Simoneau Camille R, Chen Pei-Yi, Xing Galen K, Khalid Mir M, Meyers Nathan L, Hayashi Jennifer M, Taha Taha Y, Leon Kristoffer E, Ashuach Tal, Fontaine Krystal A, Rodriguez Lauren, Joehnk Bastian, Walcott Keith, Vasudevan Sreelakshmi, Fang Xiaohui, Maishan Mazharul, Schultz Shawn, Roose Jeroen, Matthay Michael A, Sil Anita, Arjomandi Mehrdad, Yosef Nir, Ott Melanie
Gladstone Institute of Virology, San Francisco, CA, USA.
Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, CA, USA.
bioRxiv. 2022 Aug 2:2022.08.02.502100. doi: 10.1101/2022.08.02.502100.
As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.
随着严重急性呼吸综合征冠状病毒2（SARS-CoV-2）在全球范围内持续传播，需要能够准确重现对新出现的病毒变种的细胞内在反应的易处理的原发性气道细胞模型。在此，我们描述了一种过表达血管紧张素转换酶2（ACE2）受体的成人干细胞衍生的人气道类器官模型，该模型支持强劲的病毒复制，同时保持气道上皮的三维结构和细胞多样性。用SARS-CoV-2变种感染ACE2过表达（ACE2-OE）类器官，并进行单细胞RNA测序。NF-κB抑制因子α在受感染的上皮细胞中持续上调，其mRNA表达与感染水平呈正相关。共聚焦显微镜显示，受感染细胞中的IκBα表达比旁观者细胞更多，但发现NF-κB同时发生了IκBα通常会阻止的核转位。过表达一种不可降解的IκBα突变体可减少NF-κB转位并增加病毒感染。这些数据证明了ACE2-OE类器官在SARS-CoV-2研究中的功能，并确定了一个不完整的NF-κB反馈环是病毒感染的一个调节器，可能会促进炎症和严重疾病。
