De Meo Meghan L, Shahzad Muhammad H, Spicer Jonathan D
Thoracic Surgery & Upper Gastrointestinal Cancer Research Laboratories, McGill University, Montreal, QC, Canada.
Cancer Research Program, Research Institute of the McGill University Health Centre, Montreal, QC, Canada.
Methods Mol Biol. 2023;2614:71-80. doi: 10.1007/978-1-0716-2914-7_5.
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.
作为循环中数量最多的白细胞，中性粒细胞在维持体内平衡方面发挥着深远的作用。然而，在疾病背景下，中性粒细胞可增强各种病理生理机制，给患者带来灾难性后果。中性粒细胞在疾病中的作用很复杂，例如中性粒细胞胞外诱捕网形成（NETosis）等机制推动了多种病理过程的进展。NETosis过程中，中性粒细胞会排出由蛋白质修饰的DNA网络，即中性粒细胞胞外诱捕网（NETs），这会促进炎症性、非感染性和肿瘤性病理过程的发展。因此，可视化NETs的需求从未如此迫切。目前可视化NETs的方法在特异性和敏感性方面存在局限性，涉及非特异性荧光DNA染料或中性粒细胞与DNA标记物的共染色。需要改进方法以可靠地区分NETs与其他游离DNA。令人兴奋的是，最近发现了一种涉及组蛋白H3 N端切割的新型NET特异性翻译后修饰。在此，我们证明，在可视化中性粒细胞NETosis方面，这一单一标记物优于传统使用的中性粒细胞标记物髓过氧化物酶和DNA标记物组蛋白H3瓜氨酸化的共染色。这是因为这一单一标记物不仅能够识别更多的NETs，还能在NETosis的早期阶段识别其形成，具有无与伦比的能力。此外，我们还提出了中性粒细胞NETosis的分步机制，其中组蛋白H3切割事件先于组蛋白H3瓜氨酸化。综上所述，这些结果证明了一种可视化NETs的新方法，有助于继续探索它们在免疫和疾病中的多方面作用。
