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

Maarten Van Der Linden, Geertje H.A. Westerlaken, Michiel Van Der Vlist, Joris Van Montfrans, Linde Meyaard*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


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.

Original languageEnglish
Article number6529
JournalScientific Reports
Issue number1
Publication statusPublished - 1 Dec 2017


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