Source: International Congress 2015 – Advances from translational research in respiratory infections
Disease area: Respiratory infections

Abstract

Introduction: NET formation, driven by the process of NETosis, represents a novel strategy utilised by cells of the innate immune system, especially neutrophils, to trap and dispose of extracellular pathogens. Some bacterial pathogens, such as the pneumococcus appear to be adept not only at evading NETs, but also subverting them to promote persistence and dissemination.Aims and objectives: The aim of the current study was to investigate the potential of the pneumococcal toxin, Ply, to activate NETosis.Methods: Isolated human blood neutrophils were exposed to recombinant Ply (5 and 10 ng.ml^-1) for 30 and 60 min at 37°C after which NET formation was measured using the following combination of procedures: i) spectrofluorimetry using the fluorophore, sytox orange (5 mM), as well as NanoDrop® technology to detect extracellular DNA; ii) fluorescence microscopy using DAPI (nuclear stain) and anti-citrullinated histone monoclonal antibodies to visualise nets; and iii) flow cytometry using vibrant dye cycle ruby to detect loss of intracellular DNA.Results: Exposure of neutrophils to Ply resulted in significant increases in the numbers of NET-forming cells (4.3±0.9%, 14.3±0.2%, 16.5±1.5%, for the control and systems treated with 5 and 10 ng.ml^-1 Ply respectively, P <0.0001) which was paralleled by significant (P<0.05) time-related increases in the release of extracellular DNA and citrullinated histones in the setting of loss of intracellular DNA and retention of cell viability.Conclusions: Ply induces vital NETosis in human neutrophils, which may contribute to either host defence or disease severity depending on the intensity of the inflammatory response during pneumococcal infection.


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Charles Feldman (Johannesburg, South Africa), Jan-Gert Nel, Annette Theron, Chrisna Durandt, Gregory Tintinger, Roger Pool, Timothy Mitchell, Ronald Anderson, Charles Feldman. Induction of neutrophil extracellular traps (NETs) by the pneumococcal toxin, pneumolysin (Ply). Eur Respir J 2015; 46: Suppl. 59, 5041

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