Cell culture model system utilizing engineered A549 cells to express high levels of ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals

Novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an imminent global threat since its initial outbreak in December 2019. A simple in vitro model system using cell lines highly susceptible to SARS-CoV-2 infection are critical to facilitate the study of the virus cycle and to discover effective antivirals against the virus. Human lung alveolar A549 cells are regarded as a useful and valuable model for respiratory virus infection. However, SARS-CoV-2 uses the ACE2 as receptor for viral entry and the TMPRSS2 to prime the Spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a robust human lung epithelial cell-based model by transducing ACE2 and TMPRSS2 into A549 cells and show that the ACE2 enriched A549ACE2/TMPRSS2 cells (ACE2plus) and its single-cell-derived subclone (ACE2plusC3) are highly susceptible to SARS-CoV-2 infection. These engineered ACE2plus showed higher ACE2 and TMPRSS2 mRNA expression levels than currently used Calu3 and commercial A549ACE2/TMPRSS2 cells. ACE2 and TMPRSS2 proteins were also highly and ubiquitously expressed in ACE2plusC3 cells. Additionally, antiviral drugs like Camostat mesylate, EIDD-1931, and Remdesivir strongly inhibited SARS-CoV-2 replication. Notably, multinucleated syncytia, a clinical feature commonly observed in severe COVID-19 patients was induced in ACE2plusC3 cells either by virus infection or by overexpressing the Spike proteins of different variants of SARS-CoV-2. Syncytial process was effectively blocked by the furin protease inhibitor, Decanoyl-RVKR-CMK. Taken together, we have developed a robust human A549 lung epithelial cell-based model that can be applied to probe SARS-CoV-2 replication and to facilitate the discovery of SARS-CoV-2 inhibitors.

Single-cell RNA-seq analysis reveals lung epithelial cell-specific contributions of Tet1 to allergic inflammation

Background: Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. We explored the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation. Methods: A model of HDM-induced lung inflammation was established in Tet1 knockout and littermate wildtype mice. EpCAM+ lung epithelial cells were isolated. Libraries were generated using the 10X Chromium workflow and sequenced. ScRNA-seq analysis was performed using Cell Ranger, scAlign, and Seurat. Cell types were labeled using known markers. Enriched pathways were identified using Ingenuity Pathway Analysis. Transcription factor (TF) activity was analyzed by DoRothEA. Single-cell trajectory analysis was performed with Monocle to explore Alveolar type 2 (AT2) cell differentiation. Results: AT2 cells were the most abundant among the eight EpCAM+ lung epithelial cell types. HDM challenge increased the percentage of alveolar progenitor cells (AP), broncho alveolar stem cells (BAS), and goblet cells, and decreased the percentage of AT2 and ciliated cells. Bulk and cell-type-specific analysis identified genes subject to Tet1 regulation and linked to augmented lung inflammation, including alarms, detoxification enzymes and oxidative stress response genes, and gene in tissue repair. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Conclusions: Collectively, lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, with an overall effect of promoting allergen-induced lung inflammation.

Effect of curcumin on lung epithelial injury and ferroptosis induced by cigarette smoke

Cigarette smoke (CS)-caused ferroptosis was involved in the pathogenesis of COPD, but the role of ferroptosis in lung epithelial injury and inflammation is not clear. Rats were treated with CS or CUR and BEAS-2B cells were exposed to CS extract (CSE), ferrostatin-1 (Fer-1), deferoxamine (DFO), or CUR to detect reactive oxygen species (ROS) accumulation, lipid peroxidation, iron overload, and ferroptosis-related protein, which were the characteristic changes of ferroptosis. Compared with the control group, CSE-treated BEAS-2B cells had more cell death, higher cytotoxicity, and lower cell viability. The infiltration of inflammatory cell around the bronchi in the CS group of rats was more than that in the normal group. Meanwhile, CSE/CS elevated the levels of interleukin-6 and tumor necrosis factor-α in BEAS-2B cells and bronchoalveolar lavage fluid of rats. Besides, accumulative ROS and depleted glutathione was observed in vitro. In BEAS-2B cells and lung tissues of rats, CSE/CS increased malondialdehyde and iron; down-regulated solute carrier family 7, glutathione peroxidase 4, and ferritin heavy chain levels; and up-regulated transferrin receptor level. These changes were rescued by pretreatment of Fer-1 or DFO in vitro, and mitigated by CUR in vitro and in vivo. Collectively, this study reveals that ferroptosis was involved in lung epithelial cell injury and inflammation induced by CS, and CUR may alleviate CS-induced injury, inflammation, and ferroptosis of lung epithelial cell.

Fetal Tracheal Occlusion Increases Lung Basal Cells via Increased Yap Signaling

Fetal endoscopic tracheal occlusion (FETO) is an emerging surgical therapy for congenital diaphragmatic hernia (CDH). Ovine and rabbit data suggested altered lung epithelial cell populations after TO with transcriptomic signatures implicating basal cells. To test this hypothesis, we deconvolved mRNA-seq data and used quantitative image analysis in fetal rabbit lungs to showed increased basal cells and reduced ciliated cells after TO. In a fetal mouse TO model, flow cytometry showed increased basal cells, and immunohistochemistry demonstrated basal cell extension to the subpleura. Nuclear yap, a known regulator of basal cell fate, was increased in TO lung, and Yap ablation on the lung epithelium abrogated TO-mediated basal cell expansion. mRNA-seq of TO lung showed increased activity of downstream Yap genes. Human lung specimens with congenital and fetal endoscopic tracheal occlusion had clusters of subpleural basal cell that were not present in control. TO increases lung epithelial cell nuclear Yap leading to basal cell expansion.

Regulated in Development and DNA Damage Response 1 Knockdown Alleviates Lipopolysaccharide-Induced Acute Lung Injury

Regulated in Development and DNA Damage Response 1 (REDD1) knockdown can reduce the endoplasmic reticulum stress response in liver injury. However, its role on lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored. This study aimed to evaluate the effect of REDD1 on lung epithelial cells induced by LPS. Rt-qPCR and Western blot were used to detect REDD1 expression in 16HBE cells induced by LPS. The interfering REDD1 plasmid was constructed, and CCK8 was used to detect the effect of interference with REDD1 on LPS-induced lung epithelial cell activity. The expression of inflammatory factors was detected by ELISA and the apoptotic level was detected by TUNEL staining. String database was used to predict the combination of REDD1 and EP300 in lung epithelial cells, which was verified by CoIP experiment. An overexpressed plasmid of EP300 was constructed to detect the effects of EP300 on inflammatory factors and apoptosis in REDD1 lung epithelial cells. LPS-induced increased REDD1 expression in lung epithelial cells. Interference with REDD1 inhibits LPS-induced lung epithelial cell activity injury and inflammatory factor expression and inhibits LPS-induced lung epithelial cell apoptosis. After interference with REDD1, the expression of EP300 in LPS-induced lung epithelial cells was inhibited, and the overexpression of EP300 was reversed to promote the production of inflammatory factors and apoptosis. In conclusion, these results demonstrate that REDD1 knockdown alleviates LPS-induced acute lung injury.