Pharmacological inhibition of sodium-calcium exchange activates NADPH oxidase and induces infection-independent NETotic cell death.

In addition to its function of innate immunity against invading pathogens, neutrophil extracellular traps (NETs) promote thrombosis, autoimmune disease, and cancer metastasis; therefore, unnecessary exposure to the triggers of infection-independent NET generation should be avoided. We herein show that inhibition of forward-mode Na/Ca exchange by amiloride analogs, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and 5-(N-Methyl-N-isobutyl)amiloride (MIA), triggers NETotic cell death independently of infectious stimuli. Isolated human neutrophils treated with EIPA and MIA undergo NETotic cell death by an increase of intracellular Ca following activation of NADPH oxidase and the resultant upregulation of intracellular ROS. EIPA- and MIA-mediated intracellular Ca increase is attributed to the competitive binding of EIPA and MIA against Na to Na/Ca exchanger 1 (NCX1). These results demonstrate a new mechanism of infection-independent NET generation and implicate NCX1 as a physiologic regulator of intracellular calcium balance and NETotic cell death.