Adenosine Triphosphate Accumulated Following Cerebral Ischemia Induces Neutrophil Extracellular Trap Formation

In ischemic stroke, neutrophils infiltrate damaged brain tissue immediately following the ischemic insult and aggravate inflammation via various mechanisms which include neutrophil extracellular traps (NETs) formation. In the present study, we showed that adenosine triphosphate (ATP), a DAMP molecule, accumulates in the brain and induces NETosis in brain parenchyma and in circulating neutrophils (PMNs) isolated from a murine model of stroke induced by middle cerebral artery occlusion (MCAO). Expression of peptidylarginine deiminase-4 (PAD4), which induces citrullination of histones H3 (CitH3) and initiates NETosis, was significantly enhanced in brain parenchyma and blood PMNs following MCAO. ATP or BzATP (a prototypic P2X7R agonist) significantly enhanced the inductions of PAD4 and CitH3 in a P2X7R-dependent manner and intracellular Ca²⁺ influx, PKCα activation, and NADPH oxidase-dependent reactive oxygen species (ROS) production play critical roles in this ATP-P2X7R-mediated NETosis. In our MCAO animal model, NETosis was markedly suppressed by treatment with apyrase, an enzyme hydrolyzing ATP, but enhanced by co-treatment of BzATP, confirming ATP-P2X7R-mediated NETosis. Since ATP not only induced NETosis but was also extruded after NETosis, our results indicate that ATP accumulated in the ischemic brain induces NETosis, mediating a cross-talk linking NETosis with neuronal damage that might aggravate inflammation and brain damage.