Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist
Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist
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DOI：
10.1159/000521012
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发表时间：
2021-12
影响因子：
2.4
通讯作者：
Mako Okudera;Mina Odawara;Masashi Arakawa;S. Kawaguchi;K. Seya;T. Matsumiya;Riko Sato;Jiangli Ding;E. Morita;T. Imaizumi
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文献类型：
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作者：
Mako Okudera;Mina Odawara;Masashi Arakawa;S. Kawaguchi;K. Seya;T. Matsumiya;Riko Sato;Jiangli Ding;E. Morita;T. Imaizumi
简介：病毒侵入大脑会导致病毒性脑炎，这可能是致命的，并导致永久性脑损伤。血脑屏障（BBB）通过排除有害物质和微生物来保护大脑。脑微血管内皮细胞是血脑屏障的重要组成部分，然而，这些细胞中的抗病毒反应机制尚未完全阐明。锌指抗病毒蛋白（Zinc finger antiviral protein，ZAP）是一种限制多种病毒感染的分子，主要有两种亚型：ZAPL和ZAPS。Toll样受体3（TLR 3）是一种针对病毒双链RNA的模式识别受体，参与抗病毒先天免疫反应。本研究的目的是研究ZAP在培养的hCMEC/D3人脑微血管内皮细胞中的表达，所述hCMEC/D3人脑微血管内皮细胞用真正的TLR 3激动剂聚肌苷酸-聚胞苷酸（polyinosinic-polycytidylic acid，poly IC）处理。方法：体外培养hCMEC/D3细胞，用poly IC处理。ZAPL和ZAPS mRNA的表达进行了研究，使用定量逆转录-聚合酶链反应，这些分子的蛋白质表达进行了研究，使用蛋白质印迹法。使用NF-κB抑制剂SN 50检测核因子-κB（NF-κB）的作用。干扰素（IFN）-β、IFN调节因子3（IRF 3）、三联体基序蛋白25（TRIM 25）和视黄酸诱导基因I（RIG-I）在聚IC诱导的ZAPS表达中的作用用RNA干扰进行了检测。用成灶试验检测了日本脑炎病毒（JEV）的繁殖。结果：poly IC可上调ZAPS的mRNA和蛋白表达，而对ZAPL的mRNA和蛋白水平影响甚微。敲低IRF 3或TRIM 25降低了聚IC诱导的ZAPS上调，而敲低IFN-β或RIG-I不影响ZAPS上调。SN 50不影响ZAPS的表达。ZAP的敲除增强了JEV的传播。结论：hCMEC/D3细胞表达ZAPL和ZAPS，poly IC可上调ZAPS的表达。IRF 3和TRIM 25参与poly IC诱导的ZAPS上调。ZAP可能有助于脑微血管内皮细胞的抗病毒反应，并保护大脑免受JEV等病毒的侵袭。
Introduction: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). Methods: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-β, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. Results: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-β or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. Conclusion: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.