Department of Rheumatology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, The Ministry of Education Key Laboratory, National Clinical Research Center for Dermatologic and Immunologic Diseases, Beijing, China.
School of Nursing, Peking Union Medical College, Beijing, China.
Front Immunol. 2021 Feb 5;11:590622. doi: 10.3389/fimmu.2020.590622. eCollection 2020.
Neutrophil extracellular traps (NETs) are upregulated and promote thrombosis in Behçet's disease (BD). However, whether NETs promote autoinflammation in BD remains unclear. This study aimed to investigate the potential role of NETs in promoting macrophage activation in BD. Firstly, we quantified NETs by measuring double-stranded DNA (dsDNA) using PicoGreen and calculating the proportion of NETosis. Then macrophages were stimulated with BD- or healthy controls (HC)-derived NETs, and IL-8 and TNF-α production and IFN-γ CD4 T cells differentiation were measured using ELISA and flow cytometry, respectively. The protein components in NETs were analyzed by western blot. Macrophages were stimulated with Histone H4 neutralized NETs, and IL-8 and TNF-α production were measured using ELISA. The level of 8-hydroxydeoxyguanosine (8-OHdG) DNA in NETs was measured using ELISA. The levels of reactive oxygen species (ROS) in serum and neutrophils were measured using ROS probes by a microplate reader and flow cytometry. We found that circulating NETs and neutrophil-derived NETs were significantly higher in BD than HC. BD NETs stimulated macrophages produced higher levels of IL-8 and TNF-α, and promoted IFN-γ CD4 T cells differentiation. BD NETs were enriched in Histone H4, and neutralizing Histone H4 abrogated the BD NETs-mediated IL-8 production by macrophages, but not TNF-α. Also, BD neutrophils produced more 8-OHdG DNA than HC neutrophils, and the percentage of 8-OHdG DNA in dsDNA from BD neutrophils was also higher than that of HC neutrophils. The ROS levels in serum and neutrophils were both higher in BD than HC. Our findings suggested that excessive BD NETs promoted macrophages activation and facilitated IFN-γ CD4 T cells differentiation. Higher levels of Histone H4 and oxidized DNA in BD NETs might mediate macrophages hyperactivation.
中性粒细胞胞外诱捕网（NETs）在贝赫切特病（BD）中上调并促进血栓形成。然而，NETs 是否在 BD 中促进自身炎症仍不清楚。本研究旨在探讨 NETs 在促进 BD 中巨噬细胞活化中的潜在作用。首先，我们通过使用 PicoGreen 测量双链 DNA（dsDNA）并计算 NETosis 的比例来量化 NETs。然后，用 BD 或健康对照（HC）来源的 NETs 刺激巨噬细胞，分别使用 ELISA 和流式细胞术测量 IL-8 和 TNF-α 的产生和 IFN-γ CD4 T 细胞分化。通过 Western blot 分析 NETs 的蛋白成分。用Histone H4 中和 NETs 刺激巨噬细胞，使用 ELISA 测量 IL-8 和 TNF-α 的产生。用 ELISA 测量 NETs 中 8-羟基脱氧鸟苷（8-OHdG）DNA 的水平。用微板阅读器和流式细胞术通过 ROS 探针测量血清和中性粒细胞中的活性氧（ROS）水平。我们发现，BD 中的循环 NETs 和中性粒细胞来源的 NETs 明显高于 HC。BD NETs 刺激巨噬细胞产生更高水平的 IL-8 和 TNF-α，并促进 IFN-γ CD4 T 细胞分化。BD NETs 富含 Histone H4，中和 Histone H4 可阻断 BD NETs 介导的巨噬细胞产生 IL-8，但不阻断 TNF-α。此外，BD 中性粒细胞产生的 8-OHdG DNA 多于 HC 中性粒细胞，并且 BD 中性粒细胞 dsDNA 中的 8-OHdG DNA 百分比也高于 HC 中性粒细胞。BD 中的血清和中性粒细胞中的 ROS 水平均高于 HC。我们的研究结果表明，过多的 BD NETs 促进了巨噬细胞的活化，并促进了 IFN-γ CD4 T 细胞的分化。BD NETs 中更高水平的 Histone H4 和氧化 DNA 可能介导巨噬细胞的过度激活。
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