Mechanisms by which the cystic fibrosis transmembrane conductance regulator may influence SARS-CoV-2 infection and COVID-19 disease severity

Abstract

Patients with cystic fibrosis (CF) exhibit pronounced respiratory damage and were initially considered among those at highest risk for serious harm from SARS-CoV-2 infection. Numerous clinical studies have subsequently reported that individuals with CF in North America and Europe-while susceptible to severe COVID-19-are often spared from the highest levels of virus-associated mortality. To understand features that might influence COVID-19 among patients with cystic fibrosis, we studied relationships between SARS-CoV-2 and the gene responsible for CF (i.e., the cystic fibrosis transmembrane conductance regulator, CFTR). In contrast to previous reports, we found no association between CFTR carrier status (mutation heterozygosity) and more severe COVID-19 clinical outcomes. We did observe an unexpected trend toward higher mortality among control individuals compared with silent carriers of the common F508del CFTR variant-a finding that will require further study. We next performed experiments to test the influence of homozygous CFTR deficiency on viral propagation and showed that SARS-CoV-2 production in primary airway cells was not altered by the absence of functional CFTR using two independent protocols. On the contrary, experiments performed in vitro strongly indicated that virus proliferation depended on features of the mucosal fluid layer known to be disrupted by absent CFTR in patients with CF, including both low pH and increased viscosity. These results point to the acidic, viscous, and mucus-obstructed airways in patients with cystic fibrosis as unfavorable for the establishment of coronaviral infection. Our findings provide new and important information concerning relationships between the CF clinical phenotype and severity of COVID-19.

Keywords: COVID-19; SARS-CoV-2; comorbidity; cystic fibrosis transmembrane conductance regulator; mucus membrane; virus replication.

Figure 1.

SARS-CoV-2 related features of primary human airway (bronchial) epithelium (hBEC) from three non-CF individuals or three patients with CF. Human primary cells were prepared as differentiated monolayers at air-liquid interface for each data point. (A) Quantitative analyses of ACE2, TMPRSS2, and CFTR expression in CF versus non-CF primary airway epithelia determined by ddPCR. Data is shown as the ratio of target gene expression to TBP or HPRT1 internal controls and calculated as mean +/− standard deviation (SD). Each bar represents 2–4 biological replicates per patient sample, with 2 ddPCR runs per replicate. (TMPRSS2 was normalized to HPRT1 due to expression at much higher levels than CFTR or ACE2 in primary airway epithelia.) For ACE2: CF vs. non-CF, p <0.0001; for TMPRSS2 and CFTR: CF vs. non-CF, p >0.05. (B) Correlation analysis between ACE2 and TMPRSS2 mRNA levels and SARS-CoV-2 replication. (C) hBEC monolayers were grown at air liquid interface and exposed to ~300 infectious units of SARS-CoV-2. Total RNA was harvested 48 h post infection and levels of SARS-CoV-2 RNA determined by RT-qPCR. Each bar represents a different individual with 2 biological replicates and 2 wells per repeat (4 wells total), with two duplicate qPCR samples per well (8 total qPCR values per bar). Propagation is expressed as viral RNA within cells, 48 h post infection, relative to sample CF-3 and normalized to RNaseP. Standard deviation bars are shown. Note that for certain points, error bars are shorter than height of symbols. (D) Virus replication kinetics in bronchial epithelial monolayers from three non-CF individuals and three individuals with CF were determined by inoculating separate monolayers with an MOI of 0.1 of SARS-CoV-2. Samples were collected from the apical surface at 3, 12, 24, and 48 h post infection. RNA was extracted and RT-qPCR performed to detect E gRNA. In each run, dilutions of counted RNA standards were run in parallel to calculate copy numbers in the samples. To confirm active virus, RT-qPCR was performed on the inoculum and immediately after removal of the wash. Data represent means and standard error of 4 replicate wells per donor per timepoint. Values of replication are variable between patient hBEC samples due to factors such as cell confluence, passage number, monolayer resistance, effects of genes other than CFTR, epistasis, etc. Differences between WT versus CF primary cells in (C) amount to modestly higher production of SARS-CoV-2 in the CF models, but overall (WT vs. CF) failed to reach statistical significance. CF samples in Figure 1D showed a maximal increase (peak value vs. 3-hour post inoculum timepoint) of 624-fold and non-CF samples showed a 370-fold increase.

Figure 2.

Impact of washing mucosal fluid prior to infection by SARS-CoV-2. (A) Human primary airway (bronchial) epithelia (hBEC), or (B) Calu-3 cells were prepared at air-liquid interface, and treated with ~300 infectious units of SARS-CoV-2 in PBS. In A), two different WT hBEC cell models from different individuals were tested eight times, and two different CF hBEC models were tested three times, under both gently washed and unwashed conditions (11 experiments in total). Washing was conducted to remove periciliary fluid and mucus. Total RNA from cells was harvested at 48 h post infection and levels of SARS-CoV-2 RNA determined by RT-qPCR. Replication is expressed as relative viral RNA levels at 48 h, comparing unwashed vs. washed samples. The effects of washing were pronounced and, in many cases, essentially superimposable, making individual line graphs in Panel A difficult to distinguish. P value calculations by t-test included all 11 experiments (washed vs. unwashed, p < 0.0001). (C) Human primary bronchial epithelia were prepared as above at ALI (donor WT-1), then transduced with 1×10⁸ infectious units (titered on 293T cells) of GFP-expressing recombinant adenovirus. After 48 h, GFP-positive cells were counted. Data in Panels (B) and (C) represent means and standard deviations. Overall viral production in Calu-3 cells was modestly (~2.5-fold) higher than in primary airway epithelial monolayers. Tissue origin from which cells are derived (airway glandular (Calu-3) versus pulmonary surface epithelial), growth conditions (including time required to achieve mature, polarizing cells), confluency, and other factors may contribute to differences in viral replication in hBEC versus Calu-3 model systems.

Figure 3.

Effect of mucosal pH and viscosity on SARS-CoV-2 infection. (A) Human primary airway cells were prepared at air-liquid interface, and treated with approximately 2,000 infectious units of SARS-CoV-2 in HBSS at the indicated pH. Total RNA was harvested 48 h post infection and levels of SARS-CoV-2 RNA determined by RT-qPCR. Replication is expressed as viral RNA levels, relative to results at pH 7 (*p < 0.05). (B) Calu-3 cells were seeded in 96-well plates. Prior to infection, cultures were switched to medium adjusted to contain the indicated percentage (v/v) of carboxymethylcellulose. Cells were infected at an MOI of 0.01 with icSARS-CoV-2-mNG. After 48 h, monolayers were fixed and number of infected cells per well determined by fluorescence microscopy. (C) Human bronchial epithelial monolayers at air-liquid interface were treated with approximately 1,000 infectious units of SARS-CoV-2 diluted in PBS containing the indicated percentage (w/v) of carboxymethylcellulose. Error bars in (A) and (C) represent standard deviations. (**p = 0.009 at 0.5% CMC and *p = 0.048 at 1% CMC compared to control)

Figure 4.

Effect of CF or non-CF human sputum on SARS-CoV-2 infection. Primary bronchial epithelial cells were prepared as epithelial monolayers at air-liquid interface. (A) A subset of bronchial epithelial cell filters were washed to remove conditioned surface liquid and mucus. Prior to infection, 2,000 infectious units of SARS-CoV-2 were diluted 1:5 either in PBS or in freshly expectorated mucus collected from patients with cystic fibrosis or a healthy individual and added to cells in culture. Multiple comparison p values washed versus unwashed (Bonferroni): PBS: 0.57; CFsp1: 0.05; CFsp2: >0.99; Non-CFsp: 0.04. (B) Paired hBEC samples (WT/WT or F508del/F508del) were washed to remove surface liquid and mucus. SARS-CoV-2 viral stock was diluted 1:5 in PBS or sputum supernatant and 2,000 infectious units added to airway cell monolayers. PBS versus CFsp2, p = 0.01 (2-way ANOVA). (C) Prior to infection of unwashed non-CF epithelia, 1,000, 100 or 10 infectious units (IU) of SARS-CoV-2 were diluted in PBS or CF sputum supernatant 2. PBS versus CFsp2, p = 0.02; for virus dose effect, p = 0.03 (2-way ANOVA). Replication values in all panels represent viral RNA levels at 48 h. In (C), RNA levels are additionally normalized to the sample inoculated with 1,000 IU virus in PBS. Error bars in all panels represent standard deviations.