Pneumolysin activates neutrophil extracellular trap formation
Corresponding Author
J. G. Nel
Department of Haematology
Correspondence: J. G. Nel, Department of Haematology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service, PO Box 2034, Pretoria 0001, South Africa. E-mail: [email protected]Search for more papers by this authorA. J. Theron
Department of Immunology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service
South African Medical Research Council Unit for Stem Cell Research, Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria
Search for more papers by this authorC. Durandt
South African Medical Research Council Unit for Stem Cell Research, Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria
Search for more papers by this authorG. R. Tintinger
Department of Internal Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
Search for more papers by this authorT. J. Mitchell
Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
Search for more papers by this authorC. Feldman
Division of Pulmonology, Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg and Charlotte Maxeke Academic Hospital, Johannesburg, South Africa
Search for more papers by this authorR. Anderson
South African Medical Research Council Unit for Stem Cell Research, Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria
Search for more papers by this authorCorresponding Author
J. G. Nel
Department of Haematology
Correspondence: J. G. Nel, Department of Haematology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service, PO Box 2034, Pretoria 0001, South Africa. E-mail: [email protected]Search for more papers by this authorA. J. Theron
Department of Immunology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service
South African Medical Research Council Unit for Stem Cell Research, Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria
Search for more papers by this authorC. Durandt
South African Medical Research Council Unit for Stem Cell Research, Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria
Search for more papers by this authorG. R. Tintinger
Department of Internal Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
Search for more papers by this authorT. J. Mitchell
Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
Search for more papers by this authorC. Feldman
Division of Pulmonology, Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg and Charlotte Maxeke Academic Hospital, Johannesburg, South Africa
Search for more papers by this authorR. Anderson
South African Medical Research Council Unit for Stem Cell Research, Institute for Cellular and Molecular Medicine, Department of Immunology, Faculty of Health Sciences, University of Pretoria
Search for more papers by this authorSummary
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 (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® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox® Orange (5 μM); and (iii) NanoDrop® technology. These procedures were complemented by fluorescence microscopy using 4′, 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize 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.
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