Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging

Abstract A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here,...

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Autores principales:	Maarten van der Linden, Geertje H. A. Westerlaken, Michiel van der Vlist, Joris van Montfrans, Linde Meyaard
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0aff07b3d8b34ad7ac7bbd1c1165cffd
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spelling	oai:doaj.org-article:0aff07b3d8b34ad7ac7bbd1c1165cffd2021-12-02T11:53:03ZDifferential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging10.1038/s41598-017-06901-w2045-2322https://doaj.org/article/0aff07b3d8b34ad7ac7bbd1c1165cffd2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06901-whttps://doaj.org/toc/2045-2322Abstract A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.Maarten van der LindenGeertje H. A. WesterlakenMichiel van der VlistJoris van MontfransLinde MeyaardNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Maarten van der Linden Geertje H. A. Westerlaken Michiel van der Vlist Joris van Montfrans Linde Meyaard Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging
description	Abstract A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.
format	article
author	Maarten van der Linden Geertje H. A. Westerlaken Michiel van der Vlist Joris van Montfrans Linde Meyaard
author_facet	Maarten van der Linden Geertje H. A. Westerlaken Michiel van der Vlist Joris van Montfrans Linde Meyaard
author_sort	Maarten van der Linden
title	Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging
title_short	Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging
title_full	Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging
title_fullStr	Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging
title_full_unstemmed	Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging
title_sort	differential signalling and kinetics of neutrophil extracellular trap release revealed by quantitative live imaging
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/0aff07b3d8b34ad7ac7bbd1c1165cffd
work_keys_str_mv	AT maartenvanderlinden differentialsignallingandkineticsofneutrophilextracellulartrapreleaserevealedbyquantitativeliveimaging AT geertjehawesterlaken differentialsignallingandkineticsofneutrophilextracellulartrapreleaserevealedbyquantitativeliveimaging AT michielvandervlist differentialsignallingandkineticsofneutrophilextracellulartrapreleaserevealedbyquantitativeliveimaging AT jorisvanmontfrans differentialsignallingandkineticsofneutrophilextracellulartrapreleaserevealedbyquantitativeliveimaging AT lindemeyaard differentialsignallingandkineticsofneutrophilextracellulartrapreleaserevealedbyquantitativeliveimaging
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Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging

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