A Co-culture System Reveals a Cross-talk between Alveolar Epithelium and Pulmonary Microvascular Endothelium during SARS-CoV-2 Infection

SARS-CoV-2 seriously injures human alveoli and causes severe respiratory illness. Histopathologic evidences suggested alveolar-capillary barrier integrity is compromised in COVID-19 deaths, however, little is known about how it is disrupted. In this study, we investigated the effects of SARS-CoV-2 infection on alveolar epithelium and pulmonary microvascular endothelium, and tried to elucidate the cross-talk between them during viral infection. Under monoculture system, SARS-CoV-2 infection caused massive virus replication and dramatic organelles re-modeling in alveolar epithelial cells. While, as for pulmonary microvascular endothelial cells, direct viral exposure had little effect on them, but treatment with culture supernatant from infected epithelial cellls significantly damaged them, which suggested SARS-CoV-2 affected endothelium indirectly, possibly by substances released from infected alveolar epithelium. Then, we tested SARS-CoV-2 infection in an alveolar epithelium/endothelium co-culture system, and found viral infection caused global proteomic modulations and ultrastructual changes in both cell types. Especially for alveolar epithelial cells, viral infection elicited significant protein changes and structural reorganizations across many sub-cellular compartments. Among the affected organelles, mitochondrion seems to be a primary target organelle. In addition, based on proteomic analysis and EM clues, we tested several autophagy inhibitors, and discovered one of them, Daurisoline, could inhibit virus replication effectively in cells. Collectively, our study revealed the distinctive responses of alveolar epithelium and microvascular endothelium to SARS-CoV-2 infection, which will expand our understanding of COVID-19 and helpful for targeted drug development.

SARS-CoV-2（严重急性呼吸综合征冠状病毒2型）可严重损伤人体肺泡，引发重症呼吸系统疾病。组织病理学证据显示，新型冠状病毒肺炎（COVID-19）死亡病例的肺泡-毛细血管屏障完整性遭到破坏，但目前对其破坏机制仍知之甚少。本研究探究了SARS-CoV-2感染对肺泡上皮细胞与肺微血管内皮细胞的影响，并试图阐明病毒感染过程中二者之间的细胞串扰。在单层培养体系（monoculture system）下，SARS-CoV-2感染可导致肺泡上皮细胞发生大量病毒复制与显著的细胞器重构。而对于肺微血管内皮细胞，直接病毒暴露几乎未对其产生影响，但经感染的肺泡上皮细胞的培养上清液处理后，细胞会受到明显损伤，这表明SARS-CoV-2可通过感染的肺泡上皮细胞释放的物质间接作用于内皮细胞。本研究在肺泡上皮细胞/内皮细胞共培养体系（co-culture system）中验证了SARS-CoV-2感染，发现病毒感染可引发两种细胞类型的整体蛋白质组调控与超微结构改变。尤其在肺泡上皮细胞中，病毒感染可引发多个亚细胞区室出现显著的蛋白质表达变化与结构重排。在受影响的细胞器中，线粒体（mitochondrion）似乎是主要的靶标细胞器。此外，基于蛋白质组学分析与EM（电子显微镜）线索，本研究测试了多种自噬抑制剂（autophagy inhibitors），发现其中一种——蝙蝠葛苏林碱（Daurisoline）——可在细胞中有效抑制病毒复制。综上，本研究揭示了肺泡上皮细胞与微血管内皮细胞对SARS-CoV-2感染的差异化应答，这将加深我们对COVID-19的认知，并有助于靶向药物的开发。