Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage
Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage
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DOI：
10.1371/journal.ppat.1007625
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发表时间：
2019-04-01
期刊：
影响因子：
6.7
通讯作者：
Yeh, Trai-Ming
中科院分区：
文献类型：
--
作者：
Chao, Chiao-Hsuan;Wu, Wei-Chueh;Yeh, Trai-Ming
登革病毒（DENV）感染是最常见的蚊媒病毒感染，可引起一系列疾病，从自限性登革热到危及生命的登革出血热和休克综合征。血小板减少症是在轻度和重度登革热病中观察到的主要特征，并且与登革热严重程度的进展显著相关。先前的研究表明，可以分泌到患者血液中的DENV非结构蛋白1（NS 1）可以通过Toll样受体4（TLR 4）刺激免疫细胞，并可能导致内皮渗漏。然而，目前尚不清楚DENV NS 1是否可以直接诱导血小板活化或在DENV感染过程中引起血小板减少症。在这项研究中，我们首先证明了DENV而不是寨卡病毒细胞培养上清液可以诱导人血小板中的P-选择素表达和磷脂酰丝氨酸（PS）暴露，当从DENV上清液中耗尽NS 1时，这两者都被消除。使用来自DENV的所有四种血清型的重组NS 1发现了类似的结果，并且在存在抗NS 1 F（ab '）（2）、抗TLR 4抗体、TLR 4拮抗剂（球形红细菌脂多糖，LPS-Rs）和TLR 4信号传导抑制剂（TAK 242）但不存在多粘菌素B（LPS抑制剂）的情况下，这些作用被阻断。此外，DENV NS 1对血小板的活化促进了亚阈值浓度的二磷酸腺苷（ADP）诱导的血小板聚集，并增强了血小板与内皮细胞的粘附和巨噬细胞的吞噬作用。最后，我们证明了当NS 1从DENV上清液中耗尽时，在TLR 4敲除和野生型小鼠中DENV诱导的血小板减少和出血减弱。总之，这些结果表明，DENV NS 1与血小板上的TLR 4的结合可以触发其激活，这可能有助于在登革热感染过程中血小板减少和出血。作者总结在过去的50年里，登革热一直是一个持续的全球威胁，没有有效的疫苗或特异性抗病毒药物。登革热感染导致广泛的结果，从发热样症状到严重的登革出血热。血小板减少症，血小板计数减少，是在轻度和重度登革热中观察到的常见特征，并且与疾病严重程度相关。在这项研究中，我们使用登革病毒上清液或登革病毒重组NS 1蛋白刺激人分离的血小板。我们发现DENV NS 1可以通过TLR 4直接激活血小板，并可以进一步增强血小板聚集，粘附到内皮细胞和巨噬细胞的吞噬作用，这可能导致血小板减少症。我们还使用DENV诱导的出血性小鼠模型证明NS 1和TLR 4对于DENV诱导的血小板减少和出血都是关键的。我们的研究揭示了NS 1在登革感染中的新致病作用，并强调NS 1应成为开发针对登革感染的治疗药物和疫苗的关注主题。
Dengue virus (DENV) infection, the most common mosquito-transmitted viral infection, can cause a range of diseases from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and shock syndrome. Thrombocytopenia is a major characteristic observed in both mild and severe dengue disease and is significantly correlated with the progression of dengue severity. Previous studies have shown that DENV nonstructural protein 1 (NS1), which can be secreted into patients' blood, can stimulate immune cells via Toll-like receptor 4 (TLR4) and can cause endothelial leakage. However, it is unclear whether DENV NS1 can directly induce platelet activation or cause thrombocytopenia during DENV infection. In this study, we first demonstrated that DENV but not Zika virus cell culture supernatant could induce P-selectin expression and phosphatidylserine (PS) exposure in human platelets, both of which were abolished when NS1 was depleted from the DENV supernatant. Similar results were found using recombinant NS1 from all four serotypes of DENV, and those effects were blocked in the presence of anti-NS1 F(ab')(2), anti-TLR4 antibody, a TLR4 antagonist (Rhodobacter sphaeroides lipopolysaccharide, LPS-Rs) and a TLR4 signaling inhibitor (TAK242), but not polymyxin B (an LPS inhibitor). Moreover, the activation of platelets by DENV NS1 promoted subthreshold concentrations of adenosine diphosphate (ADP)-induced platelet aggregation and enhanced platelet adhesion to endothelial cells and phagocytosis by macrophages. Finally, we demonstrated that DENV-induced thrombocytopenia and hemorrhage were attenuated in TLR4 knockout and wild-type mice when NS1 was depleted from DENV supernatant. Taken together, these results suggest that the binding of DENV NS1 to TLR4 on platelets can trigger its activation, which may contribute to thrombocytopenia and hemorrhage during dengue infection.Author summary Over the past 50 years, dengue has been a continuing global threat, with no effective vaccine or specific antiviral drug. Dengue infection causes a wide range of outcomes, from fever-like symptoms to severe dengue hemorrhagic fever. Thrombocytopenia, a reduction in platelet count, is a common feature observed in both mild and severe dengue and is correlated with disease severity. In this study, we used dengue viral supernatant or DENV recombinant NS1 protein to stimulate human-isolated platelets. We found that DENV NS1 could directly activate platelets through TLR4 and could further enhance platelet aggregation, adhesion to endothelial cells and phagocytosis by macrophages, which could lead to thrombocytopenia. We also proved that both NS1 and TLR4 are critical for DENV-induced thrombocytopenia and hemorrhage using a DENV-induced hemorrhagic mouse model. Our study reveals a new pathogenic role of NS1 during dengue infection and highlights that NS1 should be a topic of attention in the development of therapeutic drugs and vaccines against dengue infection.