therapy. NETs are composed of decondensed chromatin decorated with granular proteins that function to trap extracellular pathogens; their formation requires the activation of neutrophils and release of their DNA in a process that may or may not result in neutrophil death. In a mouse model of TRALI that is neutrophil and platelet dependent, NETs appeared in the lung microvasculature and NET components increased in the plasma. We detected NETs in the lungs and plasma of human TRALI and in the plasma of patients with acute lung injury. In the experimental TRALI model, targeting platelet activation with either aspirin or a glycoprotein IIb/IIIa inhibitor decreased NET formation and lung injury. We then directly targeted NET components with a histone blocking antibody and DNase1, both of which protected mice from TRALI. These data suggest that NETs contribute to lung endothelial injury and that targeting NET formation may be a promising new direction for the treatment of acute lung injury.

Figure 1. Activated platelets induce NET formation in human neutrophils.

(A–E) Representative images from direct immunofluorescence staining of DNA (blue), histones (red), and MPO (green) on (A) normal neutrophils incubated in media, (B) showing NET formation in neutrophils treated with PMA (25 nM; as positive control of NETosis) and (D) with platelets activated by TRAP (50 μM). (C) No NET formation was apparent from treatment with platelets alone. (E) Treatment with TRAP alone produced less NET formation than in B or D. n = 6. Scale bar: 10 μm. (F) MPO-DNA ELISA was used to quantify NETs in neutrophil supernatants and is expressed as percentage increase above control (media); mean ± SD (n = 6). *P < 0.05 versus media and platelets groups; ^†P < 0.05 versus PMA and TRAP-activated platelets groups; ***P < 0.001 versus media and platelets groups.

Figure 2. NET formation is dependent on thromboxane production and MEK signaling and increases endothelial permeability.

(A) Quantification of NET release in cell supernatant (MPO-DNA ELISA). Neutrophils were pretreated with the MEK inhibitor (U0126, 10 μM) or a thromboxane receptor antagonist (SQ29548, 10 μM) for 10 minutes before the addition of platelets and TRAP. Pretreatment of neutrophils with U0126 or SQ29548 inhibited NET formation compared with that in neutrophils treated with TRAP-activated platelets. Mean ± SD (n = 6); *P < 0.05, ***P < 0.001 versus media and platelets; ^†P < 0.05 versus TRAP-activated platelets. (B–D) Representative images from direct immunofluorescence staining of DNA (blue), histone (red), and MPO (green), showing NET formation in neutrophils treated with (B) TRAP-activated platelets and less NET formation in neutrophils pretreated with (C) U0126 or (D) SQ29548. n > 6; Scale bar: 10 μm. (E) Permeability of endothelial cell monolayers (HUVEC) measured in a Transwell system. In selected experiments, HUVECs were primed with LPS (2 μg/ml) for 24 hours prior to the experiment. Permeability was measured by ¹²⁵I-albumin flux across endothelium over 1 hour and was increased in cells treated with cytomix (0.5 ng/ml) or in LPS-primed endothelium with TRAP-activated platelets or PMA. Mean ± SD (n = 12); *P < 0.05, **P < 0.01, ***P < 0.001 versus HUVECs without treatment. ^†P < 0.05, ^†††P < 0.001 versus HUVECs treated LPS and with neutrophils and TRAP-activated platelets.

Figure 3. NETs are present in TRALI mouse lungs, and aspirin decreases NET formation.

(A–D) Representative images of NET formation detected by immunofluorescence in the lung microcirculation of (A) control mice (LPS plus isotype control mAb) and (B) mice with TRALI (LPS plus H2K^d mAb, 1.0 mg/kg), (C) with or without aspirin treatment at 2 hours after mAb injection. Platelet (CD41) staining was increased in TRALI mice and localized to areas of NET formation. Aspirin treatment decreased platelet staining to levels observed in control mice. (D) Mice treated with a higher dose of H2K^d mAb (4.5 mg/kg) had NET formation at 5 minutes after mAb injection. Scale bar: 20 μm. (E) NET formation was quantified (MPO-DNA ELISA) in mouse plasma and plotted as mean ± SD (n > 4). **P < 0.01, ***P < 0.001 versus group receiving no treatment; ^††P < 0.01 versus TRALI plus DMSO group.

Figure 4. Platelets and extracellular histones are therapeutic targets in TRALI.

(A) Mice with TRALI (LPS plus H2K^d mAb) administered BWA3 mAb (10 mg/kg, i.v.) or IgG control (10 mg/kg) given immediately prior to H2K^d mAb. BWA3 mAb decreased extravascular lung water and lung vascular permeability to ¹²⁵I-labeled albumin and decreased mortality compared with that in the IgG control group. Mean ± SD (n = 9). *P < 0.05 versus IgG control group. **P < 0.01. (B) Mice with TRALI (LPS plus H2K^d mAb) administered tirofiban (0.5 μg/g, i.v.) or PBS given immediately prior to H2K^d mAb. Tirofiban decreased extravascular lung water and lung vascular permeability to ¹²⁵I-labeled albumin and decreased mortality compared with that in the PBS group. Mean ± SD (n = 7–9). **P < 0.01 versus PBS group. (C and D) MPO-DNA ELISA was used to quantify NET formation in the plasma of mice treated with (C) BWA3 mAb or (D) tirofiban compared with that in treatment controls and normal mouse plasma. Mean ± SD (n = 6). ***P < 0.001 versus control group; ^††P < 0.01 versus IgG control and PBS groups. (E and F) Lung sections stained for NET formation (DNA, histone, and MPO) and for platelets (CD41). Representative images of mice with (E) TRALI given diluent (PBS) compared with those with (F) TRALI administered tirofiban. Mice treated with tirofiban have decreased NET formation and associated platelet sequestration compared with the PBS group. (n = 6). Scale bar: 20 μm.

Figure 5. DNase1 treatment decreases TRALI and NETs.

(A) Mice treated with DNase1 (10 mg/kg, i.v.) or diluent immediately prior to H2K^d mAb injection are protected from TRALI with decreased extravascular lung water and decreased lung vascular permeability to ¹²⁵I-labeled albumin (EVPE). Mean ± SD (n = 7). **P < 0.01 versus diluent group. (B) MPO-DNA ELISA was used to quantify NET formation in the plasma of mice pretreated with DNase1 or diluent compared with that in normal mouse plasma. Mean ± SD (n = 6). **P < 0.01, ***P < 0.001 versus control group. (C and D) Lung sections stained for NET formation (DNA, histone, and MPO) and for platelets (CD41). Representative images of mice with TRALI given (C) diluent or (D) DNase1. Mice treated with DNase1 have decreased NET formation and associated platelet sequestration compared with the diluent-treated group. (n = 3). Scale bar: 20 μm. (E and F) Mice treated with DNase1 (10 mg/kg, i.v.) or diluent 5 minutes after H2K^d mAb injection are protected from TRALI with (E) decreased extravascular lung water and (F) decreased mortality compared with that in the diluent group. Mean ± SD (n = 8). *P < 0.05, **P < 0.01 versus diluent group.

Figure 6. NETs are present in human TRALI lungs and plasma.

(A and B) Human lung paraffin sections were stained for histone (red), MPO (green), and DNA (blue) and analyzed by confocal microscopy. (B) In the TRALI fatality case, we observed clumps of NET-forming neutrophils in the intravascular compartment. (A) In the TACO fatality case, neutrophils were found in mainly intra-alveolar locations, but no NET formation was detected. Scale bar: 10 μm. (C) MPO-DNA ELISA was used to quantify NET components in the plasma of patients, and the mean optical density of plasma obtained from normal, human blood donors (n = 6) was used as the control. Plasma from individuals with cardiac disease (n = 6), individuals before TRALI and after TRALI (paired samples, n = 14), and individuals before ALI and after ALI (paired samples, n = 9) are compared. Horizontal bars represent the mean; symbols represent individual samples. *P < 0.05 versus donor blood group; ***P < 0.001 versus donor blood, cardiac disease, and before TRALI groups; ^†P < 0.05 versus donor blood, cardiac disease, and before ALI groups.