TGF-β1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-β signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-β1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-β1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-β1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-β1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-β1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-β1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-β1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-β1 inhibition of ACE2 levels. Finally, TGF-β1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-β1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-β1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-β1 on the host immune responses, leading to a complex and yet incompletely understood circuitry.