癌症中的中性粒细胞胞外诱捕网（NETs），3幅图入门！

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图1. 调控自杀性中性粒细胞溶解的机制示意图。

不同的刺激 [如PMA（phorbol 12-myristate 13-acetate，佛波醇）、离子霉素、晶体、CXCL8、Fc（可结晶的片段）-受体和病原体] 诱导自杀性中性粒细胞溶解，有时称为 "NETosis"。PMA，一个经典的非生理性刺激物，直接激活蛋白激酶C（PKC）和Raf-MEK-ERK-MAP激酶途径。MAP激酶的激活将启动NADPH氧化酶复合物的形成，导致快速生成活性氧（ROS）。中性粒细胞弹性酶（NE）和骨髓过氧化物酶（MPO）有助于核膜通透性和染色质的进一步展开。肽基精氨酸脱氨酶4（PAD4）是一种Ca2+特异性酶，主要定位在细胞核内。PAD4通过将精氨酸变为瓜氨酸来修饰组蛋白，从而导致染色质解聚，在核破裂时，瓜氨酸组蛋白和核DNA一起被释放出来。释放的DNA会进一步被颗粒蛋白（NE，MPO）和细胞溶质蛋白（calpain）装饰。中性粒细胞溶解也被证明依赖于细胞周期蛋白激酶4和6（CDK4/6）。CDK4/6的激活导致视网膜母细胞瘤蛋白（Rb-P）和核纤层B（lamin B）的磷酸化，以及核包膜破裂。细胞内ROS的增加会进一步激活受体交互蛋白激酶3（RIPK3）和混合系激酶域样蛋白（MLKL），促使质膜破裂。Gasdermin D是一种成孔的细胞质蛋白，在质膜通透性的后期阶段牵涉其中。质膜破裂后，含有颗粒蛋白的瓜氨酸核DNA支架以云的形式释放在细胞外空间，导致中性粒细胞死亡。

原文

Fig. 1. Schematic representation of mechanisms regulating suicidal neutrophil cytolysis. Different stimuli [e.g., PMA [59], ionomycin [198], crystals [301], CXCL8 [204], Fc (fragment crystallizable)-receptors [243,302] and pathogens [303]] induce suicidal neutrophil lysis, sometimes called “NETosis”. PMA, a classical non-physiological stimulus, directly activates protein kinase C (PKC) and the Raf-MEK-ERK-MAP kinase pathway [66]. MAP kinases activation would initiate NADPH oxidase complex formation leading to rapid generation of reactive oxygen species (ROS) [60, 229]. Neutrophil elastase (NE) [68] and myeloperoxidase (MPO) [200] contribute to nuclear membrane permeabilization and further unfolding of chromatin [246]. Peptidyl arginine deiminase 4 (PAD4) is a Ca2+-specific enzyme primarily localized in the nucleus. PAD4 modifies histone by changing arginine to citrulline which leads to chromatin decondensation, and upon nuclear rupture, citrullinated-histone together with nuclear DNA is released [70, 304,305]. Released DNA becomes further decorated with granular (NE, MPO) [15] and cytosolic (calpain) proteins [306,307]. Neutrophil cytolysis was also shown to be dependent on cyclin kinases 4 and 6 (CDK4/6) [308]. Activation of CDK4/6 leads to phosphorylation of the retinoblastoma protein (Rb-P) and lamin B, and nuclear envelope breakdown [308]. Increased intracellular ROS would further activate receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL), contributing to the plasma membrane rupture [309,310]. Gasdermin D, a pore-formin cytoplasmic protein, is implicated during later stages of plasma membrane permeabilization [207,257]. After plasma membrane rupture, the citrullinated nuclear DNA scaffold containing granule proteins are released in the extracellular space in a cloud format leading to neutrophil death [14,59].

图 2. 调节vital中性粒细胞胞外诱捕网 (NETs) 形成的机制示意图。

不同的刺激物 [例如金黄色葡萄球菌、LPS、GM-CSF 和 C5a、甲状腺癌条件培养基 、免疫复合物迅速（30 分钟内 ) 诱导 NET 的形成。中性粒细胞的刺激导致 NADPH 氧化酶快速激活，从而导致细胞内 ROS 增加。Vital NET 形成在没有核膜破坏的情况下发生，据报道 DNA 通过囊泡离开细胞质。DNA 被颗粒蛋白（NE、MPO、PR3）修饰。中性粒细胞仍然存活以发挥进一步的功能，并利用增加的细胞内 ROS 动员细胞骨架将颗粒和线粒体运输到活化的中性粒细胞的前缘。线粒体可以在Vital NET 形成中扮演三个角色。首先，它们可以介导线粒体 ROS (mtROS) 的形成，其次，线粒体 DNA (mtDNA) 可以释放到细胞外，再者，它们为糖酵解 ATP 的产生提供支持。还有报道称，mtROS 可以通过线粒体外膜中电压依赖性阴离子通道 (VDAC) 寡聚体形成的孔隙释放短的 mtDNA 片段。mtROS 和 mtDNA 可能参与 NET 形成引起了极大的兴趣，因为 mtDNA 在人和小鼠中发挥免疫调节作用。

关于翻译这里插几句： neutrophil extracellular traps（NETs）的翻译，有些人用的是中性粒细胞胞外陷阱，我个人倾向于 中性粒细胞胞外诱捕网这个翻译，因为它的缩写刚好是NET，这样的话有点形意兼备的味道，也比较好记。NETosis有两种，分别是suicidal NETosis（肿瘤组织中主要是suicidal型[1]）和vital NETosis，根据前面suicidal 的意思，老编觉得vital翻译为重要的显然是不合适的。根据文献[1-2]（Vital NET formation occurs independently of cell death in the absence of membrane disruption within minutes after stimulation of neutrophils. Suicidal NETosis, in which neutrophils die after expelling the filaments; and vital NETosis, in which expulsion appears without altering the membrane.）或许活力型NETosis比较合适（中性粒细胞没死；可能也正因如此，有些大牛用Vital NET formation来来描述这个生物学过程，而不是vital NETosis[1]），跟ChatGPT（GPT-3.5 Turbo）想的意思差不多（见下图）。还有NETosis，这些名词到底怎么翻译才最好，就留给大牛们来做了。

▲截自： 聊天记录

原文

Fig. 2. Schematic representation of mechanisms regulating vital neutrophil extracellular traps (NET) formation. NET formation. Different stimuli [e.g., S. aureus [30,58], LPS [75,227,311], GM-CSF and C5a [16,29], conditioned media of thyroid cancer [72], immune complexes [29,290,312]] rapidly (within 30 min) induce the formation of NETs. Stimulation of neutrophils leads to rapid NADPH oxidase activation, resulting in increased intracellular ROS [16,60]. Vital NET formation occurs without nuclear membrane disruption and has been reported with DNA leaving the cytoplasm in vesicles [30,58,75]. DNA becomes decorated with granule proteins (NE, MPO, PR3) [15,16,29]. The neutrophil remains alive for further functions [46,72], and utilizes the increased intracellular ROS to mobilize the cell cytoskeleton to transport granules and mitochondria to the leading edge of activated neutrophils [73]. Mitochondria can play three roles in vital NET formation. First, they can mediate mitochondrial ROS (mtROS) formation [72, 290], and second, mitochondria DNA (mtDNA) can be released extracellularly [290,312,313], and third, they provide support for glycolytic ATP production [29]. It has also been reported that mtROS can release short mtDNA fragments via pores formed by the voltage-dependent anion channel (VDAC) oligomers in the mitochondrial outer membrane [314]. The possible involvement of mtROS and mtDNA in NET formation is of great interest because mtDNA plays an immunomodulatory role in human and mouse [54].

图 3.A)髓过氧化物酶 (MPO) 和瓜氨酸化组蛋白 H3 (H3Cit)，分别是中性粒细胞和 NET 的标志物，在乳腺癌和结肠癌患者的肝转移中含量丰富。肝脏中NETs (NET-DNA) 的 DNA 成分通过与 CCDC25（coiled-coil domain containing protein 25；是一种在癌细胞上表达的跨膜蛋白）蛋白的高亲和力相互作用，对癌细胞具有趋化作用。DNA-CCDC25 相互作用触发细胞内信号级联，促进癌细胞的定向迁移和转移的形成。在小鼠模型中，体内给予抗 CCDC25 中和抗体减少了 NET 介导的远处转移的形成。B)NET 唤醒休眠的癌细胞。在某些癌症（例如乳腺癌、前列腺癌）中，癌细胞在苏醒之前可以保持休眠状态并且在临床上检测不到达数年，如转移性癌症。有人提出炎症在癌细胞休眠和觉醒之间的转换中发挥作用。在乳腺癌和前列腺癌的小鼠模型中，休眠的癌细胞与细胞外基质 (ECM) 蛋白层粘连蛋白结合。反复滴注脂多糖或尼古丁（两种 NET 诱导剂），促进肺部嗜中性粒细胞炎症和 NET 形成。两种NET 相关蛋白酶，中性粒细胞弹性蛋白酶 (NE) 和基质金属肽酶 9 (MMP9)，依次切割层粘连蛋白，暴露表位，通过整合素激活触发休眠癌细胞的增殖。一种针对 NET 重塑层粘连蛋白的阻断抗体可减少尼古丁或 LPS 诱导的炎症，这些炎症会唤醒小鼠休眠的癌细胞。C)不同的原发性黑色素瘤和结肠癌细胞系诱导人 PMN（ polymorphonuclear neutrophil leukocyte，即neutrophil ）和 PMN-MDSC（ polymorphonucler myeloid-derived suppressor cells, 多形核髓源性抑制细胞 ）形成 NET。Reparixin 是一种 CXCR1/CXCR2 趋化因子受体拮抗剂，可抑制 NET 形成。NETs 包裹和包裹肿瘤细胞并保护它们免受 CD8+ T 细胞和自然杀伤 (NK) 细胞介导的细胞毒性。D)人中性粒细胞胞外诱捕网 (NETs) 调节血管生成。PMA 激活的人嗜中性粒细胞释放 NET，从而诱导人肺动脉内皮细胞 (HPAEC) 的促血管生成反应（例如，增加毛细管长度、新芽数量和新芽面积）。这些作用被 DNase 1 或髓过氧化物酶 (MPO) 抑制剂抑制。NET 还增加了 HPAEC 的增殖活性。此外，单独或联合使用血管生成素 1 (ANGPT1) 和血管生成素 2 (ANGPT2) 可诱导人嗜中性粒细胞释放 NET。ANGPT 诱导的 NET 形成被 PAD4 抑制剂 GSK484 和 NADPH 氧化酶抑制剂抑制。血管生成素介导的 NETs 增加了人血管内皮细胞 (HUVEC) 管的长度和环的数量。

原文

Fig. 3.A) Myeloperoxidase (MPO) and citrullinated histone H3 (H3Cit), markers for neutrophils and NET, respectively, are abundant in the liver metastases of patients with breast and colon cancers [125]. The DNA components of NETs (NET-DNA) in the liver is chemotactic for cancer cells by interacting with high affinity with the coiled-coil domain containing protein 25 (CCDC25), which is a transmembrane protein expressed on cancer cells. DNA CCDC25 interaction triggers an intracellular signaling cascade promoting directional migration of cancer cells and the formation of metastasis. In mouse models, the administration in vivo of a neutralizing antibody anti-CCDC25 reduced the formation of NET-mediated distant metastases [125]. B) NET awaken dormant cancer cells. In several cancers (e.g., breast, prostate) cancer cells can remain dormant and clinically undetectable for years before awakening, as metastatic cancer [127,128]. It has been suggested that inflammation plays a role in the switch between dormancy and awakening of cancer cells [129,130]. In mouse models of breast and prostate cancers, dormant cancer cells bind to the extracellular matrix (ECM) protein laminin. Repeated instillation of lipopolysaccharide [54,131] or nicotine, two inducers of NETs [247], promote neutrophil inflammation and NET formation in the lung. Two NET-associated proteases, neutrophil elastase (NE) and matrix metallopeptidase 9 (MMP9), sequentially cleave laminin exposing an epitope that triggers proliferation of dormant cancer cells through integrin activation. A blocking antibody against NET-remodeled laminin reduces nicotine- or LPS-induced inflammation from awakening dormant cancer cells in mice [131]. C) Different primary melanoma and colon carcinoma cell lines induce NET formation from human PMNs and PMN-MDSCs [116]. Reparixin, a CXCR1/ CXCR2 chemokine receptor antagonists, inhibits NET formation. NETs wrap and coat tumor cells and shields them from the cytotoxicity, mediated by CD8+ T cells and natural killer (NK) cell [116]. D) Human neutrophil extracellular traps (NETs) modulate angiogenesis. PMA-activated human neutrophils release NETs, which induce pro-angiogenic responses (e.g., increased capillary tube length, number of sprouts, and sprouting area) of human pulmonary artery endothelial cells (HPAECs). These effects are inhibited by DNase 1 or a myeloperoxidase (MPO) inhibitor. NETs also increases the proliferative activity of HPAECs [152]. In addition, angiopoietin 1 (ANGPT1) and angiopoietin 2 (ANGPT2) alone or combined induce NET release from human neutrophils. NET formation induced by ANGPTs is inhibited by the PAD4 inhibitor GSK484, and by a NADPH oxidase inhibitor. Angiopoietin-mediated NETs increase human vascular endothelial cell (HUVEC) tube length and the number of loops [155].

参考文献

1. Fang Q, Stehr A M, Naschberger E, et al. No NETs no TIME: Crosstalk between neutrophil extracellular traps and the tumor immune microenvironment[J]. Frontiers in Immunology, 2022, 13.

2. Guillotin F, Fortier M, Portes M, et al. Vital NETosis vs. suicidal NETosis during normal pregnancy and preeclampsia[J]. Frontiers in Cell and Developmental Biology, 2022, 10.

主要译自：Seminars in Cancer Biology (10.1016/j.semcancer.2021.07.011)，仅作学术分享使用；侵删

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