The primary objective of the current study was to investigate the potential of the
pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap
(NET) formation in vitro.
Isolated human blood neutrophils were exposed to recombinant Ply (2.5-20 ng.ml-1)
for 30-90 min at 37°C and NET formation measured using the following procedures
to detect extracellular DNA: i) flow cytometry using Vybrant Dye Cycle Ruby; ii)
spectrofluorimetry using the fluorophore, Sytox® Orange (5 μM); iii) and NanoDrop®
technology. These procedures were complemented by fluorescence microscopy
using DAPI (nuclear stain) in combination with anti-citrullinated histone monoclonal
antibodies to visualise nets.
Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min),
statistically significant (p<0.05) dose- and time-related increases in the release of
cellular DNA impregnated with both citrullinated histone and myeloperoxidase.
Microscopy revealed that NETosis appeared to be restricted to a subpopulation of
neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems
(10 and 20 ng.ml-1 ) were 4.3(4.2), 14.3(9.9) and 16.5(7.5) respectively (n=4,
p<0.0001 for comparison of the control with both Ply-treated systems). Ply-induced
NETosis occurred in the setting of retention of cell viability, and apparent lack of
involvement of reactive oxygen species and Toll-like receptor 4.
In conclusion, Ply induces vital NETosis in human neutrophils, a process which may
either contribute to host defence or worsen disease severity depending on the
intensity of the inflammatory response during pneumococcal infection.