A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes
File(s)
Accepted version
Accepted version
Author(s)
Type
Journal Article
Abstract
A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.
Date Issued
2016-02-27
Date Acceptance
2016-02-15
Citation
Autoimmunity Reviews, 2016, 15 (6), pp.577-584
ISSN
1873-0183
Publisher
Elsevier
Start Page
577
End Page
584
Journal / Book Title
Autoimmunity Reviews
Volume
15
Issue
6
Copyright Statement
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
(http://creativecommons.org/licenses/by/4.0/).
License URL
Sponsor
Imperial College Healthcare NHS Trust- BRC Funding
Grant Number
RDA04 79560
Subjects
Autoimmune disease
Neutrophil extracellular traps
Neutrophils
Immunology
1107 Immunology
Publication Status
Published