For cultivation of hAELVI cells, cell culture flasks were coated prior to application of cells using huAEC Coating solution (inSCREENex, INS-SU-1018-100ml). hAELVi cells were cultivated in huAEC Medium (inSCREENex, INS-ME-1013-500ml). Prior to seeding of hAELVi cells on transwell filters (ThinCert, pore size 0.4 µm, Greiner), inserts were coated by adding huAEC coating solution according to the manufactures protocol. Approximately 2.3 x 105 (12-well: Fig. 1b-e and Fig. 2a-e) or 0.7 x 105 cells (24-well: Fig. 2f-g) were seeded into the apical chamber of the filter insert and initially incubated at 37°C and 5% CO2 under liquid-liquid-conditions for 3 days following further cultivation under ALI up to 28 days. Based on the analyses of barrier integrity, morphological analysis and expression of cellular host factors, the polarized hAELVi cells were used for infection experiments after an incubation period of at least 21 days under ALI. Vero E6 cells were propagated in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with 2mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1x non-essential amino acids and 1 mM sodium pyruvate. MDCKII cells were cultivated in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were incubated at 37°C with 5% CO2 in a humidified atmosphere.
Infection of hAELVi cells and virus titration on Vero E6 cells
Cells were infected with SARS-CoV-2 D614G (hCoV-19/Germany/BW-RKI-N-0001/2020, GISAID accession: EPI_ISL_481253), SARS-CoV-2 Delta (ENA project PRJEB50616; sequence ID IMSSC2-206-2021-00148), SARS-CoV-2 Omicron (hCoV-19/Germany/BE-RKI-I-353502/2021, GISAID accession: EPI_ISL_7116918), SARS-CoV Frankfurt-1 (Genbank accession number FJ429166.1), MERS-CoV EMC/2012 (Genbank: JX869059) or Influenza A/Panama/2007/1999 virus (Genbank: DQ487333-DQ487340). For infection, cells were washed with D-PBS once and inoculated with virus diluted in D-PBS/0.3% BA in the apical chamber. Following incubation for 1 h at 37°C, cells were washed apically with D-PBS and fresh medium was added to the basolateral compartment of the filter insert. For replication analysis, 10% of the basolateral supernatant was harvested at indicated time points and refilled with fresh culture medium. To collect apical samples 50µl (24-well) or 100µl D-PBS (12-well), respectively, were used to perform apical washes at 37°C for 30 min. Supernatants were stored at -80°C until titration by standard Plaque Assay on Vero E6 cells (CoV) or MDCKII cells (influenza A virus) using Avicel overlay was performed to quantify infectious virus particles.
SARS-CoV-2 viruses were isolated from naso- or oropharyngeal swabs on VeroE6 or Caco-2 cells in the context of routine diagnostics by RKI units FG17 and ZBS1, which were approved by the ethics committees of Charité-Universitätsmedizin Berlin Ethical Board (Reference EA2/126/11) and the Berlin Medical Association (Eth-40/20).
Preparation of cell lysates and immunoblot analysis
Cells were washed twice with ice-cold D-PBS before lysis buffer (10 mM Tris/HCl (pH 7,5), 150 mM NaCl, 0.5 mM EDTA, 1% NP-40 including protease inhibitor) was added to the apical chamber of the filter insert and incubated for at least 30 min at 4°C. Cells lysates were centrifuged at 15000 x g and 4°C for 10 min and supernatants were stored at -20°C until further processing. 50 µg of each sample was separated by reducing SDS-PAGE under denaturing conditions and transferred onto nitrocellulose membrane by semi-dry western blotting Detection of ACE2 was performed by incubation with anti-hACE2 antibody (R&D Systems (AF933); 1:500) and suitable secondary antibody coupled to horseradish-peroxidase (HRP) (1:10,000; Agilent Technologies, Santa Clara, USA). Equal loading of samples was controlled with immunostaining of actin. SuperSignal™WestDura Extended Duration Substrate was added to the membrane and the resulting chemiluminescence was detected using an Advanced Fluorescence Imager (Intas).
Basolateral supernatants of infected cells were collected at indicated time points and stored at -80°C until further processing. If necessary, samples were diluted in the corresponding culture medium prior to ELISA measurement. Samples were analyzed using the R&D DuoSet ELISA Kits DY9345 (IFNα2), DY814 (IFNβ), DY7246 (IFNλ1), DY1587 (IFNλ2), DY5259 (IFNλ3), DY279 (CCL2), DY270 (CCL3), DY271 (CCL4), DY275 (CXCL1), DY254 (CXCL5), DY214 (G-CSF), DY206 (IL-6), DY208 (IL-8), DY289 (MIF), DY201 (IL-1β), DY266 (CXCL10) according to the manufacturer’s instructions. For collective heat map presentation of all analyzed cytokines, mean values of x-fold inductions relative to mock-infection of three independent experiments were calculated for each cytokine. Individual concentrations of all cytokines are shown in Supplementary Fig. 4.
For quantification of host factor expression cell lysates were prepared as previously described, total protein amount was measured using -BCA-Kit- according to the manufacturer’s instructions and 100µg of protein lysate was analyzed using the R&D DuoSet ELISA Kits DY933-05 (ACE2), DY1180 (DPP4) and the commercially available ELISA kit ABIN6960140 (TMPRSS2) according to the corresponding manufacturers protocol.
Detection of viral antigen by laser-scanning confocal fluorescence microscopy
Cells were washed once with D-PBS, fixed with 3.7% formaldehyde in D-PBS for 15 min at RT and further incubated for 10 min at RT with 10 mM ammonium chloride/D-PBS. Subsequently, cells were permeabilized for 7 min with 0.5% TritonX-100/D-PBS at RT, followed by blocking of cells with 3% BSA/D-PBS for at least 1 h at RT. Between each of these steps, the samples were washed twice with D-PBS.
For staining of specific proteins, filter inserts were stamped out with 6 mm biopsy punches and incubated with primary antibodies diluted in 3% BSA/D-PBS at 4°C overnight. After washing three times with D-PBS for at least 5 min, cells were incubated with secondary fluorescence conjugated antibodies for 1 h at RT in the dark. Cells were subsequently washed twice with D-PBS and nucleus stained by incubating cells with DAPI for 10 min. Fluorescence laser-scanning microscopy was performed using a Zeiss LSM 780 confocal microscope with corresponding ZEN software and a Plan-Achromate 20x (NA 0.8) objective.
Thin section electron microscopy (EM)
hAELVi cells were fixed on their filter substrate with a mixture of 1% formaldehyde and 2.5% glutaraldehyde in HEPES (0.05 M, pH 7.4) for at least 2 h at RT. Filters were punched out with a skin punch and were post-fixed with osmium tetroxide, tannic acid, uranyl acetate and embedded in epon21. Ultrathin sections were collected on naked grids and examined with a transmission electron microscope (Tecnai Spirit, Thermo Fisher Scientific Inc.) operated at 120 kV acceleration voltage. Images were recorded with a CMOS camera (Phurona, EMSIS). Overview images of the epithelium were recorded from carbon coated resin block-faces after sectioning with a diamond knife using a scanning electron microscope (Teneo VS, Thermo Fisher Scientific Inc.) and the T1 detector at 2 kV.
Determination of transepithelial electrical resistance (TEER) and Transepithelial Transport of Fluorescein in hAELVi cell monolayers
To determine TEER values, the apical chamber of cultured hAELVi cells was refilled to LCC levels with DPBS and the MillicellR ERS (electrical resistance system) volt was used for measurement. To obtain Ω*cm2 the surface area of the transwell insert was multiplied with the measured TEER values. To assess the transport of sodium fluorescein across the cellular monolayer, fresh culture medium was added to the basolateral acceptor chamber and culture medium containing 1mg/ml sodium fluorescein was added into the apical chamber of the insert. Every 30 minutes for 3h one fifth of the basolateral culture medium was taken and directly replaced with fresh huAEC medium. Optical density of samples was determined at 486nm including a sodium fluorescein standard curve (0, 5, 10, 15, 20, 30, 40, 50 µg/ml).
All statistical analyses were done by using non-paired, non-parametric Kruskal-Wallis test (* p < 0.05) and performed using GraphPad Prism Software Version 9.1.0. Results were presented as mean ± standard error (SEM). For heat map presentation, mean values of x-fold inductions relative to mock-infection of three independent experiments were calculated for each cytokine.