CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells

Abstract

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

Keywords: ACE-2; CFTR; CFTR inhibitor; SARS-CoV-2 virus; cystic fibrosis; human bronchial epithelial cells.

Figure 1

SARS-CoV-2 replication capability is different in WT and ΔF CFBE41o- cells: (a) Time course measurement of viral load in the supernatant of CFBE41o- cells infected with SARS-CoV-2 by commercial qualitative real-time RT-PCR (Seegene) able to detect several viral gene targets. The data are presented as the mean ± SD from independent experiments (n = 4; *** p < 0.001). (b) Measurement of ORF-1ab mRNA expression normalized to GAPDH (used as a housekeeping control) in cells infected with RNA extracted from SARS-CoV-2-infected CFBE41o- cells. The data are presented as the mean ± SD from independent experiments (n = 3; * p < 0.05, ** p < 0.01). (c) Time course measurement of viral load in the supernatant of WT and ΔF CFBE41o- cells infected with the BetaCoV/Australia/VIC01/2020 SARS-CoV-2 strain. The data were obtained from semiquantitative PCR (Seegene) of cellular supernatant and are expressed as the copies/µL ratio. (d) Total S1 protein quantification by Western blot analysis in CFBE41o- cells. Samples analyzed were harvested from 0 to 72 hpi with SARS-CoV-2. β-Actin was used as a loading control. The blot is representative of n = 3.

Figure 2

ACE-2 expression levels in CFBE41o- cells determined by different techniques: (a) Flow cytometry of ACE-2 cell membrane expression in uninfected ΔF and WTCFBE41o- cells. The data are representative of n = 3. (b) MFI of ACE-2 in uninfected ΔF and WT CFBE41o- cells. The data are presented as the mean ± SD from independent experiments (n = 3). (c) Total ACE-2 protein quantification by Western blot analysis in CFBE41o- cells. The samples analyzed were harvested from 0 to 72 hpi with SARS-CoV-2. β-Actin was used as a loading control. The data are from a Western blot representative of n = 3. (d) Measurement of ACE-2 mRNA expression normalized to GAPDH (used as a housekeeping control) in RNA extracted from SARS-CoV-2-infected CFBE41o- cells. The data are presented as the mean ± SD from independent experiments (n = 3).

Figure 3

Immunofluorescence confocal microscopy showing the expression of ACE-2 (red) in uninfected (a) and infected CFBE41o- WT cells obtained at 24 (b), 48 (c), 72 (d) hpi. Cell nuclei were counterstained with TO-PRO 3 (blue). Scale bar: 20 µm.

Figure 4

Immunofluorescence confocal microscopy showing the expression of ACE-2 (red) in uninfected (a) and infected CFBE41o- ΔF cells obtained at 24 (b), 48 (c), 72 (d) hpi. Cell nuclei were counterstained with TO-PRO 3 (blue). Scale bar: 20 µm.

Figure 5

SARS-CoV-2 replication and ACE-2 expression in WT and KO 16HBE14o- cells: (a) Time course measurement of viral load in the supernatant of 16HBE14o- cells infected with SARS-CoV-2 analyzed by qualitative real-time RT-PCR (Seegene) able to detect several viral gene targets. The data are presented as the mean ± SD from independent experiments (n = 4; ** p < 0.01). (b) Flow cytometry analysis of ACE-2 cell membrane expression in uninfected WT and KO 16HBE14o- cells. The data are representative of n = 3 measurements. (c) MFI of ACE-2 in uninfected WT and KO 16HBE14o- cells. The bars represent the mean ± SD from independent experiments (n = 3; * p < 0.05). (d) Total ACE-2 protein quantification by Western blot analysis in 16HBE14o- cells. The samples analyzed were harvested from 0 to 72 hpi with SARS-CoV-2. β-Actin was used as a loading control. The data are representative of n = 3.

Figure 6

Effect of CFTR inhibition in CFBE41o- WT cells via IOWH-032 treatment compared to WT and ΔF CFBE41o- cells: (a) Time course measurement of viral load in the supernatant of CFBE41o- cells infected with SARS-CoV-2 analyzed by qualitative real-time RT-PCR (Seegene). The data are presented as the mean ± SD from independent experiments (n = 3; **** p < 0.0001). (b) Total ACE-2 protein quantification by Western blot analysis in CFBE41o- cells. Samples were harvested from CFBE41o- WT cells exposed or not to IOWH-032 as well as CFBE41o- ΔF cells at different time points of SARS-CoV-2 infection. β-Actin was used as a loading control. The data are representative of n = 3 experiments.

Figure 7

SARS-CoV-2 replication in wt/wt-CFTR and F508del/F508del-CFTR MucilAir™: (a) Apical viral production was assessed in washes of the apical side at 24, 48, 72 hpi of both wt/wt-CFTR and F508del/F508del-CFTR MucilAir™ by commercial qualitative real-time RT-PCR (Seegene). The data are presented as the mean ± SD from independent experiments (n = 3; ** p < 0.01; **** p < 0.0001). (b) Trans-epithelial resistance measurements in wt/wt-CFTR and F508del/F508del-CFTR MucilAir™ (TEER in Ω·cm²) between the apical and basal sides at each time point. The data are presented as the mean ± SD from independent experiments (n = 3). (c) Basolateral SARS-CoV-2 quantification in wt/wt-CFTR and F508del/F508del-CFTR MucilAir™. Basolateral SARS-CoV-2 viral quantification was assessed in the basolateral medium at 24, 48, 72 hpi of both wt/wt-CFTR and F508del/F508del-CFTR MucilAir™ by commercial qualitative real-time RT-PCR (Seegene) able to detect several viral gene targets. The data are presented as the mean ± SD from independent experiments (n = 3; **** p < 0.0001).

Figure 8

SARS-CoV-2 replication cycle in MucilAir™ is driven by CFTR modulation: (a) Apical viral production was assessed in washes of the apical side at 24, 48, 72 hpi of both wt/wt-CFTR and F508del/F508del-CFTR MucilAir™ with and without CFTR-modulator/inhibitor treatment by commercial qualitative real-time RT-PCR (Seegene). The data are presented as the mean ± SD from independent experiments (n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (b) Trans-epithelial resistance (TEER in Ω·cm²) between the apical and basal sides was measured at each time point in both wt/wt-CFTR and F508del/F508del-CFTR MucilAir™ with and without VX-661+VX-445, for F508del/F508del-CFTR, or IOWH-032, for wt/wt-CFTR, chronic treatments. The data are presented as the mean ± SD from independent experiments (n = 3).