CPHEN-017: Comprehensive phenotyping of neutrophil extracellular traps (NETs) on peripheral human neutrophils

With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSC^highFSC^highCD15⁺CD66b⁺. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR⁺7-AAD⁺) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIR^dim7-AAD⁺) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins – myeloperoxidase (MPO) and neutrophil elastase (NE) – were utilized to identify neutrophil-appendant NET events (SSC^highFSC^highCD15⁺CD66b⁺Zombie NIR^dim7-AAD⁺MPO⁺NE⁺). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.