Abstract
As the most abundant leukocyte in circulation, the neutrophil plays a far-reaching role in maintaining homeostasis. Within the context of disease, however, neutrophils can potentiate various pathophysiological mechanisms with disastrous consequences for patients. The role of the neutrophil in disease is complex with mechanisms like NETosis driving the progression of several pathologies. NETosis involves neutrophils extruding protein-decorated DNA webs called neutrophil extracellular traps (NETs), which facilitate the progression of inflammatory, non-infectious, and neoplastic pathologies. The need to visualize NETs has thus never been greater. Current approaches for visualizing NETs are limited in specificity and sensitivity, involving non-specific fluorescent DNA dyes or co-stains of neutrophil and DNA markers. Improved methodologies are needed to robustly distinguish NETs from other cell-free DNA. Excitingly, a novel NET-specific posttranslational modification involving cleavage on the N-terminus of histone H3 has recently been identified. Here, we demonstrate that this single marker is superior to the conventional use of the co-stain of the neutrophil marker, myeloperoxidase, and, the DNA marker, histone H3 citrullination in visualizing neutrophil NETosis. This is due to this single marker’s unparalleled ability to identify, not only more NETs but also their formation at earlier stages of NETosis. Moreover, we additionally propose a stepwise mechanism of neutrophil NETosis in which a histone H3 cleavage event precedes histone H3 citrullination. Taken together, these results demonstrate a novel method for visualizing NETs, allowing for continued exploration of their multifaceted roles in immunity and disease.
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Acknowledgments
Jonathan D. Spicer is supported by the Clinician Scientist Junior 2, Fonds de recherche du Québec—Santé (FRQS). Meghan L. De Meo is supported by the Bourse de formation de maîtrise du Fonds de recherche du Québec—Santé (FRQS). We would like to thank Dr. Arturo Zychlinsky’s laboratory for their generosity in sharing their novel H3 clipping antibody with us. We would also like to thank the Molecular Imaging Platform for their assistance in acquiring these high-resolution images.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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De Meo, M.L., Shahzad, M.H., Spicer, J.D. (2023). Visualizing NETosis Using a Novel Neutrophil Extracellular Trap-Specific Marker. In: Ursini-Siegel, J. (eds) The Tumor Microenvironment. Methods in Molecular Biology, vol 2614. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2914-7_5
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DOI: https://doi.org/10.1007/978-1-0716-2914-7_5
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