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

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-an...

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Autores principales: Chicca, Ilaria J., Milward, Michael R., Chapple, Iain Leslie C., Griffiths, Gareth, Benson, Rod, Dietrich, Thomas, Cooper, Paul R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844942/
https://www.ncbi.nlm.nih.gov/pubmed/29556228
http://dx.doi.org/10.3389/fimmu.2018.00337
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author Chicca, Ilaria J.
Milward, Michael R.
Chapple, Iain Leslie C.
Griffiths, Gareth
Benson, Rod
Dietrich, Thomas
Cooper, Paul R.
author_facet Chicca, Ilaria J.
Milward, Michael R.
Chapple, Iain Leslie C.
Griffiths, Gareth
Benson, Rod
Dietrich, Thomas
Cooper, Paul R.
author_sort Chicca, Ilaria J.
collection PubMed
description 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 optimize 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 optimized 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 characterization 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.
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spelling pubmed-58449422018-03-19 Development and Application of High-Content Biological Screening for Modulators of NET Production Chicca, Ilaria J. Milward, Michael R. Chapple, Iain Leslie C. Griffiths, Gareth Benson, Rod Dietrich, Thomas Cooper, Paul R. Front Immunol Immunology 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 optimize 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 optimized 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 characterization 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. Frontiers Media S.A. 2018-03-05 /pmc/articles/PMC5844942/ /pubmed/29556228 http://dx.doi.org/10.3389/fimmu.2018.00337 Text en Copyright © 2018 Chicca, Milward, Chapple, Griffiths, Benson, Dietrich and Cooper. http://creativecommons.org/licenses/by/4.0/ 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.
spellingShingle Immunology
Chicca, Ilaria J.
Milward, Michael R.
Chapple, Iain Leslie C.
Griffiths, Gareth
Benson, Rod
Dietrich, Thomas
Cooper, Paul R.
Development and Application of High-Content Biological Screening for Modulators of NET Production
title Development and Application of High-Content Biological Screening for Modulators of NET Production
title_full Development and Application of High-Content Biological Screening for Modulators of NET Production
title_fullStr Development and Application of High-Content Biological Screening for Modulators of NET Production
title_full_unstemmed Development and Application of High-Content Biological Screening for Modulators of NET Production
title_short Development and Application of High-Content Biological Screening for Modulators of NET Production
title_sort development and application of high-content biological screening for modulators of net production
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844942/
https://www.ncbi.nlm.nih.gov/pubmed/29556228
http://dx.doi.org/10.3389/fimmu.2018.00337
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