Peptides and reagents

Recombinant SARS-CoV-2 E Protein was purchased from Novoprotein (Shanghai, China), and the target gene was expressed with a β-barrel protein platform and 6 × His tag at the N-terminus,60 and dissolved in the solution of 20 mM Tris-HCl and 200 mM NaCl. SARS-CoV-2 Spike S1 and S2 recombinant protein were purchased from Sino Biological (Beijing, China). SARS-CoV E protein was purchased from Glpbio (USA); rolipram from Sigma Aldrich (USA); C29, Resatorvid, SP600125, T-5224 and EMD638683 from MedChemExpress (MCE, USA).

Cell culture

The human bronchial epithelial cell line BEAS-2B and 16HBE14o- were cultured as previously described.35,58 Briefly, BEAS-2B cells, 16HBE14o- cells, and CFBE41o- cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM, Hyclone, USA) or Minimum Essential Medium (MEM, Corning, USA) supplemented with 1% (vol/vol) penicillin−streptomycin (Hyclone, USA) and 10% (vol/vol) fetal bovine serum (Gibco, USA), maintaining in 5% CO2 at 37 °C. Cells were cultured to 80% confluence, and serum-starved overnight before E protein stimulation. BEAS-2B cells were inoculated in the 24-well cell culture plate and stimulated with 400 μL SARS-CoV-2 E protein (50 μg/mL) for 12 h. After stimulation, the E protein could be observed in cell lysates and was co-localized with ER marker calreticulin (Supplementary Fig. 5d, e). For additional experiments, cells were pre-treated with several inhibitors for 1 hr before E protein challenge. Besides, SGK1 gene knockout (KO) BEAS-2B cells and empty vector control BEAS-2B cells were constructed as previously described.35

TER measurement

TER values of epithelial cell monolayers were measured to evaluate the tight junction permeability.61 Briefly, 16HBE14o- cells were seeded into plate inserts (12 mm diameter, 3.0 μm pore size, BIOFIL JET), and the TER of 16HBE14o- monolayers was measured in the presence or absence of SARS-CoV-2 E protein or S protein, using the Millicell-ERS Electrical Resistance System (Millipore, USA). The TER values were obtained by subtracting the intrinsic resistance of the membrane, and multiplied by the surface area of the filter (1.12 cm2). After stimulation, the E protein significantly decreased the TER at a concentration of 50 μg/mL (Supplementary Fig. 5f).

Cell permeability assay

FD4 was used to evaluate the paracellular permeability of airway epithelium.62 Briefly, 16HBE14o- cells were cultured in plate inserts (3.0 μm pore size) until a confluent monolayer was formed. FD4 (Sigma, USA) at a concentration of 1 mg/ml was added to the upper chambers accompanied with or without SARS-CoV-2 E protein (50 μg/mL) stimulation. The fluorescence intensity of FD4 passage at indicated times was detected by fluorescence microplate reader (Synergy H1, BioTek, USA), with excitation and emission wavelengths at 490 nm and 520 nm, respectively. The fluorescence intensity of serial standard dilutions ranging from 0–50 μg/mL was also obtained from the fluorescence reader and a standard curve was made to determine the FD4 concentration of the samples. After stimulation, the E protein resulted in a significant increase in the paracellular flux of FD4 at a concentration of 50 μg/mL (Supplementary Fig. 5g).

Bacterial culture and monolayer invasion assay

Pseudomonas aeruginosa strain PAO1 was by courtesy of Dr. Lei Ni (Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, P. R. China). Bacteria were grown on solid Luria–Bertani (LB) medium (1.5% agar) at 37 °C for 18 h and bacterial colonies were scraped off and routinely grown in LB broth at 37 °C with shaking (200 rpm) overnight. Bacterial cultures were resuspended in MEM medium with no antibiotics until an optical density at a wavelength of 600 nm was 1.0, corresponding to ~1×109 colony-forming units (CFU) per ml.

The penetration of PAO1 through the 16HBE14o- cell monolayer was evaluated to determine the effect of SARS-CoV-2 E protein on the epithelial tight junction permeability and the invasiveness of respiratory pathogens. Briefly, 16HBE14o- cells were seeded on plate inserts to form a monolayer with tight junctions, monitored by measurement of TER values. 16HBE14o- cells were then infected apically with PAO1 at a multiplicity of infection (MOI) of 20,63 with or without SARS-CoV-2 E protein stimulation. 2 h later, the PAO1 penetrated to the basolateral medium were counted through plating serially diluted medium onto LB agar plates.

Measurement of bronchoalveolar epithelial permeability

Male ICR mice, weighing 25–30 g, were obtained from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). Mice were anesthetized with tribromoethanol [0.2 ml/10 g body weight of a 1.25% (vol/vol) solution] and intratracheally instilled with 50 μl E protein (200 μg/ml) or an equal volume of normal saline for 6 h. Mice were next injected intravenously via the tail vein with FD4 (5 mg/ml; 10 mg/kg body weight). 15 min later, the blood was collected and stored at 4 °C overnight, and the serum was isolated by centrifugation at 600 g for 5 min. Meanwhile, using a tracheal cannula, the BALF was collected via bronchoalveolar lavage for three times with 600 μl of phosphate buffered saline (PBS) per wash. The fluorescence intensity of serum (10-fold diluted by PBS) or BALF samples was detected as described above. Finally, the ratio of fluorescence in BALF and serum was calculated to assess pulmonary epithelial permeability. All procedures were performed in strict accordance with the animal use protocols approved by the Sun Yat-sen University Institutional Animal Care and Use Committee (Guangzhou, China).

RNA sequencing

Total RNA was extracted by using TRIzol reagent (Invitrogen, USA) and the quality of RNA was assessed using agarose gel electrophoresis and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Next, cDNA libraries were constructed and sequenced on the Illumina HiSeq2500 platform by Gene Denovo Biotechnology (Guangzhou, China). Differentially expressed genes (DEGs) between two different groups were identified using DESeq2 software. A false discovery rate (FDR) below 0.05 and a log2 (fold change) ≥ 1 was considered significant.

Quantitative real-time PCR

The total RNA extracted from airway epithelial cells or mouse lung samples by using SteadyPure Universal RNA Extraction Kit (Accurate Biology, China) was reverse-transcribed with HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, China). Real-time PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, China). The levels of target genes were normalized to that of GAPDH for cells, and HPRT1 for lung tissues, and the relative expression was calculated using the 2-∆∆Ct algorithm, as described previously.64 The sequences of primers used in Real-Time PCR reactions are shown in Supplementary Table 1.

Western blotting

The total protein was extracted from airway epithelial cells or mouse lung specimens by using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, China). Western blotting analysis for the target proteins was performed as described previously.64 The primary antibody against ZO-1 (1:500, #61-7300) and Occludin (1:500, #71-1500) were purchased from Thermo Fisher Scientific (USA). The antibodies against p38 MAPK (1:1000, #8690), phospho-p38 MAPK (1:1000, #4511), p44/42 MAPK (Erk1/2, 1:1000, #4695), phospho-p44/42 MAPK (Erk1/2, 1:1000, #4370), SAPK/JNK (1:1000, #9252), phospho-SAPK/JNK (1:1000, #4668), c-Jun (1:1000, #9165), phospho-c-Jun (1:1000, #3270), and phospho-IκB-α (1:1000, #2859) were purchased from Cell Signaling Technology (USA). The antibody against phospho-SGK1 (1:1000, #36-002) was purchased from Sigma-Aldrich (USA). The antibodies against Claudin-4 (1:1000, #ab53156), TLR2 (1:1000, ab68159), CFTR (1:1000, #ab2784) and GAPDH (1:1000, #ab8245) were from Abcam (UK). The antibody against TLR4 (1:1000, #AF7017) was purchased from Affinity Biosciences (USA). The antibody against His-Tag (1:10000, #66005-1-Ig) was purchased from Proteintech (USA). The antibody against SARS-CoV-2 Envelope (1:1000, #GTX636915) was purchased from GeneTex (USA). The antibodies against SARS-CoV-2 Spike S1 (1:1000, #40591-R235) and Spike S2 (1:1000, #40590-T62) were from Sino Biological (China).

Co-immunoprecipitation assay

Airway epithelial cells were lysed in cell lysis buffer for Western blotting and co-immunoprecipitation (Co-IP) assay (Beyotime, China) for 20 min. Cell lysates were next centrifuged at 12000 g for 5 min. The supernatant was incubated with 10 μg His-tagged E protein (Novoprotein, China) and Anti-His Tag Magnetic Beads (Abbkine, China) at 4 °C overnight on a rocking platform. The magnetic beads were, by using a magnetic separator, washed with the ice-cold PBS followed by removal of the supernatant. The collected magnetic beads were boiled for 5 min in the 1 × sodium dodecyl sulfate (SDS) loading buffer solution and Western blotting analysis was performed.

Immunofluorescence staining

Airway epithelial cells were fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 5 min. The mouse lungs were fixed in 10% formalin, embedded in paraffin and cut into 4 μm sections. Fixed cells or lung tissues were then blocked in 3% bovine serum albumin (BSA) for 1 hr, and incubated with anti-Envelope (GeneTex, #GTX636915 1:500), anti-calreticulin (Abcam, #ab22683, 1:200), anti-ZO-1 (Thermo Fisher Scientific, #61-7300, 1:50), anti-Occludin (Thermo Fisher Scientific, #71–1500, 1:100), anti-Claudin-4 (Abcam, #ab53156, 1:200), anti-phospho-JNK (Affinity, #AF3318, 1:200), anti-PDE4D (Abcam, #ab171750, 1:200), anti-phospho-SGK1 (#36–002, 1:200, Millipore), at 4 °C overnight. Then, cells or lung sections were incubated with Donkey anti-Mouse IgG (H + L), Alexa FluorTM 488 (Thermo Fisher Scientific, #A-21206, 1:500) or Donkey anti-Rabbit IgG (H + L), Alexa Fluor™ 568 (Thermo Fisher Scientific, #A-10042, 1:500) for 1 hr at room temperature. Finally, the cell nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI, D9542, Sigma, USA) and visualized using confocal microscopy (TCS-SP5, Leica, Germany).

SARS-CoV-2 infection in mice

Adenovirus 5-human ACE2 (Ad5-hACE2) transgenic BALB/c mice 6–8 weeks old were infected with 1×105 plaque-forming units (PFU) of SARS-CoV-2, as previously described.65 The mice were sacrificed at day 4 post infection, and the lung samples were fixed in formalin and embedded in paraffin for further immunofluorescence assay. All work with SARS-CoV-2 was strictly confined in the Biosafety Level 3 Laboratory of Guangzhou Customs Technology Center (Guangzhou, China). All experimental procedures were approved by the Institutional Animal Care and Use Committees of the Guangzhou Medical University (Guangzhou, China).

Mouse model of SARS-CoV-2 E protein stimulation

Male ICR mice, weighing 25–30 g, were obtained from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). Mice were anesthetized with tribromoethanol [0.2 ml/10 g body weight of a 1.25% (vol/vol) solution]. For the E protein-treated groups, different concentrations of SARS-CoV-2 E protein was administered by intratracheal instillation dissolved in 50 μl saline, and mice in the control group were instilled with an equal volume of saline. EMD638683 (10 mg/kg) or rolipram (10 mg/kg) was injected intraperitoneally 1 hr before E protein stimulation. After 24 h, all mice were sacrificed by CO2 asphyxiation, and lung tissue samples were collected for subsequent experiments. All procedures were performed in strict accordance with the animal use protocols approved by the Sun Yat-sen University Institutional Animal Care and Use Committee (Guangzhou, China).

Lung histopathology

The lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin sections (4 μm) of lungs were stained with hematoxylin-eosin (HE) to visualize histopathological changes and to assess the extent of inflammation induced by E protein. HE staining images were obtained with an Optical Microscope (Nikon, Tokyo, Japan).

Glutathione S-transferase (GST) pull-down assay

SARS-CoV-2 E protein cDNA was cloned in-frame with GST in the prokaryotic expression vector pGEX-4T-1, and the fusion protein was expressed in Escherichia coli. GST fusion proteins were subsequently purified via affinity chromatography with glutathione-Sepharose and immobilized on GST resin. GST protein (negative control) and GST-tagged SARS-CoV-2 E protein were used to pull down His-tagged TLR2 or TLR4 recombinant protein (Zoonbio Biotechnology, China). After washing with PBS, the bound proteins were eluted with elution buffer and protein interactions were verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis.

Ussing chamber measurements

Electrophysiological analyses of airway epithelial cells were performed by using short circuit current (ISC) technique, as described previously.11 Briefly, 16HBE14o- cells were grown on permeable supports (Millipore, USA) and mounted in an Ussing chamber. The transepithelial ISC through 16HBE14o- cell monolayers was measured with an epithelial voltage clamp (MODEL VCC MC6, Physiologic Instruments, USA). Forskolin was added apically to activate CFTR as previously described.66

Isolation and culture of primary mouse airway epithelial cells

Mouse airway epithelial cells were cultured as described previously.67 Briefly, male ICR mice were euthanized using CO2 asphyxia and the trachea was isolated and immersed into Hanks’ balanced salt solution containing 1% (v/v) penicillin−streptomycin (Hyclone, USA). Subsequently, the tissues were digested with 0.25% (w/v) trypsin (Gibco, USA) overnight at 4 °C. The digested trachea was removed and the epithelial cells were collected by centrifugation at 400 g for 4 min. Cells were cultured in DMEM/Nutrient Mixture F-12 (Hyclone, USA) medium supplemented with 3% (v/v) fetal bovine serum (Gibco, USA) and 1% (v/v) penicillin−streptomycin (Hyclone, USA) at 37 °C in an atmosphere of 5% CO2. On day 3, the cells were harvested for subsequent experiments.

Intracellular Cl− measurement

Airway epithelial cells were seeded on glass cover-slips, and the concentration of intracellular Cl− was monitored through recording the changes of fluorescence intensity of N-(ethoxycarbonylmethyl)−6-methoxyquinolinium bromide (MQAE), a chloride-sensitive indicator dye as described previously.35,64

cAMP enzyme-linked immunosorbent assay

The content of intracellular cAMP was measured by performing enzyme-linked immunosorbent assay (ELISA) with the cAMP Parameter Assay Kit (R&D Systems, USA), according to the manufacturer’s protocol.

Isolation and culture of primary human airway epithelial cells (hPAECs)

The hPAECs were isolated from the bronchial brushing specimens obtained from the outpatient clinics of the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). All study participants provided written informed consent, and this study was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University. Upon collection, the airway epithelial cells were detached from the brush and subsequently centrifuged at 500 g for 5 min. The cells were then re-suspended in PneumaCult™-Ex Plus Medium (STEMCELL) containing 1% (v/v) penicillin and streptomycin, and cultured at 37 °C in a humidified atmosphere of 5% CO2.

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Differences between two groups were assessed with Student’s two-tailed t-test, and for multiple comparisons, one-way ANOVA followed by Bonferroni was used. Statistical analyses were conducted using Origin Pro software (OriginLab Corporation, MA, USA). P < 0.05 was considered statistically significant for the comparisons.