IL ‐ 17A Promotes NETs Formation via the PKCζ – ERK – ROS – PAD4 Pathway in a Mouse Model of Ischemic Stroke

ABSTRACT Aims Interleukin‐17A (IL‐17A) aggravates poststroke neurological damage and enhances neutrophil extracellular traps (NETs) formation, yet the underlying mechanism remains unclear. This study aimed to elucidate how IL‐17A regulates NETs generation after ischemic stroke. Methods Using a mouse middle cerebral artery occlusion (MCAO) model, we administered the Peptidylarginine deiminase 4 (PAD4) inhibitor GSK484 or an IL‐17A‐neutralizing antibody (IL‐17mAb). Infarct volume and neurological function were assessed, and protein expression of PAD4, myeloperoxidase (MPO), citrullinated histones H3 (CitH3), protein kinase Cζ (PKCζ), and phosphorylated extracellular signal‐regulated kinase (p‐ERK) was evaluated. IL‐17A − / − mice and primary neutrophils were used to further validate the signaling pathway. Results Inhibiting PAD4 with GSK484 can significantly reduce infarct size, improve neurological outcomes, and decrease PAD4, MPO, and CitH3 protein levels. Subsequently, we found that IL‐17mAb treatment can reduce the expression of PAD4, MPO, and CitH3 in the peri‐infarct area, as well as the expression of PKC‐ζ and p‐ERK. Similarly, the expression of PAD4 and CitH3 decreased in the peri‐infarct area of IL‐17A − / − MCAO mice. Finally, we verified IL‐17A induced PAD4 upregulation via the PKCζ–ERK–ROS axis in vitro. Conclusion IL‐17A promotes NETs formation by upregulating PAD4 through the PKCζ–ERK–ROS pathway, exacerbating ischemic brain injury. Targeting this axis may offer a novel therapeutic strategy for stroke.