SARS-CoV-2 induces transcriptional signatures in human lung epithelial cells that promote lung fibrosis

Abstract

Background: Severe acute respiratory syndrome (SARS)-CoV-2-induced coronavirus disease-2019 (COVID-19) is a pandemic disease that affects > 2.8 million people worldwide, with numbers increasing dramatically daily. However, there is no specific treatment for COVID-19 and much remains unknown about this disease. Angiotensin-converting enzyme (ACE)2 is a cellular receptor of SARS-CoV-2. It is cleaved by type II transmembrane serine protease (TMPRSS)2 and disintegrin and metallopeptidase domain (ADAM)17 to assist viral entry into host cells. Clinically, SARS-CoV-2 infection may result in acute lung injury and lung fibrosis, but the underlying mechanisms of COVID-19 induced lung fibrosis are not fully understood.

Methods: The networks of ACE2 and its interacting molecules were identified using bioinformatic methods. Their gene and protein expressions were measured in human epithelial cells after 24 h SARS-CoV-2 infection, or in existing datasets of lung fibrosis patients.

Results: We confirmed the binding of SARS-CoV-2 and ACE2 by bioinformatic analysis. TMPRSS2, ADAM17, tissue inhibitor of metalloproteinase (TIMP)3, angiotensinogen (AGT), transformation growth factor beta (TGFB1), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) A and fibronectin (FN) were interacted with ACE2, and the mRNA and protein of these molecules were expressed in lung epithelial cells. SARS-CoV-2 infection increased ACE2, TGFB1, CTGF and FN1 mRNA that were drivers of lung fibrosis. These changes were also found in lung tissues from lung fibrosis patients.

Conclusions: Therefore, SARS-CoV-2 binds with ACE2 and activates fibrosis-related genes and processes to induce lung fibrosis.

Keywords: Angiotensin-converting enzyme 2; Coronavirus; Lung fibrosis; SARS-CoV-2.

Fig. 1

In silico modelling showing that SARS-CoV-2 may bind to ACE2 and associates with interacting molecules. a The spike (S) protein of SARS-CoV is predicted to bind with ACE2 protein and represented front and back images of protein structure using p-hipster. The predicted network of ACE2 gene (b) and protein (c) interacting molecules identified using bioinformatic analysis

Fig. 2

mRNA transcripts and protein levels of ACE2 and its interacting molecules in human in the lungs and gut. a mRNA expression of ACE2, TMPRSS2. ADAM17, TIMP3, TGFB1, CTGF. VEGFA and FN1 in all human organs and b in the lung and gastrointestinal tract using GTEx Portal. c ACE2, TMPRSS2, ADAM17, AGT, TGFB1, VEGFA, CRGF and FN proteins in human lung using immunohistochemistry from Pathology Atlas database (1x scale bar is 200 μm, 16x scale bar is 100 μm)

Fig. 3

Single cell RNA-sequencing analysis of ACE2, TMPRSS2. ADAM17, TIMP3, AGT, TGFB1, CTGF. VEGFA and FN1 mRNAs in human airways and lungs. a Single cells were isolated from human bronchial biopsy (n = 4) and subjected to RNA-sequencing. Cells were clustered using a graph-based shared nearest neighbour clustering approach and visualised using a t-distributed Stochastic Neighbor Embedding (tSNE) plot from UCSC cell browser. b mRNA expression of ACE2, TMPRSS2. ADAM17, TIMP3, TGFB1, CTGF, VEGFA and FN1 in different cells from human airways. c Single cells were isolated from human lungs (n = 12) and the cells were clustered using tSNE plot from UCSC cell browser. d mRNA expression of ACE2, TMPRSS2. ADAM17, TIMP3, TGFB1, CTGF, VEGFA and FN1in different cells from human lungs

Fig. 4

SARS-CoV-2 infection induces Ace2 gene and its interacting factors in human alveolar but not bronchial epithelial cells. Human bronchial epithelial cells (HBEC) and alveolar epithelial (A549) cells were infected with SARS-CoV-2 for 24 h, and control cells received medium only. The mRNA expression of ACE2 (a), TMPRSS2 (b), ADAM17 (c), TIMP3 (d), AGF (e), TGFB1 (f), CTGF (g), VEGFA (h) and FN1 (i) in HBECs and alveolar epithelia cells. n = 2–3, *P < 0.05, **P < 0.01 compared to control cells

Fig. 5

Single cell RNA-sequencing analysis of ACE2, TMPRSS2. ADAM17, TIMP3, TGFB1, CTGF, VEGFA and FN1 mRNA in human lungs from patients with idiopathic pulmonary fibrosis (IPF). a Single cells were isolated from cryobiopsy samples in one IPF patient. They were subjected to RNA-sequencing and data clustered using a graph-based shared nearest neighbour clustering approach and visualised using a t-distributed Stochastic Neighbor Embedding (tSNE) plot from UCSC cell browser. b mRNA expression of ACE2, TMPRSS2, ADAM17, TIMP3, TGFB1, CTGF, VEGFA and FN1 in different cells from human airways. c Single cells were isolated from human lungs from IPF patients (n = 8), subjected to RNA-sequencing and data clustered using tSNE plot from UCSC cell browser. d mRNA expression of ACE2, TMPRSS2, ADAM17, TIMP3, AGT, TGFB1, CTGF, VEGFA and FN1 in different cells from human lungs

Fig. 6

mRNA expression of ACE2 and selected interacting factors in lung tissues from IPF patients. mRNA expression of ACE2 (a), TMPRSS2 (b), TIMP3 (c), TGFB1 (d), CTGF (e). VEGFA (f) and FN1 (g) in lung tissues from IPF patients (n = 13) and lung healthy control (n = 11) were extracted from an existing microarray dataset (GSE2052). *P < 0.05 compared to lung healthy controls

Fig. 7

mRNA expression of ACE2 and selected interacting factors in lung tissues from IPF and other ILD patients. mRNA expression of ACE2 (a), TMPRSS2 (b), ADAM17 (c), TIMP3 (d), AGF (e), TGFB1 (f), CTGF (g). VEGFA (h) and FN1 (i) in lung tissues from IPF (n = 8), other ILD patients (n = 23) and lung healthy control (n = 15) were extracted from an existing microarray dataset (GSE10667). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to lung healthy controls. #P < 0.05, ##P < 0.01 compared to other ILD patients