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

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, CFTR_inh-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 CFTR_inh-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.