Oral Presentation Australasian Cytometry Society 44th Annual Conference and Workshop

Comprehensive immunophenotyping of Neutrophil Extracellular Traps (NETs). (24541)

Ceridwyn Jones 1 , Anne Camille La Flamme 1 , Peter Larsen 2 , Kathryn Hally 2
  1. Victoria University of Wellington, Wellington, New Zealand
  2. University of Otago, Wellington, New Zealand

Introduction. 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, and there is considerable interest in how these structures drive disease processes in humans. Our ability to advance research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 10-marker flow cytometry panel for analysing NETosis on human peripheral neutrophils.

Methods. This panel was optimized on healthy neutrophils in vitro stimulated with 1.9-13.3 nM phorbol 12-myristate 13-acetate (PMA; a known NET-inducing agonist) for 2 hours at 37°C/5% CO2. Isolated neutrophils were also either left unstimulated or incubated with 0.5 µg/mL fibroblast-stimulating lipopeptide (FSL)-1 (a known neutrophil activator that does not induce NETosis) or 10% DMSO (positive control for apoptosis and necrosis). Neutrophils were then stained for 30 minutes at 4°C and analysed.

Results. PMA stimulation caused shedding of CD16, a marker commonly used for identifying neutrophils. Thus, neutrophils were alternatively identified as SSChighFSChighCD15+CD66b+. Necrotic neutrophils (Annexin-V++Zombie-NIR++) were removed from all conditions. Myeloperoxidase (MPO) and neutrophil elastase (NE) were utilized to identify NETs (MPO+NE+) appendant to neutrophils. PMA induced a dose-dependent increase in NET+-neutrophils (13.3-32.6%), while NETs were largely absent on unstimulated or FSL-1-stimulated neutrophils (<6%). Across PMA concentrations, 23-63% of NET+-neutrophils were also positive for citrullinated histone H3, a marker of peptidyl arginine deiminase-4 (PAD4)-dependent NETosis. NETosis was similarly induced on both Annexin-V- and Annexin-V+ neutrophils. NET+-neutrophils had substantially increased expression of CD11b (3.6±1.8-fold increase over non-NETotic neutrophils) and CD66b (2.7±1.8-fold), and slightly increased expression of CD15 (1.8±0.2-fold) and CD11c (1.5±0.3-fold), all of which are markers of neutrophil activation.

Conclusion. This flow cytometry panel provides a novel tool for examining NETosis in human disease.