title: Pneumolysin activates neutrophil extracellular trap formation.
abstract: 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.
authors.affiliation: Department of Haematology.
authors.forename: J G
authors.initials: JG
authors.lastname: Nel
authors.affiliation: 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.
authors.forename: A J
authors.initials: AJ
authors.lastname: Theron
authors.affiliation: 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.
authors.forename: C
authors.initials: C
authors.lastname: Durandt
authors.affiliation: Department of Internal Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
authors.forename: G R
authors.initials: GR
authors.lastname: Tintinger
authors.affiliation: Department of Haematology.
authors.forename: R
authors.initials: R
authors.lastname: Pool
authors.affiliation: Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.
authors.forename: T J
authors.initials: TJ
authors.lastname: Mitchell
authors.affiliation: Division of Pulmonology, Department of Internal Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg and Charlotte Maxeke Academic Hospital, Johannesburg, South Africa.
authors.forename: C
authors.initials: C
authors.lastname: Feldman
authors.affiliation: 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.
authors.forename: R
authors.initials: R
authors.lastname: Anderson
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001426:Bacterial Proteins / Q000494:pharmacology; D002470:Cell Survival; D002956:Citrulline / Q000276:immunology; D004247:DNA / Q000819:agonists / Q000276:immunology* / Q000378:metabolism; D065206:Extracellular Traps / Q000276:immunology*; D015870:Gene Expression; D006657:Histones / Q000235:genetics / Q000276:immunology; D006801:Humans; D007211:Indoles; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D009195:Peroxidase / Q000235:genetics / Q000276:immunology; D061251:Primary Cell Culture; D017382:Reactive Oxygen Species / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D013296:Streptococcus pneumoniae / Q000737:chemistry; D013301:Streptolysins / Q000494:pharmacology*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D006657:Histones; D007211:Indoles; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013301:Streptolysins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C032290:plY protein, Streptococcus pneumoniae; D002956:Citrulline; C007293:DAPI; D004247:DNA; D009195:Peroxidase
keywords: NETosis; Toll-like receptor 4; calcium; chronic granulomatous disease; neutrophils; pneumolysin; reactive oxygen species