College of Veterinary Medicine, Northeast Agricultural University, Harbin, China.
J Cell Physiol. 2021 May;236(5):3688-3699. doi: 10.1002/jcp.30105. Epub 2020 Oct 12.
As an important immune mechanism of neutrophils, the release of Web-like chromatin structures known as neutrophil extracellular traps (NETs) can rapidly locate and capture invading pathogens, which has received sustained attention. There are still some fundamental questions surrounding established studies on the mechanism of balance between reactive oxygen species (ROS) dependent release and neutrophil antioxidant response. Glutathione peroxidase 3 (GPx3) is an important antioxidant protein and has been identified can regulate the immune response. However, the effect of GPx3 on the NETs formation and microRNA in this process remain poorly understood. In the present study, we used chicken peripheral blood neutrophils treated with Phorbol-12-myristate-13-acetate (PMA) for 3 h as NETs formation model. The result of morphological observation showed that GPx3 inactivation compromised the release of NETs. Further analysis revealed that knockdown of GPx3 significantly disturbed oxidative balance by inhibiting antioxidant enzymes activity and increasing H O content. Quantitative analysis of NETs-related genes found that the phosphorylation level of mitogen-activated protein kinase (MAPK) pathway genes (ERK, JNK, and p38) and expression of phosphoinositide-3-kinase (PI3K)/AKT pathway genes (PI3K and AKT) were suppressed with the downregulation of GPx3. Meanwhile, we identified that miR-1696 can target GPx3 expression by using dual luciferase reporter system. Additionally, overexpression of miR-1696 can not only inhibit the formation of NETs by restraining the expression of GPx3, interfering with the generation of ROS and activation of the MAPK and PI3K/AKT pathways, but also reducing the release of PMA-induced NETs promoted by overexpression of GPx3. These results provide evidence that miR-1696 targeted GPx3 activities in neutrophils could be used to regulate the NETs formation stimulated by PMA.
作为中性粒细胞的一种重要免疫机制，网状染色质结构的释放，即中性粒细胞胞外诱捕网（NETs）的释放，可以快速定位和捕获入侵的病原体，这一机制受到了持续关注。尽管目前已经有大量研究对活性氧（ROS）依赖的释放和中性粒细胞抗氧化反应之间平衡的机制进行了探讨，但仍有一些基本问题尚未解决。谷胱甘肽过氧化物酶 3（GPx3）是一种重要的抗氧化蛋白，已被证实可以调节免疫反应。然而，GPx3 对 NETs 形成和这一过程中的 microRNA 的影响仍知之甚少。在本研究中，我们使用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）处理鸡外周血中性粒细胞 3 小时作为 NETs 形成模型。形态学观察结果表明，GPx3 失活会损害 NETs 的释放。进一步分析显示，GPx3 的敲低通过抑制抗氧化酶活性和增加 H2O2 含量显著扰乱了氧化平衡。NETs 相关基因的定量分析发现，丝裂原活化蛋白激酶（MAPK）途径基因（ERK、JNK 和 p38）和磷酸肌醇 3-激酶（PI3K）/蛋白激酶 B（AKT）途径基因（PI3K 和 AKT）的磷酸化水平随着 GPx3 的下调而受到抑制。同时，我们通过双荧光素酶报告系统证实 miR-1696 可以靶向 GPx3 的表达。此外，miR-1696 的过表达不仅可以通过抑制 GPx3 的表达来抑制 NETs 的形成，干扰 ROS 的产生以及 MAPK 和 PI3K/AKT 途径的激活，还可以减少由 GPx3 过表达促进的 PMA 诱导的 NETs 的释放。这些结果为 miR-1696 靶向中性粒细胞中 GPx3 的活性以调节 PMA 刺激的 NETs 形成提供了证据。
