School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
Department of Surgery and Anaesthesia, University of Otago, Wellington, New Zealand.
Cytometry A. 2024 Aug;105(8):639-652. doi: 10.1002/cyto.a.24851. Epub 2024 Jun 12.
With the recent discovery of their ability to produce neutrophil extracellular traps (NETs), neutrophils are increasingly appreciated as active participants in infection and inflammation. NETs are characterized as large, web-like networks of DNA and proteins extruded from neutrophils, and there is considerable interest in how these structures drive disease in humans. Advancing research in this field is contingent on developing novel tools for quantifying NETosis. To this end, we have developed a 7-marker flow cytometry panel for analyzing NETosis on human peripheral neutrophils following in vitro stimulation, and in fresh circulating neutrophils under inflammatory conditions. This panel was optimized on neutrophils isolated from whole blood and analyzed fresh or in vitro stimulated with phorbol 12-myristate 13-acetate (PMA) or ionomycin, two known NET-inducing agonists. Neutrophils were identified as SSCFSCCD15CD66b. Neutrophils positive for amine residues and 7-Aminoactinomycin D (7-AAD), our DNA dye of choice, were deemed necrotic (Zombie-NIR7-AAD) and were removed from downstream analysis. Exclusion of Zombie-NIR and positivity for 7-AAD (Zombie-NIR7-AAD) was used here as a marker of neutrophil-appendant DNA, a key feature of NETs. The presence of two NET-associated proteins - myeloperoxidase (MPO) and neutrophil elastase (NE) - were utilized to identify neutrophil-appendant NET events (SSCFSCCD15CD66bZombie NIR7-AADMPONE). We also demonstrate that NETotic neutrophils express citrullinated histone H3 (H3cit), are concentration-dependently induced by in vitro PMA and ionomycin stimulation but are disassembled with DNase treatment, and are present in both chronic and acute inflammation. This 7-color flow cytometry panel provides a novel tool for examining NETosis in humans.
最近发现中性粒细胞能够产生中性粒细胞胞外诱捕网（NETs），人们越来越认识到中性粒细胞是感染和炎症的积极参与者。NETs 的特征是从中性粒细胞中挤出的大的、网状的 DNA 和蛋白质网络，人们对这些结构如何在人类中引发疾病非常感兴趣。该领域的研究进展取决于开发用于定量 NETosis 的新工具。为此，我们开发了一种 7 色流式细胞术面板，用于分析体外刺激后人类外周血中性粒细胞中的 NETosis，以及在炎症条件下新鲜循环的中性粒细胞中的 NETosis。该面板在全血分离的中性粒细胞上进行了优化，并进行了新鲜分析或体外刺激，使用了两种已知的 NET 诱导激动剂佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）或离子霉素。中性粒细胞被鉴定为 SSCFSCCD15CD66b。带正电荷的胺残基和我们首选的 DNA 染料 7-氨基放线菌素 D（7-AAD）的中性粒细胞被认为是坏死的（Zombie-NIR7-AAD），并从下游分析中删除。这里排除 Zombie-NIR 和 7-AAD 的阳性（Zombie-NIR7-AAD）被用作中性粒细胞相关 DNA 的标志物，这是 NETs 的一个关键特征。两种 NET 相关蛋白 - 髓过氧化物酶（MPO）和中性粒细胞弹性蛋白酶（NE） - 的存在用于识别中性粒细胞相关的 NET 事件（SSCFSCCD15CD66bZombie NIR7-AADMPONE）。我们还证明 NETotic 中性粒细胞表达瓜氨酸化组蛋白 H3（H3cit），可被体外 PMA 和离子霉素刺激浓度依赖性诱导，但用 DNA 酶处理后会解体，并存在于慢性和急性炎症中。这种 7 色流式细胞术面板为研究人类 NETosis 提供了一种新工具。
