Core Facilities-Proteomics Laboratory, Istituto Giannina Gaslini, Genoa, Italy.
Laboratory of Molecular Nephrology, Istituto Giannina Gaslini, Genoa, Italy.
PLoS One. 2019 Jul 8;14(7):e0218946. doi: 10.1371/journal.pone.0218946. eCollection 2019.
Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.
中性粒细胞胞外诱捕网（NET）的形成是中性粒细胞对感染的反应的一部分，但过度或不适当的 NETosis 可能会触发自身抗体的产生，并在自身免疫性疾病中导致器官损伤。自发形成 NET 的中性粒细胞并不常见，因此需要通过选择适当的刺激物在体外诱导 NET 的形成，以研究其结构。在本工作中，通过蛋白质组学分析研究了不同刺激物诱导产生的 NET 的蛋白质组成和翻译后修饰。用 PMA、A23187、大肠杆菌 LPS 或未处理的方法刺激来自健康供体的中性粒细胞；3 小时后，洗涤细胞，用 DNAse 处理，收集上清液进行质谱分析。通过无监督层次聚类分析对数据进行分析。我们鉴定了任何来源或仅一种刺激物诱导的 NET 中包含的蛋白质：LPS 诱导和自发 NET 在蛋白质组成上存在差异，而 PMA 和 A23187 诱导的 NET 则更为相似。在我们检查的翻译后修饰中，蛋氨酸亚砜化特别频繁，尤其是在 PMA 和 LPS 诱导的 NET 中。髓过氧化物酶是修饰最广泛的蛋白质。因此，蛋白质组学分析表明，不同刺激物诱导的 NET 在蛋白质组成和翻译后修饰方面存在异质性，这表明在不同条件下诱导的 NET 可能具有不同的生物学效应。
