Subjects and sample collection
The 1×1-cm-sized nasal mucosal tissue samples from the middle turbinate of the subjects (N=4) who underwent septoplasty under general anesthesia in the Department of Otorhinolaryngology Seoul National University Hospital (Seoul, Korea) were obtained for real-time PCR. Also, 1×1-cm-sized human lung parenchymal tissues of the subjects (N=4) were obtained from the subjects referred to the Department of Thoracic and Cardiovascular Surgery Seoul National University Hospital, primarily for pneumonectomy.
Nasal mucus S. epidermidis characterization
Mucus from the middle turbinate of healthy volunteers was collected individually using sterile 3M Quick swabs (3M Microbiology Products, St. Paul, MN, USA) from four subjects using a rigid 0-degree endoscope in an operating room. The swabs with mucus were fixed in a fixative solution and transported immediately to the laboratory for identification and subsequent microbial analysis. For bacterial colony isolation, the mucus was plated on Lysogeny Broth (LB) plates. After two days of incubation, bacterial colonies were obtained from the LB plates, and the species of each colony was identified using GS-FLX 454 pyrosequencing and 16S rRNA gene amplification.13 Four S. epidermidis strains were isolated from four individuals.
Normal human nasal epithelial (NHNE) cells were cultured as described previously . Brieﬂy, passage-2 NHNE cells (1 x 105 cells/culture) were seeded in 0.25 ml of culture medium on Transwell-Clear culture inserts (24.5 mm, with a 0.45-mm pore size; Costar Co., Cambridge, MA, USA). Cells were cultured in a 1:1 mixture of basal epithelial growth medium and DMEM containing previously described supplements. Cultures were grown while submerged for the ﬁrst 9 days. The culture medium was changed on Day 1 and every other day thereafter. An air–liquid interface (ALI) was created on Day 9 by removing the apical medium and feeding the cultures from the basal compartment only. The culture medium was changed daily after establishment of the ALI. The antifungal agent fungizone (1 ml / 1000 ml media) (Life Technologies, Grand Island, NY, USA) was added after filtering the media. All experiments described here used cultured nasal epithelial cells at 14 days after ALI.
Single-cell RNA sequencing (scRNA-seq)
Library construction was performed using 10X Chromium Single Cell 3’ reagent kits v3.1. Samples were sequenced using the Illumina NovaSeq 6000 platform, and preliminary sequencing results were converted to FASTQ files using the Cell Ranger pipeline. We followed the 10x Genomics standard sequence protocol by trimming the barcode and unique molecular identifier (UMI) end to 26 bp and the mRNA end to 98 bp. Then, the FASTQ files were aligned to the human reference genome (GRCh38). Subsequently, we applied Cell Ranger for preliminary data analysis and generated a file that contained a barcode table, a gene table, and a gene expression matrix. We used the WinSeurat v2.1 (Ebiogen Inc., Seoul, Korea) based on Seurat version 3 for QC, analysis, and exploration of single-cell RNA-seq data [22, 23]. Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc., Seoul, Korea).
NHNE cells were infected with S. epidermidis for 4, 8, or 24 h, and total RNA was isolated using TRIzol (Life Technology, Seoul, Korea). cDNA was synthesized from 3 μg of RNA with random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Perkin Elmer Life Sciences, Waltham, MA, USA and Roche Applied Science, Indianapolis, IN, USA). Amplification was performed using the TaqMan Universal PCR master mix (PE Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Briefly, 12 μl amplification reactions contained 2 μl of cDNA (reverse transcription mixture), oligonucleotide primers (final concentration of 800 nM), and TaqMan hybridization probe (200 nM). Real-time PCR probes were labeled at the 5’ end with carboxyfluorescein (FAM) and at the 3’ end with the quencher 5-carboxytetramethylrhodamine (5-TAMRA). To quantify cellular viral level and host gene expression, cellular RNA was used to generate cDNA. Primers for femA, SERPINE1, SERPINE2, TMPRSS2, and ACE2 were purchased from Applied Biosystems (Foster City, CA, USA). Real-time PCR was performed using the PE Biosystems ABI PRISM® 7700 Sequence Detection System. Thermocycler parameters were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Target mRNA levels were quantified using target-specific primer and probe sets for femA, SERPINE1, SERPINE2, TMPRSS2, and ACE2. All PCR assays were quantitative and utilized plasmids containing the target gene sequences as standards. All reactions were performed in triplicate, and all real-time PCR data were normalized to the level of glyceraldehyde phosphate dehydrogenase (GAPDH, 1´106 copies) to correct for variations between samples.
Western blot analysis
Protein level of ACE2 was assessed using western blot analysis, and the monoclonal antibody of ACE2 was purchased from Cell Signaling Technology (Beverly, MA, USA). The NHNE cells and were lysed with 2X lysis buffer (250 mM Tris-Cl (pH6.5), 2% SDS, 4% β-mercaptoethanol, 0.02% bromophenol blue, and 10% glycerol). Cell lysate (30 μg of protein) was electrophoresed in 10% SDS gels and transferred to polyvinylidene difluoride membranes in Tris-buffered saline (TBS; 50 mM Tris-Cl (pH 7.5), 150 mM NaCl) for 1 h at room temperature. The membrane was incubated overnight with primary antibody (1:500) in Tween-Tris buffered saline (TTBS; 0.5% Tween-20 in TBS). After washing with TTBS, the blot was incubated for 1 h at room temperature with secondary anti-rabbit antibody (1:1000, Cell Signaling, Beverly, MA, USA) in TTBS and was visualized using an ECL system (Amersham, Little Chalfont, UK).
Human nasal mucosa was obtained from the middle turbinate of healthy subjects who had nasal surgery due to nasal obstruction, and paraffin block slides were prepared for immunostaining. Immunohistochemical analysis was performed using ACE2 antibody (1:200, Cell Signaling Technology, MA, USA) to determine protein level in the nasal mucosa. Briefly, 5-μm sections were fixed in acetone for 10 min at room temperature (RT). Non-specific protein staining was blocked with goat serum. Slides were treated with 0.5% hydrogen peroxidase to eliminate endogenous peroxidase for 10 min at RT and incubated with primary antibody overnight at RT. After washing with Tris-buffered saline (TBS, pH 7.5), slides were incubated with horseradish peroxidase-conjugated secondary antibody (Thermo, Asheville, NC, USA) for 30 min at RT. Chromogen (3-amino-9-ethylcarbazole) was applied for visualization. Glass cover slides were mounted and examined with optical microscopy and ACE2 protein was detected with DAB (3,3'-diaminobenzidine) chromogen staining. The same procedures were performed using non-immunized mouse IgG (purified IgG, Sigma) instead of primary antibody as a negative control.
For in vitro study, at least three independent experiments were performed with cultured cells from each donor, and the results are presented as the mean value ± standard deviation (SD) of triplicate cultures. Differences between treatment groups were evaluated by analysis of variance (ANOVA) with a post hoc test. We present the in vivo results of real-time PCR, plaque assays, and ELISA as mean value ± SD from five individual mice. Statistical analyses were performed using GraphPad Prism software (version 5; GraphPad Software, La Jolla, CA, USA). A p-value <0.05 was considered statistically significant.