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Increased intracellular Cl concentration mediates neutrophil extracellular traps formation in atherosclerotic cardiovascular diseases

Abstract

Neutrophil extracellular traps (NETs) play crucial roles in atherosclerotic cardiovascular diseases such as acute coronary syndrome (ACS). Our preliminary study shows that oxidized low-density lipoprotein (oxLDL)-induced NET formation is accompanied by an elevated intracellular Cl concentration ([Cl]i) and reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression in freshly isolated human blood neutrophils. Herein we investigated whether and how [Cl]i regulated NET formation in vitro and in vivo. We showed that neutrophil [Cl]i and NET levels were increased in global CFTR null (Cftr−/−) mice in the resting state, which was mimicked by intravenous injection of the CFTR inhibitor, CFTRinh-172, in wild-type mice. OxLDL-induced NET formation was aggravated by defective CFTR function. Clamping [Cl]i at high levels directly triggered NET formation. Furthermore, we demonstrated that increased [Cl]i by CFTRinh-172 or CFTR knockout increased the phosphorylation of serum- and glucocorticoid-inducible protein kinase 1 (SGK1) and generation of intracellular reactive oxygen species in neutrophils, and promoted oxLDL-induced NET formation and pro-inflammatory cytokine production. Consistently, peripheral blood samples obtained from atherosclerotic ApoE−/− mice or stable angina (SA) and ST-elevation ACS (STE-ACS) patients exhibited increased neutrophil [Cl]i and SGK1 activity, decreased CFTR expression, and elevated NET levels. VX-661, a CFTR corrector, reduced the NET formation in the peripheral blood sample obtained from oxLDL-injected mice, ApoE−/− atherosclerotic mice or patients with STE-ACS by lowering neutrophil [Cl]i. These results demonstrate that elevated neutrophil [Cl]i during the development of atherosclerosis and ACS contributes to increased NET formation via Cl-sensitive SGK1 signaling, suggesting that defective CFTR function might be a novel therapeutic target for atherosclerotic cardiovascular diseases.

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Fig. 1: OxLDL increased [Cl]i and decreased CFTR expression in neutrophils during NET formation, and 100 mM [Cl]i induces NET formation.
Fig. 2: CFTR dysfunction induces NET formation by regulating neutrophil [Cl]i.
Fig. 3: Increased neutrophil [Cl]i resulting from CFTR deficiency aggravates oxLDL-induced NET formation, whereas VX-661 reverses it.
Fig. 4: Phosphorylated SGK1 levels are augmented in neutrophils upon stimulation with high [Cl]i or oxLDL.
Fig. 5: Higher neutrophil [Cl]i promotes ROS production during NET formation.
Fig. 6: Elevated neutrophil [Cl]i, NET burden, and proinflammatory cytokine levels in the peripheral blood of atherosclerotic ApoE−/− mice on a high-fat diet.
Fig. 7: Elevated NET burden in peripheral blood of SA and STE-ACS patients with alterations of neutrophil CFTR, [Cl]i, and SGK1 activity.

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos. 82073848, 81773722, 81903687, 61672547, 81773721, and 81803522); the Science and Technology Program of Guangzhou City (No. 201803010092; China); Guangdong Natural Science Foundation (No. 2020A1515010045; China); Guangdong Provincial Department of Science and Technology (No. 2017A020215104; China).

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GLW, BZ, and HH designed the study. HH, CL, ML, and JW performed the experiments and analyzed the data. YZ, YSL, ZCL, ZHL, RH, RMW, and YYG assisted with the experiments. GLW, HH, and BZ wrote the manuscript. All authors have approved the final article.

Corresponding authors

Correspondence to Bin Zhang or Guan-lei Wang.

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The authors declare no competing interests.

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Han, H., Liu, C., Li, M. et al. Increased intracellular Cl concentration mediates neutrophil extracellular traps formation in atherosclerotic cardiovascular diseases. Acta Pharmacol Sin (2022). https://doi.org/10.1038/s41401-022-00911-9

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  • DOI: https://doi.org/10.1038/s41401-022-00911-9

Keywords

  • atherosclerotic cardiovascular diseases
  • neutrophil extracellular traps
  • intracellular chloride
  • CFTR
  • oxLDL
  • SGK1

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