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. 2022 Mar;102(3):263-271.
doi: 10.1038/s41374-021-00683-6. Epub 2021 Nov 3.

c-Abl kinase regulates neutrophil extracellular trap formation and lung injury in abdominal sepsis

Avin Hawez  1 , Zhiyi Ding  1 , Dler Taha  1 , Raed Madhi  1 , Milladur Rahman  1 , Henrik Thorlacius  2
Affiliations

c-Abl kinase regulates neutrophil extracellular trap formation and lung injury in abdominal sepsis

Avin Hawez et al. Lab Invest. 2022 Mar.

Abstract

Sepsis is associated with exaggerated neutrophil responses although mechanisms remain elusive. The aim of this study was to investigate the role of c-Abelson (c-Abl) kinase in neutrophil extracellular trap (NET) formation and inflammation in septic lung injury. Abdominal sepsis was induced by cecal ligation and puncture (CLP). NETs were detected by electron microscopy in the lung and by confocal microscopy in vitro. Plasma levels of DNA-histone complexes, interleukin-6 (IL-6) and CXC chemokines were quantified. CLP-induced enhanced phosphorylation of c-Abl kinase in circulating neutrophils. Administration of the c-Abl kinase inhibitor GZD824 not only abolished activation of c-Abl kinase in neutrophils but also reduced NET formation in the lung and plasma levels of DNA-histone complexes in CLP mice. Moreover, inhibition of c-Abl kinase decreased CLP-induced lung edema and injury. Administration of GDZ824 reduced CLP-induced increases in the number of alveolar neutrophils. Inhibition of c-Abl kinase also markedly attenuated levels of CXC chemokines in the lung and plasma as well as IL-6 levels in the plasma of septic animals. Taken together, this study demonstrates that c-Abl kinase is a potent regulator of NET formation and we conclude that c-Abl kinase might be a useful target to ameliorate lung damage in abdominal sepsis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. c-Abl kinase activity in neutrophils. Phosphorylation of c-Abl kinase in circulating neutrophils were examined by western blot as described in Materials and Methods.
Animals were treated with GZD824 (5 mg/kg) or vehicle prior to CLP induction. Mice treated with saline (sham) or GZD824 alone without CLP. Samples were collected 24 h after induction of CLP. Data are presented as mean values ± standard error of the mean (SEM) and n = 5. #P < 0.05 vs. Sham and *P < 0.05 vs. Vehicle + CLP.
Fig. 2
Fig. 2. NET formation in sepsis.
A Scanning electron microscopy showing extracellular web-like structures in the lung from CLP mice. Scale bar = 5 μm. B Transmission electron microscopy of the indicated area of interest from Fig. 2A incubated with gold-labeled antibody against citrullinated histone 3 (large gold particles) and anti-elastase (small gold particles) antibodies. Scale bar = 0.25 μm. All images are representative of five independent experiments. C DNA–histone complex formation. Animals were treated with GZD824 (5 mg/kg) or vehicle prior to CLP induction. Mice treated with saline (sham) or GZD824 alone without CLP. Samples were collected 24 h after induction of CLP. Data are presented as mean values ± standard error of the mean (SEM) and n = 5. #P < 0.05 vs. Sham and *P < 0.05 vs. Vehicle + CLP.
Fig. 3
Fig. 3. NET formation in neutrophils.
NETs were generated from isolated neutrophils by TNF-α-stimulation, co-incubated with or without GZD824 (500 nM). Non-stimulated neutrophils served as a control. A Levels of citrullinated histone 3 and MPO in isolated neutrophils detected by flow cytometry and B DNA-histone complexes in the supernatant determined by ELISA. Data represent means ± SEM and n = 5. #P < 0.05 versus control mice and *P < 0.05 versus Vehicle + TNF-α. C Neutrophils were immune-stained with antibodies to citrullinated histone 3 (H3cit), myeloperoxidase (MPO), and DAPI nuclear stain. Representative confocal fluorescence microscopy images from four independent experiments. Scale bar indicates 10 μm.
Fig. 4
Fig. 4. ROS formation in neutrophils.
Quantification of ROS formation in isolated neutrophils by flow cytometry. Neutrophils were stimulated by TNF-α with or without GZD824 (500 nM). Nonstimulated neutrophils served as a control. A Representative histogram of ROS generation and B aggregate data. Data are presented as mean values ± standard error of the mean (SEM) and n = 5. #P < 0.05 versus control mice and *P < 0.05 versus Vehicle + TNF-α.
Fig. 5
Fig. 5. Representative haematoxylin & eosin sections of the lung.
A Animals were treated with saline or B GZD824 alone. Separate mice were pretreated with C vehicle or D 5 mg/kg of GZD824 prior to CLP induction. E Lung injury score and F edema formation were quantified as described in Materials and Methods. Data are presented as mean values ± standard error of the mean (SEM) and n = 5. #P < 0.05 vs. Sham and *P < 0.05 vs. Vehicle + CLP. Samples were harvested 24 h after CLP induction. Scale bar indicates 100 μm.
Fig. 6
Fig. 6. c-Abl kinase regulates CLP-induced infiltration of neutrophils in the lung.
A Number of BALF neutrophils were determined 24 h after CLP induction. Pulmonary levels of B CXCL1 and C CXCL2. Animals were treated with GZD824 (5 mg/kg) or vehicle prior to CLP induction. Mice treated with saline (sham) or GZD824 alone without CLP. Mice treated with PBS served as sham animals. Data are presented as mean values ± standard error of the mean (SEM) and n = 5. #P < 0.05 vs. Sham and *P < 0.05 vs. Vehicle + CLP.
Fig. 7
Fig. 7. c-Abl kinase regulates CLP-induced systemic inflammation.
Plasma levels of A IL-6, B CXCL1, and C CXCL2. Animals were treated with GZD824 (5 mg/kg) or vehicle prior to CLP induction. Mice treated with PBS (sham) or GZD824 alone without CLP. Data are presented as mean values ± standard error of the mean (SEM) and n = 5. #P < 0.05 vs. Sham and *P < 0.05 vs. Vehicle + CLP.
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