An autonomous intrapulmonary inflammatory feedback response, mediated by surfactants, operates as an inherent viral defense mechanism: evidence from acute SARS-CoV-2 infection [Prox, Distal, and Whole Lung Organoid scRNA-seq]

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles neonatal RDS – a condition attributable to the deficiency of the surface tension reducing factor surfactant. Using a novel multi-cell type, human-induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that surfactant proteins (SP) A, B, C and D are dynamically regulated by SARS-CoV-2 infection. In the absence of SP-B (including through the study of LOs from SP-B deficient patients), resistance to infection was impaired and inflammatory cytokine/chemokine production and interferon (IFN) responses were modulated in lung cells of non-hematopoietic origin and without a circulatory system, suggesting specialization of viral inflammatory responses by human lung cells. Such cells can be infected independent of ACE2 and TMPRSS2 expression via what we propose is a non-canonical endocytotic route. Single cell RNA sequencing revealed broad susceptibility of many lung cell types with differential expression of BSG, cathepsins, and ACE2 that all facilitate viral entry. SARS-CoV-2 triggers a rapid loss of lung cell viability via apoptosis that impairs viral dissemination evident in the presence of Bcl-xL antagonism. Treatment with exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion mutation restored resistance to SARS-CoV-2 infection and improved cell viability. These findings suggest an unrecognized specialized intrinsic intra-pulmonary inflammation-based feedback loop of developmental origin that autonomously, without the intercession of hematopoietic derivatives, attempts to restore lung homeostasis after infection by inhibiting viral dissemination, averting lung epithelial demise, and dampening inflammatory cascades -- an additional inherent host-pathogen defense system. Given that SP-B is central to this response, an antiviral role may now be ascribed to this structural component that heretofore had been regarded as necessary only for surface tension reduction. Overall design: Single-cell transcriptomics comparison of normal proximal, distal, and whole and SARS-CoV-2 infected proximal organoids by scRNA-seq

严重急性呼吸综合征冠状病毒2型（Severe Acute Respiratory Syndrome Coronavirus-2，SARS-CoV-2）可引发类似新生儿呼吸窘迫综合征（neonatal RDS）的急性呼吸窘迫综合征（ARDS）——该病症由降低表面张力的肺表面活性物质缺乏所致。本研究借助一种经原代肺细胞验证的新型多细胞类型人诱导多能干细胞（human-induced pluripotent stem cell，hiPSC）源性肺类器官（lung organoid，LO）系统，发现肺表面活性物质相关蛋白（surfactant proteins，SP）A、B、C、D的表达会受SARS-CoV-2感染的动态调控。在SP-B缺乏的情况下（包括通过SP-B缺陷患者的肺类器官开展研究），细胞对感染的抵抗能力受损，且非造血起源、无循环系统的肺细胞中，炎性细胞因子/趋化因子的产生与干扰素（IFN）应答均发生异常调控，这提示人肺细胞的病毒炎性反应存在特异性调控机制。此类细胞可通过我们推测的非经典内吞途径实现感染，且该过程不依赖血管紧张素转换酶2（ACE2）与跨膜丝氨酸蛋白酶2（TMPRSS2）的表达。
单细胞RNA测序（single cell RNA sequencing，scRNA-seq）结果显示，多种肺细胞类型均表现出广泛易感性，且BSG、组织蛋白酶与ACE2的差异表达均可促进病毒入侵。SARS-CoV-2可通过细胞凋亡快速诱导肺细胞活力丧失，该过程会削弱病毒播散，且这一效应在Bcl-xL拮抗剂存在时更为显著。给予外源性肺表面活性物质、重组SP-B，或对SP-B缺失突变进行基因组校正，均可恢复细胞对SARS-CoV-2的感染抵抗能力，并改善细胞活力。
上述研究结果表明，存在一种此前未被识别的、源自发育起源的肺内特异性炎性反馈环路：该环路无需造血衍生物的干预即可自主发挥作用，通过抑制病毒播散、阻止肺上皮细胞死亡以及减弱炎性级联反应，在感染后协助恢复肺内稳态——这是一种额外的固有宿主-病原体防御系统。鉴于SP-B在该应答过程中发挥核心作用，我们可将抗病毒功能赋予这一此前仅被认为仅具备降低表面张力功能的结构蛋白。
整体实验设计：通过单细胞RNA测序（scRNA-seq），对正常近端肺类器官、正常远端肺类器官、完整肺类器官以及SARS-CoV-2感染的近端肺类器官进行单细胞转录组学比较。