Tian Xiaoyan, Liu Bingxin, Li Linrun, Yuan Meng, You Qiao, Zhang Rui, Chen Deyan, Cheng Min, Zheng Nan, He Miao, Wu Zhiwei
Center for Public Health Research, Medical School, Nanjing University, 22 Hankou Rd, Nanjing, 210093, China.
Medical School, The Drum Tower Hospital, Nanjing University, Nanjing, China.
Cell Commun Signal. 2025 Apr 14;23(1):183. doi: 10.1186/s12964-025-02195-y.
Enterovirus 71 (EV71) is a major etiologic pathogen for hand-foot-and-mouth disease (HFMD) in young children. Severe cases of EV71 infection could lead to neurological complications and even death, while the mechanism inducing neurological complications remains poorly understood. In this study, we firstly proved that microvesicles (MVs) could carry EV71 virions and mediate a higher efficiency in infection. Utilizing an in vitro blood-brain barrier (BBB) model, we observed that MVs containing virions (MVsEV71) could cross the BBB with greater efficiency compared to EV71 alone. Through in vivo imaging, we confirmed the ability of MVs to cross the BBB. qPCR assays showed a higher copy number of EV71 in both blood and brain samples in the mice treated with MVsEV71 compared to those treated with free EV71. Also, our investigation unveiled that MVsEV71 infection of animals induced cerebral hemorrhage and more severe inflammatory infiltration in the brain compared to animals infected by EV71 in vivo. Furthermore, we found a reduction in the expression of junction proteins such as zonula occludens-1 (ZO-1) and occludin. Moreover, the uptake of MVs by brain cells was examined using chemical inhibitor to block the endocytic pathway. Our experiments elucidated that the internalization of MVs occurred via a non-clathrin-dependent mechanism and a portion of the internalized MVs proceeded to enter lysosomes. In addition, we identified damaged mitochondria as the "cargo" of MVs, which facilitated MVsEV71 crossing the BBB and inducing cellular apoptosis. Meanwhile, MVsEV71 crossing the BBB further induced mitochondrial damaged and activated NOX4-derived ROS pathway in U251 cells. Taken together, these findings suggested that MVs transported EV71 virions across the BBB, while damaged mitochondria facilitated this process and aggravated the brain injury. Overall, these observations provide new insights into EV71-induced neurogenic complications and present a novel therapeutic target for the treatment of viral encephalitis.
肠道病毒71型（EV71）是幼儿手足口病（HFMD）的主要病原体。EV71感染的严重病例可导致神经系统并发症甚至死亡，而诱发神经系统并发症的机制仍知之甚少。在本研究中，我们首先证明微泡（MVs）可以携带EV71病毒粒子并介导更高的感染效率。利用体外血脑屏障（BBB）模型，我们观察到含有病毒粒子的微泡（MVsEV71）比单独的EV71能更高效地穿过血脑屏障。通过体内成像，我们证实了微泡穿过血脑屏障的能力。qPCR分析显示，与游离EV71处理的小鼠相比，MVsEV71处理的小鼠血液和脑样本中EV71的拷贝数更高。此外，我们的研究发现，与体内感染EV71的动物相比，MVsEV71感染动物会导致脑出血和更严重的脑内炎症浸润。此外，我们发现紧密连接蛋白如闭合蛋白-1（ZO-1）和闭合蛋白的表达降低。此外，使用化学抑制剂阻断内吞途径来检测脑细胞对微泡的摄取。我们的实验表明，微泡的内化是通过非网格蛋白依赖机制发生的，并且一部分内化的微泡会进入溶酶体。此外，我们确定受损线粒体是微泡的“货物”，这有助于MVsEV71穿过血脑屏障并诱导细胞凋亡。同时，MVsEV71穿过血脑屏障进一步诱导U251细胞中的线粒体损伤并激活NOX4衍生的ROS途径。综上所述，这些发现表明微泡将EV71病毒粒子转运穿过血脑屏障，而受损线粒体促进了这一过程并加重了脑损伤。总体而言，这些观察结果为EV71诱导的神经源性并发症提供了新的见解，并为病毒性脑炎的治疗提出了新的治疗靶点。
