Name: Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists (Video) | JoVE Journal
Uploaded: 2024-10-18T14:20:00.000+00:00
Duration: 11 min 32 s
Description: Learn a real-time microscopy method for quantifying human neutrophil extracellular trap release and assessing NETosis antagonist pharmacology. This high-throughput technique enables studying innate immune defense mechanisms.

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The authors would like to thank Paul Vink for managing part of the project regarding the live imaging microscopy method.

Real-Time Microscopy for Studying NETosis Kinetics and Screening Inhibitors

<ol><li>Brinkmann, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+kill+bacteria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Neutrophil extracellular traps kill bacteria.</a> Science. 303 (5663), 1532-1535 (2004).</li>
<li>De Bont, C., Pruijn, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Citrulline+is+not+a+major+determinant+of+autoantibody+reactivity+to+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22Philos+Trans+R+Soc+Lond+B+Biol+Sci%22%5BJournal%5D)">Citrulline is not a major determinant of autoantibody reactivity to neutrophil extracellular traps.</a> Philos Trans R Soc Lond B Biol Sci. 378 (1890), 20220249(2023).</li>
<li>Urban, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+contain+calprotectin%2C+a+cytosolic+protein+complex+involved+in+host+defense+against+Candida+albicans%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.</a> PLoS Pathog. 5 (10), e1000639(2009).</li>
<li>Hoppenbrouwers, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+induction+of+NETosis%3A+Comprehensive+live+imaging+comparison+and+systematic+review%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review.</a> PLoS One. 12 (5), e0176472(2017).</li>
<li>Kenny, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+stimuli+engage+different+neutrophil+extracellular+trap+pathways%5BTitle%5D)+AND+%22ELife%22%5BJournal%5D)">Diverse stimuli engage different neutrophil extracellular trap pathways.</a> ELife. 6, e24437(2017).</li>
<li>Parker, H., Dragunow, M., Hampton, M. B., Kettle, A. J., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Requirements+for+NADPH+oxidase+and+myeloperoxidase+in+neutrophil+extracellular+trap+formation+differ+depending+on+the+stimulus%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus.</a> J Leukoc Biol. 92 (4), 841-849 (2012).</li>
<li>Neeli, I., Radic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Opposition+between+PKC+isoforms+regulates+histone+deimination+and+neutrophil+extracellular+chromatin+release%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release.</a> Front Immunol. 4, 38(2013).</li>
<li>Bosmann, M., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein-based+therapies+for+acute+lung+injury%3A+targeting+neutrophil+extracellular+traps%5BTitle%5D)+AND+%22Expert+Opin+Ther+Targets%22%5BJournal%5D)">Protein-based therapies for acute lung injury: targeting neutrophil extracellular traps.</a> Expert Opin Ther Targets. 18 (6), 703-714 (2014).</li>
<li>Papayannopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+in+immunity+and+disease%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Neutrophil extracellular traps in immunity and disease.</a> Nat Rev Immunol. 18 (2), 134-147 (2018).</li>
<li>Shiratori-Aso, S., Nakazawa, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+involvement+of+NETs+in+ANCA-associated+vasculitis%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">The involvement of NETs in ANCA-associated vasculitis.</a> Front Immunol. 14, 1261151(2023).</li>
<li>Byrd, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%2C+B+cells%2C+and+type+I+interferons+contribute+to+immune+dysregulation+in+hidradenitis+suppurativa%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Neutrophil extracellular traps, B cells, and type I interferons contribute to immune dysregulation in hidradenitis suppurativa.</a> Sci Transl Med. 11 (508), eaav5908(2019).</li>
<li>Kimball, A. S., Obi, A. T., Diaz, J. A., Henke, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+emerging+role+of+NETs+in+venous+thrombosis+and+immunothrombosis%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">The emerging role of NETs in venous thrombosis and immunothrombosis.</a> Front Immunol. 7, 236(2016).</li>
<li>O'Neil, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+trap-associated+carbamylation+and+histones+trigger+osteoclast+formation+in+rheumatoid+arthritis%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">Neutrophil extracellular trap-associated carbamylation and histones trigger osteoclast formation in rheumatoid arthritis.</a> Ann Rheum Dis. 82 (5), 630-638 (2023).</li>
<li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+peptidylarginine+deiminase+4+%28PAD4%29+inhibitor+BMS-P5+blocks+formation+of+neutrophil+extracellular+traps+and+delays+progression+of+multiple+myeloma%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">A novel peptidylarginine deiminase 4 (PAD4) inhibitor BMS-P5 blocks formation of neutrophil extracellular traps and delays progression of multiple myeloma.</a> Mol Cancer Ther. 19 (7), 1530-1538 (2020).</li>
<li>Chirivi, R. G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-citrullinated+protein+antibodies+as+novel+therapeutic+drugs+in+rheumatoid+arthritis%5BTitle%5D)+AND+%22J+Clin+Cell+Immunol%22%5BJournal%5D)">Anti-citrullinated protein antibodies as novel therapeutic drugs in rheumatoid arthritis.</a> J Clin Cell Immunol. S6, 006(2013).</li>
<li>Van der Linden, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-citrullinated+histone+monoclonal+antibody+CIT-013%2C+a+dual+action+therapeutic+for+neutrophil+extracellular+trap-associated+autoimmune+diseases%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Anti-citrullinated histone monoclonal antibody CIT-013, a dual action therapeutic for neutrophil extracellular trap-associated autoimmune diseases.</a> MAbs. 15 (1), 2281763(2023).</li>
<li>Chirivi, R. G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+ACPA+inhibits+NET+formation%3A+a+potential+therapy+for+neutrophil-mediated+inflammatory+diseases%5BTitle%5D)+AND+%22Cell+Mol+Immunol%22%5BJournal%5D)">Therapeutic ACPA inhibits NET formation: a potential therapy for neutrophil-mediated inflammatory diseases.</a> Cell Mol Immunol. 18 (6), 1528-1544 (2021).</li>
<li>Van der Linden, M., Westerlaken, G. H. A., Van Der Vlist, M., Van Montfrans, J., Meyaard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+signalling+and+kinetics+of+neutrophil+extracellular+trap+release+revealed+by+quantitative+live+imaging%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Differential signalling and kinetics of neutrophil extracellular trap release revealed by quantitative live imaging.</a> Sci Rep. 7 (1), 6529(2017).</li>
<li>Hoffmann, J. H. O., Schaekel, K., Gaiser, M. R., Enk, A. H., Hadaschik, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interindividual+variation+of+NETosis+in+healthy+donors%3A+introduction+and+application+of+a+refined+method+for+extracellular+trap+quantification%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification.</a> Exp Dermatol. 25 (11), 895-900 (2016).</li>
<li>Silva, L. M., Moutsopoulos, N., Bugge, T. H., Doyle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+and+quantification+of+neutrophil+extracellular+trap+formation%5BTitle%5D)+AND+%22Curr+Protoc%22%5BJournal%5D)">Live imaging and quantification of neutrophil extracellular trap formation.</a> Curr Protoc. 1 (7), e157(2021).</li>
<li>De Bont, C. M., Koopman, W. J. H., Boelens, W. C., Pruijn, G. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulus-dependent+chromatin+dynamics%2C+citrullination%2C+calcium+signalling+and+ROS+production+during+NET+formation%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Mol+Cell+Res%22%5BJournal%5D)">Stimulus-dependent chromatin dynamics, citrullination, calcium signalling and ROS production during NET formation.</a> Biochim Biophys Acta Mol Cell Res. 1865 (11 Pt A), 1621-1629 (2018).</li>
<li>Gajendran, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alleviation+of+arthritis+through+prevention+of+neutrophil+extracellular+traps+by+an+orally+available+inhibitor+of+protein+arginine+deiminase+4%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Alleviation of arthritis through prevention of neutrophil extracellular traps by an orally available inhibitor of protein arginine deiminase 4.</a> Sci Rep. 13 (1), 3189(2023).</li>
<li>Biron, B. M., Chung, C. -S., O'Brien, X. M., Chen, Y., Reichner, J. S., Ayala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cl-Amidine+prevents+histone+3+citrullination+and+neutrophil+extracellular+trap+formation%2C+and+improves+survival+in+a+murine+sepsis+model%5BTitle%5D)+AND+%22J+Innate+Immun%22%5BJournal%5D)">Cl-Amidine prevents histone 3 citrullination and neutrophil extracellular trap formation, and improves survival in a murine sepsis model.</a> J Innate Immun. 9 (1), 22-32 (2017).</li>
<li>Perdomo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+activation+and+NETosis+are+the+major+drivers+of+thrombosis+in+heparin-induced+thrombocytopenia%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia.</a> Nat Commun. 10 (1), 1322(2019).</li>
<li>Lood, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps+enriched+in+oxidized+mitochondrial+DNA+are+interferogenic+and+contribute+to+lupus-like+disease%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease.</a> Nat Med. 22 (2), 146-153 (2016).</li>
<li>Kraaij, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+method+for+high-throughput+detection+and+quantification+of+neutrophil+extracellular+traps+reveals+ROS-independent+NET+release+with+immune+complexes%5BTitle%5D)+AND+%22Autoimmun+Rev%22%5BJournal%5D)">A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.</a> Autoimmun Rev. 15 (6), 577-584 (2016).</li>
<li>De Buhr, N., Von Köckritz-Blickwede, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+neutrophil+extracellular+traps+become+visible%5BTitle%5D)+AND+%22J+Immunol+Res%22%5BJournal%5D)">How neutrophil extracellular traps become visible.</a> J Immunol Res. 2016, 1-13 (2016).</li>
<li>Brinkmann, V., Laube, B., Abu Abed, U., Goosmann, C., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutrophil+extracellular+traps%3A+How+to+generate+and+visualize+them%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Neutrophil extracellular traps: How to generate and visualize them.</a> J Vis Exp. (36), e1724(2010).</li>
<li>Zhao, W., Fogg, D. K., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+image-based+quantitative+method+for+the+characterization+of+NETosis%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">A novel image-based quantitative method for the characterization of NETosis.</a> J Immunol Methods. 423, 104-110 (2015).</li>
<li>Gavillet, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+assay+for+direct+quantification+of+neutrophil+extracellular+traps+in+blood+samples%5BTitle%5D)+AND+%22Am+J+Hematol%22%5BJournal%5D)">Flow cytometric assay for direct quantification of neutrophil extracellular traps in blood samples.</a> Am J Hematol. 90 (12), 1155-1158 (2015).</li>
<li>Sil, P., Yoo, D., Floyd, M., Gingerich, A., Rada, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+throughput+measurement+of+extracellular+DNA+release+and+quantitative+NET+formation+in+human+neutrophils+in+vitro%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">High throughput measurement of extracellular DNA release and quantitative NET formation in human neutrophils in vitro.</a> J Vis Exp. (112), e52779(2016).</li>
<li>Gupta, S., Chan, D. W., Zaal, K. J., Kaplan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+real-time+imaging+technique+to+quantify+NETosis+and+distinguish+mechanisms+of+cell+death+in+human+neutrophils%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">A high-throughput real-time imaging technique to quantify NETosis and distinguish mechanisms of cell death in human neutrophils.</a> J Immunol. 200 (2), 869-879 (2018).</li>
<li>D'Cruz, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pseudokinase+MLKL+activates+PAD4-dependent+NET+formation+in+necroptotic+neutrophils%5BTitle%5D)+AND+%22Sci+Signal%22%5BJournal%5D)">The pseudokinase MLKL activates PAD4-dependent NET formation in necroptotic neutrophils.</a> Sci Signal. 11 (546), eaao1716(2018).</li>
<li>Nash, G., Abbitt, K., Tate, K., Jetha, K., Egginton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+the+mechanical+and+adhesive+behaviour+of+human+neutrophils+on+cooling+in+vitro%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">Changes in the mechanical and adhesive behaviour of human neutrophils on cooling in vitro.</a> Pflugers Arch. 442 (5), 762-770 (2001).</li>
<li>Hundhammer, T., Gruber, M., Wittmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paralytic+impact+of+centrifugation+on+human+neutrophils%5BTitle%5D)+AND+%22Biomedicines%22%5BJournal%5D)">Paralytic impact of centrifugation on human neutrophils.</a> Biomedicines. 10 (11), 2896(2022).</li>
<li>Krémer, V., Godon, O., Bruhns, P., Jönsson, F., De Chaisemartin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+methods+determine+human+neutrophil+responses+after+stimulation%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Isolation methods determine human neutrophil responses after stimulation.</a> Front Immunol. 14, 1301183(2023).</li>
<li>Freitas, M., Porto, G., Lima, J. L. F. C., Fernandes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+activation+of+human+neutrophils+in+vitro.+The+importance+of+the+anticoagulant+used+during+blood+collection%5BTitle%5D)+AND+%22Clin+Biochem%22%5BJournal%5D)">Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection.</a> Clin Biochem. 41 (7-8), 570-575 (2008).</li>
</ol>

The authors are employees of Citryll and have financial interests.

Since the discovery of NETs in 2004, many strategies have been developed to experimentally investigate NET release in vitro, with immunofluorescence microcopy being the most common technique to quantify NETosis27,28. Although microscopy is useful to visualize NET release, it has limitations since non-automated microscopic quantification of fixed time point images is rather inaccurate and can suffer from observer bias. Another technique used to study NET release is multispectral imaging flow cytometry29,30, which measures large numbers of netting neutrophils and adopts unbiased analysis but focuses on neutrophils in the early stages of NETosis and does not quantify neutrophils that have released their NETs. Many studies on NETosis kinetics use a DNA dye to quantify NET release in combination with a fluorescence plate reader31. This technique is unable to distinguish NETs from activated or dying neutrophils with intracellular DNA staining and, therefore, is unsuitable for quantification of NET release and studying NETosis antagonists. The above emphasizes that approaches currently in use to study NETosis are valuable but have their limitations.
The real-time microscopy method described in this study addresses many issues found in previously reported techniques. It offers unbiased, semi-automated, high-throughput, reproducible, and accurate NET quantification. Indeed, this approach minimizes pipetting artifacts, like the stretched morphology of NETs18, and it is able to distinguish NETosis from necrosis and apoptosis through the distinct morphology and kinetics of DNA release18,32. In addition, this approach provides the opportunity to study different biochemical pathways for regulated cell death, like NET formation in necroptotic neutrophils, which generate structures that meet the functional and phenotypical criteria of NETs33.
There are several critical steps in this protocol that need to be followed for successful quantification of NETs. First, the correct number of cells per well is important for accurate NET quantification. When neutrophil density is too high, cells and NETs will overlap with adjacent cells and NETs, which makes them hard to distinguish, and as a result, quantification will become inaccurate. Second, it is a prerequisite to use a low concentration of non-toxic plasma membrane impermeable DNA binding dye to stain NETs when secreted into the extracellular environment. Plasma membrane permeable DNA binding dyes can easily induce neutrophil activation or cell death. Third, multiple images per well must be scanned for a representative overview of NET release amongst the heterogeneous neutrophil population. Fourth, the phase contrast image of neutrophils at t = 0 is necessary to correct for the number of neutrophils per image and calculate the percentage of netting neutrophils.
Although this real-time, high-throughput microscopy approach has many advantages over other NET detection assays, there are limitations to this method since, as far as we are aware, there are no fluorescent dyes available to detect additional NET components to confirm the release of NETs. Fluorescent-labeled antibodies for detecting additional NET components can be used but could have unwanted effects since antibody-immune complexes influence neutrophil activation and possibly NETosis. Therefore, we prefer not to use additional antibodies in this assay setup and recommend using well-known NETosis inducing stimuli instead. When not yet established NETosis stimuli are used, we advocate the use of an ELISA to detect DNA complexed with NET-specific proteins such as citrullinated histones prior to the live imaging assay. Secondly, intra- and inter-assay variation may result from donor-to-donor variability, with neutrophils from healthy donors responding differently to various NETosis stimuli due to heterogeneity within the healthy population. To minimize assay variation, neutrophils must be isolated within 1 h from freshly drawn blood and used immediately in the experiment since neutrophils are short-lived and cannot be cryopreserved. Furthermore, it is important that a neutrophil isolation protocol is validated and adopted to minimize neutrophil activation. Neutrophils are sensitive cells and can change their responsiveness to stimuli during the purification process due to mechanical and other types of stress. Neutrophil activation status and NET release can be influenced by the type of needle, blood collection tubes used, incubation temperature, centrifugation speed and the time from blood collection to neutrophil isolation34,35,36,37. An additional neutrophil isolation method that can be considered is described by Kr&#233;mer et al.36 using a negative immunomagnetic selection method without red blood cell lysis. This method resembles untouched neutrophils in whole blood and might be suitable to prevent any neutrophil activation during the purification process. All the above should serve to caution the field that data from different research groups need to be compared with great care.
Overall, the described real-time high-throughput microscopy method enables accurate quantification of NETs in a reproducible and efficient manner and can be used to study the characteristics, magnitude, and kinetics of NET release and enables the investigation of the activity of NETosis antagonists. As an example of the latter, we used the humanized anti-citrullinated histone H2A and H4 monoclonal antibody CIT-013, which is currently in clinical development.

Neutrophils are abundantly present in blood and migrate to tissue upon infection or inflammation. They play an important role in the innate immune defense by using extensive weaponry to protect the host against microbes. Neutrophils kill pathogens via phagocytosis, degranulation, generation of reactive oxygen species (ROS), and the release of decondensed chromatin termed neutrophil extracellular traps (NETs) by a process called NETosis1. NETs are extracellular structures of chromatin decorated with, amongst others, granular proteins and calprotectin2,3, and are released upon stimulation with a broad spectrum of molecules4. NETosis can be broadly categorized into two main pathways: NADPH oxidase dependent or independent5,6,7. In addition, arginine citrullination of N-terminal histone tails by peptidyl arginine deiminase 4 (PAD4) has been closely linked to NETosis and promotes chromatin decondensation which &#65279;ultimately leads to expulsion of decondensed chromatin into the extracellular environment.
Although NET release is involved in pathogen elimination, numerous studies have shown that abnormal and prolonged NET release is linked to the development of various inflammatory disorders, including acute lung injury8, rheumatoid arthritis (RA)9, vasculitis10, and hidradenitis suppurativa11. &#65279;The detrimental role of NETs in disease is multifaceted since NETs are proinflammatory, are a source of autoantigens, are cytotoxic to surrounding tissues, trigger immunothrombosis, and promote osteoclast differentiation and bone erosion9,12,13. &#65279;Pharmacological inhibition of the NETosis pathway by small molecule PAD4 inhibitors demonstrates that NETosis-targeting therapeutics have potential as treatments for diseases in which NET accumulation is an important driver of the pathogenesis14. Instead of targeting the PAD4 enzyme, we used a first-in-class NETosis inhibiting humanized anti-citrullinated histone monoclonal antibody, CIT-013, which specifically binds to citrullinated histones H2A and H415. CIT-013 has a unique dual mechanism of action by inhibiting NET release and enhancing macrophage-mediated NET phagocytosis16. CIT-013 and precursor molecules have shown therapeutic efficacy in multiple mouse models of NET-associated inflammation17.
To study NET release, different methods have been developed over the years, such as but not limited to 1) DNA detection using a plasma membrane impermeable DNA tracer in combination with an immunofluorescent plate reader, 2) enzyme-linked immunosorbent assay (ELISA)-based detection of DNA and DNA-complexed with NET-specific proteins in supernatants, 3) co-localization of NET associated molecules with extracellular DNA via immunohistochemistry, and 4) flow cytometry approaches to detect netting neutrophils. All these methods have their own advantages and limitations. We developed a real-time high-throughput approach for microscopic quantification of human NET release, which uses a plasma membrane impermeable DNA dye16,18. The described method allows investigation of NETosis kinetics and characteristics in an easy, reliable, and reproducible manner and enables assessment of the pharmacology of NETosis antagonists such as CIT-013.

<table><tbody><tr><td>A-23187 Free Acid (Calcimycin)</td><td>Thermo Fisher</td><td>A1493</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Anti-Hen egg lysozyme (control antibody; cIgG)</td><td>CrownBio</td><td>C0001</td><td></td></tr><tr><td>APC-Cy7-conjugated mouse anti-human CD45 antibody (Clone 2D1)</td><td>Biolegend</td><td>368515</td><td>Use at 1 &amp;micro;g/mL final concentration</td></tr><tr><td>BD FACSCantoTM II system + FACSDiva software (version 8.0.1)&amp;nbsp;</td><td>BD</td><td>n/a</td><td>Flow cytometry system</td></tr><tr><td>Bovine Serum Albumin (BSA) Fraction V</td><td>Roche</td><td>10735108001</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M)</td><td>VWR</td><td>E506</td><td></td></tr><tr><td>Calcium pyrophosphate disposition disease (CPPD) crystals</td><td>InvivoGen</td><td>lrl-cppd</td><td></td></tr><tr><td>Cellometer Auto T4 Bright Field Cell Counter + analysis software (version 3.3.9.5)</td><td>Nexcelom Bioscience</td><td>n/a</td><td>Bright field cell counter</td></tr><tr><td>CIT-013</td><td>Citryll B.V.</td><td>n/a</td><td></td></tr><tr><td>Costar black 96-well plate, clear bottom&amp;nbsp;</td><td>Corning</td><td>3603</td><td>96-well plate #1</td></tr><tr><td>Dextran T500</td><td>Pharmacosmos</td><td>551005009006</td><td></td></tr><tr><td>DPBS (1x)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Serena</td><td>S-FBS-SA-015</td><td></td></tr><tr><td>Ficoll&amp;nbsp;</td><td>GE Healthcare</td><td>17-1440-02</td><td>Density gradient solution</td></tr><tr><td>FITC-conjugated mouse anti-human CD66b antibody (Clone G10F5)</td><td>eBioscience</td><td>17-0666-42</td><td>Use at 1.5 &amp;micro;g/mL final concentration</td></tr><tr><td>Fixable viability dye eFluor 506</td><td>eBioscience</td><td>65-0866-14</td><td></td></tr><tr><td>Flow cytometry analysis software</td><td>FLowJo</td><td>Version 10.8.0</td><td></td></tr><tr><td>fMLP</td><td>Sigma Aldrich</td><td>47729-10MG-F</td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Human serum albumin (HSA)</td><td>VWR</td><td>31020</td><td></td></tr><tr><td>Human TruStain FcX&amp;nbsp;</td><td>Biolegend</td><td>422302</td><td>Fc receptor block</td></tr><tr><td>IBIDI 96-well plate, clear bottom&amp;nbsp;</td><td>IBIDI</td><td>89626</td><td>96-well plate #2</td></tr><tr><td>IncuCyte SX3 + analysis software (version 2022B Rev1)&amp;nbsp;</td><td>Satorius</td><td>n/a</td><td>Live cell microscopic analysis system</td></tr><tr><td>Monosodium urate crystals</td><td>InvivoGen</td><td>tlrl-msu</td><td></td></tr><tr><td>Mouse anti-histone H3 (citrulline R2 + R8 + R17) antibody&amp;nbsp;</td><td>Cayman</td><td>17939</td><td>Clone 11D3; referred to as #1</td></tr><tr><td>Mouse anti-histone H4 (K8Ac + K12Ac + K16Ac) antibody (Clone KM-2)</td><td>Absolute Antibodies</td><td>Ab01681-2.0</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>Sigma Aldrich</td><td>E5134</td><td></td></tr><tr><td>NaCl (0.9%)</td><td>B. Braun</td><td>25900</td><td></td></tr><tr><td>Penicillin (5000 U/mL) - Streptomycin (5000 &amp;micro;g/mL)</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>PerCP-Cy5.5-conjugated mouse anti-human CD16 antibody (Clone 3G8)</td><td>Biolegend</td><td>302027</td><td>Use at 0.33 &amp;micro;g/mL final concentration</td></tr><tr><td>PMA</td><td>Sigma Aldrich</td><td>P1585</td><td></td></tr><tr><td>Polyclonal rabbit anti-HSA antibody</td><td>Sigma Aldrich</td><td>A0433-2ml</td><td></td></tr><tr><td>Poly-L-Lysine (0.01%)</td><td>Sigma Aldrich</td><td>P4832</td><td></td></tr><tr><td>Potassium bicarbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>237205</td><td></td></tr><tr><td>Rabbit anti-histone H3 (citrulline R2 + R8 + R17) antibody&amp;nbsp;</td><td>Abcam</td><td>ab281584</td><td>Multiclonal; referred to as #2</td></tr><tr><td>RPMI 1640 with GlutaMAXTM supplement&amp;nbsp;</td><td>Gibco</td><td>61870-010</td><td>Culture medium containing L-glutamine</td></tr><tr><td>RPMI 1640 without phenol red&amp;nbsp;</td><td>Gibco</td><td>11835-030</td><td>Culture medium without phenol red</td></tr><tr><td>Sodium azide (NaN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Gibco</td><td>15230204</td><td></td></tr><tr><td>Sytox Green Nucleid Acid Stain&amp;nbsp;</td><td>Thermo Fisher</td><td>S7020</td><td>DNA dye</td></tr><tr><td>Trypan blue solution (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379</td><td></td></tr><tr><td>Vacutainer blood tubes Li-Heparin (17 IU/mL)</td><td>BD</td><td>367526</td><td></td></tr></tbody></table>

real-time, high-throughput microscopic quantification of human neutrophil extracellular trap release and assessing the pharmacology of antagonists

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

Real-time high-throughput microscopy, in combination with an impermeable DNA dye allows to study the kinetics, characteristics, and underlying pathways of NETosis and enables the assessment of potential inhibitors of NET release. With this approach, NETs were defined as structures positive for DNA dye with a significantly larger surface area compared to the surface area of healthy neutrophils (Figure 1A), indicating that chromatin has been expelled into the extracellular environment18. The surface area-based analysis allowed us to distinguish between NETs and activated neutrophils with compromised plasma membrane integrity, showing bright intracellular DNA staining (Figure 1A).
Calcium ionophore (A23187) and PMA are commonly used to induce NETosis in vitro. Despite being non-physiological stimuli, they are valuable because they activate distinct NETosis pathways and ensure consistent NETosis induction with low variability between donors. A23187 triggers calcium influx, leading to PAD4 activation and release of NETs rich in citrullinated histones, while PMA activates the NADPH oxidase complex, resulting in reactive oxygen species (ROS) production and the subsequent release of NETs with low levels of citrullinated histones5,16,21. The speed and magnitude of the NETosis response depend on the concentration of each stimulus (Figure 1B,C), with A23187 inducing faster NETosis, and PMA resulting in a higher proportion of neutrophils releasing NETs (Figure 1D-F). NETs resulting from A23187 stimulation (Figure 1D; red arrow) were distinct from PMA-induced NETs (Figure 1D; yellow arrow) by being more diffuse beyond the neutrophil plasma membrane, while PMA-induced NETs remained more adjacent to the neutrophil plasma membrane. In addition, A23187 stimulation resulted in permeable non-netting DNA dye-positive neutrophils (Figure 1D; blue arrow), which did not expel their DNA into the extracellular space. The detection of permeable non-netting DNA dye-positive neutrophils was dependent upon the concentration of A23187 (Figure 1G) and almost absent regardless of the concentration of PMA used to stimulate NETosis (Figure 1D,H). Besides using the non-physiological NETosis stimuli A23187 and PMA, this assay is also suitable for studying disease-relevant triggers of NETosis. As an example, neutrophils activated with soluble immune complexes (sIC) or crystals present in calcium pyrophosphate disposition disease (CPPD) showed significantly increased NET release compared to no stimulus. A trend towards elevated NET levels was observed when neutrophils were activated with coated immune complexes (cIC), fMLP, and monosodium urate (MSU) crystals (Figure 1I); however, for these stimuli, we observed considerable donor-to-donor variation.
Pharmacological inhibition of the NETosis pathway and NETosis-associated enzymes has shown that NETosis-targeting therapeutics could be effective treatments for diseases where NET accumulation significantly drives pathology14,17,22,23,24. This real-time microscopy NET assay is an easy, reliable, and reproducible approach to studying NETosis antagonists in a high-throughput manner. To exemplify this, we have used a first-in-class humanized monoclonal antibody, CIT-013, that targets citrullinated histone H2A and H4 with high affinity15,16. A23187 activates the NETosis pathway, resulting in NETs containing citrullinated histones which are subsequently targeted by CIT-013 to perform its NETosis inhibitory function16. Indeed, NET release in response to A23187 was completely inhibited by CIT-013 (Figure 2A and Supplementary Video 1), ﻿with an IC50 of 4.6 nM (Figure 2B). The NETosis-inhibitory capacity of CIT-013 is unique since other commercial antibodies targeting different (non-) citrullinated histones were not able to inhibit NET release (Figure 2C).
Previously, we have shown that a highly similar precursor molecule of CIT-013 (differing two amino acids but binding similar epitopes with equal affinity) blocks NETosis in response to physiological stimuli such as activated platelets, gout synovial fluid as well as RA synovial fluid17. Here, we show that NET release induced by sIC can be inhibited by CIT-013 (Figure 2D). The therapeutic relevance of inhibiting NETosis induced by this stimulus is highlighted by SLE, RA, and other autoimmune diseases, where autoantibodies in serum or synovial fluid support the formation of IC, which triggers NETosis25,26.
Together, these data demonstrate that this real-time high-throughput microscopy approach is suitable for studying the kinetics and characteristics of NET release and allows the study of inhibitors of NETosis. Although this method has been optimized for the use of human neutrophils, with modifications it might also be suitable to study neutrophils from other species. The data generated with this assay is a cornerstone of the rationale for CIT-013 as a potent and efficacious therapy for NET-driven diseases.
<img alt="Neutrophil activation; NET formation graphs, microscopy images; A23187/PMA-induced neutrophil response." class="xfigimg" src="/files/ftp_upload/67258/67258fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Real-time high-throughput microscopy for studying NET release. Neutrophils isolated from the blood of healthy volunteers were stimulated with A23187 or PMA to trigger the NETosis pathway. NET release was visualized with real-time high-throughput microscopy using a plasma membrane impermeable DNA dye and quantified based on the surface area with the live cell microscopy analysis system software. (A) Analysis of the surface area of non-stimulated healthy neutrophils (gray), extracellular NETs, and permeable non-netting neutrophils with intracellular DNA staining (green). (B,C) Quantification of NET release over time from neutrophils stimulated with the indicated A23187 or PMA concentrations (n = 2). (D) Representative images of NET release in response to A23187 (red arrows) and PMA (yellow arrows) at different time points. Examples of permeable non-netting neutrophils are indicated with blue arrows. (E) Quantification of NET release over time presented as a percentage of NET confluency (n = 5). Statistics were performed on t = 240 min. (F) Quantification of NET release over time presented as a percentage of netting neutrophils (n = 2). (G, H) Quantification of permeable non-netting neutrophils over time from neutrophils that have been stimulated with the indicated A23187 or PMA concentrations (n = 2). (I) Quantification of NET release at t = 240 min induced by soluble immune complexes (sIC), coated IC (cIC), fMLP, monosodium urate (MSU) crystals, and crystals present in calcium pyrophosphate disposition disease (CPPD) (n = 8-28). Results are reported as mean ± standard error of the mean. ﻿**P &lt; 0.01, and ****P &lt; 0.0001, Repeated measures one-way ANOVA with Dunnett's multiple comparisons test (B), Kruskal-Wallis test with Dunn's multiple comparisons test (I). Panels A-F has been modified with permission from van der Linden et al.16. <a href="https://www.jove.com/files/ftp_upload/67258/67258fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="NET release analysis; graphs A-D; concentration-dependent inhibition and efficacy study; data plots." class="xfigimg" src="/files/ftp_upload/67258/67258fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: CIT-013 inhibits NET release. (A) Quantification of A23187-induced NET release at t = 240 min in the absence (No Ab) or presence of CIT-013 or isotype control antibody (cIgG). (B) ﻿Dose-dependent inhibition of A23187-induced NET release with CIT-013 at t = 240 min (n = 3). Data were normalized to cIgG (set as 100% NET release). (C) Quantification of A23187-induced NET release at t = 240 min in the presence of indicated concentrations of CIT-013, anti-histone H4 antibody, anti-citrullinated histone H3 antibody #1, or anti-citrullinated histone H3 antibody #2 (n = 6). (D) Quantification of NET release at t = 240 min induced by soluble immune complexes (ICs) in the presence of CIT-013 or cIgG. Results are reported as mean ± standard error of the mean. ****P &lt; 0.0001, Repeated measures one-way ANOVA with Tukey's multiple comparisons (A) or ﻿two-tailed Wilcoxon matched-pairs signed rank test (D). Panels A, B, and D have been modified with permission from van der Linden et al.16. <a href="https://www.jove.com/files/ftp_upload/67258/67258fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>96-well plate #1</td>
			<td>96-well plate #2</td>
		</tr>
		<tr>
			<td>Volume 0.001% poly-L-lysine solution per well</td>
			<td>50 µL</td>
			<td>100 µL</td>
		</tr>
		<tr>
			<td>Volume of neutrophil suspension per well</td>
			<td>50 µL</td>
			<td>87.5 µL</td>
		</tr>
		<tr>
			<td>Number of neutrophils per well</td>
			<td>2 x 104 cells</td>
			<td>3.5 x 104 cells</td>
		</tr>
		<tr>
			<td>Volume 4x concentrated (= 80 nM) DNA dye in NET assay buffer per well</td>
			<td>50 µL</td>
			<td>87.5 µL</td>
		</tr>
		<tr>
			<td>Volume 4x concentrated NETosis stimuli in NET assay buffer per well</td>
			<td>50 µL</td>
			<td>87.5 µL</td>
		</tr>
		<tr>
			<td>Volume 4x concentrated NETosis antagonists in NET assay buffer per well</td>
			<td>50 µL</td>
			<td>87.5 µL</td>
		</tr>
	</tbody>
</table>
Table 1: Volumes and cell numbers optimized for different 96-well imaging plates.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Concentration work solution (4x concentrated)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Calcium ionophore (A23187)</td>
			<td>50 µM</td>
			<td>12.5 µM</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>16 nM</td>
			<td>4 nM</td>
		</tr>
		<tr>
			<td>fMLP</td>
			<td>4 µM</td>
			<td>1 µM</td>
		</tr>
		<tr>
			<td>Monosodium urate (MSU) crystals</td>
			<td>400 µg/mL</td>
			<td>100 µg/mL</td>
		</tr>
		<tr>
			<td>Calcium pyrophosphate disposition disease (CPPD) crystals</td>
			<td>400 µg/mL</td>
			<td>100 µg/mL</td>
		</tr>
		<tr>
			<td rowspan="3">Soluble immune complexes (sIC)</td>
			<td colspan="2">1. Add 5 µg/mL human serum albumin (HSA) in DPBS to 282.5 µg/mL polyclonal rabbit anti-HSA antibody in DPBS.</td>
		</tr>
		<tr>
			<td colspan="2">2. Incubate for at least 90 min at 37 °C.</td>
		</tr>
		<tr>
			<td colspan="2">3. Homogenize by vortexing and add 50 µL sIC solution to the corresponding wells.</td>
		</tr>
		<tr>
			<td rowspan="10">Coated immune complexes (cIC)</td>
			<td colspan="2">1. Add 10 µg/mL HSA in DPBS in the corresponding wells of the 96-well plate.</td>
		</tr>
		<tr>
			<td colspan="2">2. Incubate overnight at 4 °C.</td>
		</tr>
		<tr>
			<td colspan="2">3. Wash the wells 3 times with 200 µL 0.05% Tween-20 in DPBS (referred to as PBS/0.05%Tween hereafter).</td>
		</tr>
		<tr>
			<td colspan="2">4. Block the wells with 200 µL 1% (w/v) bovine serum albumin in PBS/0.05% Tween (referred to as blocking buffer hereafter).</td>
		</tr>
		<tr>
			<td colspan="2">5. Incubate for 120 min at room temperature and gentle agitation (400 rpm).</td>
		</tr>
		<tr>
			<td colspan="2">6. Wash the wells 3 times with 200 µL PBS/0.05% Tween.</td>
		</tr>
		<tr>
			<td colspan="2">7. Add 50 µL polyclonal rabbit anti-HSA antibody in blocking buffer to the corresponding wells.</td>
		</tr>
		<tr>
			<td colspan="2">8. Incubate for 60 min at room temperature and gentle agitation (400 rpm).</td>
		</tr>
		<tr>
			<td colspan="2">9. Wash the wells 3 times with 200 µL PBS/0.05% Tween.</td>
		</tr>
		<tr>
			<td colspan="2">10. Finally, wash the wells 3 times with 200 µL DPBS. Wells are now ready for step 4.7 in the protocol.</td>
		</tr>
	</tbody>
</table>
Table 2: Recommended concentrations for NETosis stimuli.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Concentration work solution (4x concentrated)</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Anti-Hen egg lysozyme antibody (control antibody; cIgG)</td>
			<td>80 nM</td>
			<td>20 nM</td>
		</tr>
		<tr>
			<td>CIT-013</td>
			<td>80 nM</td>
			<td>20 nM</td>
		</tr>
	</tbody>
</table>
Table 3: Recommended concentrations for NETosis antagonist.
Supplementary Video 1. Neutrophils were stimulated with A23187 in the presence of cIgG (left) or CIT-013 (right), and NET release was visualized over time using the plasma membrane impermeable DNA dye. NET release is inhibited in the presence of CIT-013. The movie is an overlay of DNA dye (green) and phase contrast. This video was obtained with permission from van der Linden et al.16. <a href="https://www.jove.com/files/ftp_upload/67258/20240726_Supplemental%20video%201.zip" target="_blank">Please click here to download this Video.</a>

This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in ...

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Learn a real-time microscopy method for quantifying human neutrophil extracellular trap release and assessing NETosis antagonist pharmacology. This high-throughput technique enables studying innate immune defense mechanisms.

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists (Video) | JoVE Journal

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists

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