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Front. Immunol. | doi: 10.3389/fimmu.2018.00337

Development and Application of High Content Biological Screening for Modulators of NET Production

 Ilaria J. Chicca1, 2*, Michael R. Milward1, Iain L. Chapple1, Gareth Griffiths2, Rod Benson2, Thomas Dietrich1 and  Paul R. Cooper1*
  • 1Institute of Clinical Sciences, School of Dentistry, University of Birmingham, United Kingdom
  • 2Imagen Therapeutics Ltd, United Kingdom

Neutrophil extracellular traps (NETs) are DNA-based antimicrobial web-like structures whose release is predominantly mediated by reactive oxygen species (ROS); their purpose is to combat infections. However, unbalanced NET production and clearance is involved in tissue injury, circulation of auto-antibodies and development of several chronic diseases. Currently, there is lack of agreement regarding the high-throughput methods available for NET investigation. This study therefore aimed to develop and optimise a High-Content Analysis (HCA) approach, which can be applied for the assay of NET production and for the screening of compounds involved in the modulation of NET release. A suitable paraformaldehyde fixation protocol was established to enable HCA of neutrophils and NETs. Bespoke and in-built bioinformatics algorithms were validated by comparison with standard low-throughput approaches for application in HCA of NETs. Subsequently, the optimised protocol was applied to High-Content Screening (HCS) of a pharmaceutically derived compound library to identify modulators of NETosis. Of 56 compounds assessed, 8 were identified from HCS for further characterisation of their effects on NET formation as being either inducers, inhibitors or biphasic modulators. The effects of these compounds on naïve neutrophils were evaluated by using specific assays for the induction of ROS and NET production, while their modulatory activity was validated in phorbol 12-myristate 13-acetate stimulated neutrophils. Results indicated the involvement of glutathione reductase, Src family kinases, molecular-target-of-Rapamycin and mitogen-activated-protein-kinase pathways in NET release. The compounds and pathways identified may provide targets for novel therapeutic approaches for treating NET-associated pathologies.

Keywords: NETs, HCA, HCS, mTOR, nuclear decondensation, MAPK, Src family kinases, Glutathione Reductase

Received: 26 Jul 2017; Accepted: 06 Feb 2018.

Edited by:

Masaaki Murakami, Institute for Genetic Medicine, Hokkaido University, Japan

Reviewed by:

Keigo Nishida, Suzuka University of Medical Science, Japan
Ikuma Nakagawa, Hokkaido University, Japan  

Copyright: © 2018 Chicca, Milward, Chapple, Griffiths, Benson, Dietrich and Cooper. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Ilaria J. Chicca, University of Birmingham, Institute of Clinical Sciences, School of Dentistry, 5 Mill Pool Way, Birmingham, B5 7EG, United Kingdom,
Prof. Paul R. Cooper, University of Birmingham, Institute of Clinical Sciences, School of Dentistry, 5 Mill Pool Way, Birmingham, B5 7EG, United Kingdom,