State-of-the-art analytical methods of viral infections in human lung organoids.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human alveolar-like organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human alveolar-like organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human alveolar-like organoids based on material from different donors.

Human lungs show limited permissiveness for SARS-CoV-2 due to scarce ACE2 levels but virus-induced expansion of inflammatory macrophages.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive. METHODS: Spectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 "gain-of-function" experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validate ex vivo results. RESULTS: We provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead, ex vivo infected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in "inflammatory alveolar macrophages", comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection. CONCLUSIONS: Collectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.