(509) CFTR positively regulates ACE2 expression and SARS-CoV-2 viral entry into airway epithelial cells: implications for patients with Cystic Fibrosis

Background: Respiratory viral infections (i.e. respiratory syncytial and influenza A viruses) have been previously associated with deterioration of pulmonary function and exacerbations in CF patients [1]. Conversely, CF patients infected by SARS-CoV-2 are not experiencing worse clinical outcomes compared to the general population, except for lung-transplanted patients, who require increased oxygen therapy and hospitalization, and exhibit a 6-fold increased death risk. Interestingly, several studies reported that CF patients positive to SARS-CoV-2 show a lower case fatality rate compared to the general population instead [2-4]. Therefore, we hypothesized that loss of CFTR protein expression/function may constitute an advantage against COVID-19 severe outcome.

Methods: We investigated the expression of Angiotensin-converting enzyme 2 (ACE2) and CD13 (ANPEP) mRNA and protein, in several CF and non-CF airway epithelial cells, including polarized CFBE41o-, 16HBE14o-, Calu-3 and ALI-differentiated primary nasal and bronchial epithelia. Cell susceptibility to SARS-CoV-2 infection was assayed by infecting single cells with a MOI of 0.1 for 2h. Twenty-four, 48 and 72 hours after infection, RNA was isolated from both cells and supernatants and SARS-CoV-2 titration was obtained using a TaqMan assay by qPCR/dPCR. IL-6 levels were quantified by ELISA in supernatants from primary airway epithelial cells stimulated with 1mg/ml SARS-CoV-2 Spike protein, for 12h.

Results: The expression of SARS-CoV-2 receptor, ACE2, is significantly downregulated in CF cells both in terms of mRNA and protein. Consistently, we observed that the expression of the aminopeptidase CD13, which has been found co-expressed with ACE2 in several tissues and acts as co-receptor for other human coronaviruses, is equally downregulated. We reported that F508del mutated CFTR channel promotes the mislocalization of ACE2, which is almost completely retained into the ER, similarly to unfolded CFTR, thus suggesting an association between these two proteins. Most importantly, reduced ACE2 expression in CF cells is associated with reduced SARS-CoV-2 viral entry and replication. Eventually, spike-induced IL-6 levels were significantly reduced in ALI-differentiated primary airway epithelia obtained from CF patients, consistently with the reduced ACE2 expression.

Conclusions: This study clarify why SARS-CoV-2 infection does not promote particularly severe outcomes in CF patients, unless they have received lung transplantation. Furthermore, our results showcase CFTR as a regulator of ACE2 and SARS-CoV-2 viral entry and replication.

Acknowledgements: This study was supported by the Società Italiana Fibrosi Cistica and Pfizer cybergrant #61509709.

References: 1. Billard L, Le Berre R, Pilorgé L, Payan C, Héry-Arnaud G, Vallet S. Viruses in cystic fibrosis patients' airways. Crit Rev Microbiol. 2017;43(6):690-708.

2. Corvol H, de Miranda S, Lemonnier L, Kemgang A, Reynaud Gaubert M, Chiron R, et al. First Wave of COVID-19 in French Patients with Cystic Fibrosis. J Clin Med. 2020;9(11).

3. Mondejar-Lopez P, Quintana-Gallego E, Giron-Moreno RM, Cortell-Aznar I, Ruiz de Valbuena-Maiz M, Diab-Caceres L, et al. Impact of SARS-CoV-2 infection in patients with cystic fibrosis in Spain: Incidence and results of the national CF-COVID19-Spain survey. Respir Med. 2020;170:106062.

4. Naehrlich L, Orenti A, Dunlevy F, Kasmi I, Harutyunyan S, Pfleger A, et al. Incidence of SARS-CoV-2 in people with cystic fibrosis in Europe between February and June 2020. J Cyst Fibros. 2021.