Abstract
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.
Highlights
Neutrophils are key components of the innate immune response and migrate to infected peripheral tissues to combat invading microorganisms [1]
The fixation agent directly interacts with the stimulated Neutrophil extracellular traps (NETs) potentially protecting them from further downstream mechanical disruption that may result from subsequent steps in the procedure
Several automated and high-throughput techniques have previously been proposed for NET analysis [21, 23, 24, 44], many of those approaches exhibit a number of shortcomings including lack of/limited reproducibility and the limitation of single parameter analysis
Summary
Neutrophils are key components of the innate immune response and migrate to infected peripheral tissues to combat invading microorganisms [1]. Well-known antimicrobial strategies employed by neutrophils include phagocytosis, degranulation of antimicrobial peptides and reactive oxygen species (ROS) [2]. Another strategy neutrophils utilize for clearing or limiting infections in tissues is via their release of neutrophil extracellular traps (NETs) [3]. The formation and physiological function of NETs were initially described as a neutrophil antimicrobial strategy targeted against both Gram-positive and Gram-negative bacteria [3]. Fundamental steps in NET formation include the production of ROS, the de-condensation of nuclear chromatin modulated by the peptidylarginine deiminase 4 enzyme, the mixing of DNA with granule proteins and, the active extrusion of the NETs through the outer cell membrane [4, 5]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
