The nasal symbiont Staphylococcus epidermidis shapes the cellular environment to decrease expression of SARS-CoV-2 entry factors in nasal epithelium

Emerging evidence indicates that severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) targets the human nasal epithelium via the principal entry factors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which are highly expressed in the nasal epithelium. However, little is known about suppressive biologics against SARS-CoV-2 entry factors. Here, we report that the nasal commensal Staphylococcus epidermidis altered the host transcriptional response against SARS-CoV-2 in the nasal epithelium by reducing ACE2 and TMPRSS2 gene expression in concert with an increase in serine-peptidase inhibitors. Our data reveal that ACE2 was more abundantly expressed in nasal epithelial (NHNE) cells than bronchial epithelial cells, and inoculation with S. epidermidis reduced ACE2 transcription in NHNE cells. Our data also show that TMPRSS2 mRNA was signicantly decreased in NHNE cells and that S. epidermidis colony number in human nasal mucus was inversely correlated with ACE2 and TMPRSS2 gene expression in the nasal mucosa. In addition, levels of the serine-peptidase inhibitors SERPINE1 and SERPINE2 were signicantly increased by S. epidermidis, and this accompanied reduction of TMPRSS2 transcription in nasal epithelial cells.


Results
Our data reveal that ACE2 was more abundantly expressed in nasal epithelial (NHNE) cells than bronchial epithelial cells, and inoculation with S. epidermidis reduced ACE2 transcription in NHNE cells. Our data also show that TMPRSS2 mRNA was signi cantly decreased in NHNE cells and that S. epidermidis colony number in human nasal mucus was inversely correlated with ACE2 and TMPRSS2 gene expression in the nasal mucosa. In addition, levels of the serine-peptidase inhibitors SERPINE1 and SERPINE2 were signi cantly increased by S. epidermidis, and this accompanied reduction of TMPRSS2 transcription in nasal epithelial cells.

Conclusion
These results characterize the S. epidermidis-regulated host transcriptional response restricting SARS-CoV-2 entry to the nasal epithelium via downregulation of receptors and host protease for SARS-CoV-2 cellular invasion coupled with SERPINE1 and SERPINE2 induction.

Background
At present, the world is suffering from a pandemic infection of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019  and leads to acute respiratory distress syndrome or viral pneumonia with severe damage to the lungs [1,2]. Currently, research on development of vaccines against SARS-CoV-2 is ongoing worldwide, and interest in effective SARS-CoV-2 therapeutics is increasing rapidly [3,4]. To succeed in development of a therapeutic or vaccine against SARS-CoV-2, knowledge of the exact target cells where SARS-CoV-2 enter the host and the mechanism of infection in the respiratory tract is essential.
Many respiratory virus families require maturation cleavage of viral surface glycoproteins after binding to a speci c receptor, generally realized by host serine proteases. The cellular infectivity of a respiratory virus to the respiratory epithelium depends on the distribution of receptors and activity of host serine proteases in the respiratory tract [5][6][7]. It is becoming increasingly apparent that nasal epithelial cells are the primary target of SARS-CoV-2, and the nasal epithelium is regarded as a portal for initial infection and/or transmission of SARS-CoV-2 to the respiratory tract [8,9]. SARS-CoV-2 employs angiotensin converting enzyme 2 (ACE2) as a receptor for internalization, and the binding a nity of the spike (S) protein of SARS-CoV-2 to ACE2 was found to be a major determinant of SARS-CoV-2 nasal epithelium cellular infection [10,11]. Host proteases are involved in cellular invasion by SARS-CoV-2, and transmembrane serine protease 2 (TMPRSS2) is indicated as the principal host protease to mediate cleavage of SARS-CoV-2 S protein in nasal epithelial cells [8]. In this regard, it is of immediate interest to determine whether localized suppression of ACE2 in the nasal epithelium restricts cellular invasion of SARS-CoV-2 and inhibits viral replication in the respiratory tract. Likewise, suppression of TMPRSS2driven SARS-CoV-2 S protein activation might provide a new therapeutic approach to prevent SARS-CoV-2-caused respiratory infection.
The respiratory microbiome is constantly exposed to inhaled pathogens and is known to in uence the course of host immune responses. Inhaled pathogens including respiratory viruses encounter the host immune system through the nasal passage, and the microbial characteristics of the nasal mucus directly impact initiation of the innate immune response [12,13]. Thus, insights into the human nasal mucus microbiome can provide fundamental information regarding defense mechanisms against respiratory virus infection, and understanding of microbiome-regulated immune factors contributes to discovery of new concepts of viral infection control [14].
Our previous study identi ed Staphylococcus epidermidis as the most abundant constituent in human nasal mucus and showed that S. epidermidis accelerated clearance of in uenza virus from the nasal epithelium through interferon-related immune responses [13]. Here, we investigated if S. epidermidis plays a role in reinforcing the antiviral innate immune response at the nasal epithelium to determine any contribution to suppressing SARS-CoV-2 infection that targets nasal epithelial cell as onset of infection.
We investigated the correlation between S. epidermidis and SARS-CoV-2 entry factors, which are mainly distributed on nasal epithelial cells, and found that the human nasal commensal S. epidermidis impeded entry of SARS-CoV-2 into nasal epithelial cells by reducing the expression of ACE2 and TMPRSS2. Mechanistically, we show that S. epidermidis protects the nasal epithelium from spread of SARS-CoV-2 by enhancing the activity of serine protease inhibitors. The current ndings provide evidence of nasal microbiome-altered cellular environments associated with disturbance of SARS-CoV-2 entry factors in the human nasal epithelium.

Results
In particular, ACE2 has been detected in both nasal and bronchial epithelium, and ACE2 gene expression has been recently reported largely in nasal epithelial cells including secretory cells and ciliated cells, which are central to SARS-CoV-2 pathogenesis in the upper airway [8]. We evaluated ACE2 RNA expression in human nasal mucosa (N=4) and lung tissue (N=4) and compared it to that of DPPIV, which encodes a known viral receptor for MERS-CoV, and ST6GAL1 and ST3GAL4, which are important for synthesis of α(2,6)-linked and α(2,3)-linked sialic acids recognized by in uenza virus [15,16]. Real-time PCR revealed that mRNA expression of DPPIV in lung parenchymal tissue was signi cantly higher than in the nasal mucosa, and neither mRNA expression of ST6GAL1 or ST3GAL4 was signi cantly different in human nasal mucosa and lung tissue. Unlike the expression of other viral receptors, the mean level of ACE2 mRNA was higher in the human nasal mucosa (1.7x10 9 ) than in lung tissue (4.8x10 7 ) (Fig. 1a). Immunohistochemistry (IHC) for ACE2 protein was performed using human nasal mucosa of middle turbinate to determine whether ACE2 protein is mainly present in nasal mucosa (Additional le 1: Figure  S1). Although the expression of ACE2 protein was observed in a part of submucous gland, IHC results showed that positive DAB (3,3'-diaminobenzidine) staining of ACE2 protein was highly increased in the nasal epithelium relative to the subepithelial area of the human nasal mucosa (Fig. 1b).
To further characterize the expression of viral receptors in nasal epithelial cells, we examined ACE2, DPPIV, ST6GAL1, and ST3GAL4 expression within air-liquid interface cultures of normal human nasal epithelial (NHNE) cells, and the results were compared to those of normal human bronchial epithelial (NHBE) cells. Based on results using this in vitro system, we con rmed increased ACE2 mRNA expression in nasal epithelial cells, and mRNA level of ACE2 was higher in NHNE than NHBE cells (Fig. 1c). We found that DPPIV expression was higher in NHBE cells, and no signi cantly different expression of ST6GAL1 and ST3GAL4 was observed between NHNE and NHBE cells. To clarify the cell subsets targeted by SARS-CoV-2 in the human nasal epithelium, we investigated gene expression of ACE2 depending on nasal epithelial cell subset through single-cell RNA sequencing (scRNA-seq). We con rmed increased normalized ACE2 expression in both suprabasal cells and secretory-like NHNE cells (Fig. 1d). These data suggest that the nasal epithelium is the primary target of SARS-CoV-2 transmission, and SARS-CoV-2 infection is spread to the respiratory tract after intracellular entry via nasal epithelial cells.
To determine the correlation between the abundant nasal commensal S. epidermidis and entry factors of SARS-CoV-2 in the nasal epithelium, NHNE cells from ve healthy subjects were inoculated with S. epidermidis isolated from healthy human nasal mucus, for 24 h at a multiplicity of infection (MOI) of 0.25 (Additional le 1: Figure S2). We performed Gene Ontology (GO) enrichment analysis of scRNA-seq data using cell lysates from the S. epidermidis-inoculated NHNE cells to con rm the effect of S. epidermidis in restricting host entry factors of SARS-CoV-2. The terms associated with virus receptor activity were analyzed, and top signi cant terms included "entry into host cells," "entry into other organism involved in symbiotic interaction," "viral entry into host," and "viral life cycle" (Fig. 2a). The terms associated with serine-type peptidase activity were also examined, and the results revealed top signi cant terms of "virus receptor activity," "serine-type endopeptidase inhibitor activity," "serine-type peptidase activity," "peptidase activity," and "receptor binding" (Fig. 2b).
We used scRNA-seq to characterize the response of the nasal epithelium to S. epidermidis inoculation. Signi cant gene populations (fold change > or < 1.5 and normalized data (log2) > or < 2.0) in S. epidermidis-inoculated NHNE cells were compared to those from NHNE cells without S. epidermidis inoculation. The scatter plot data of genes associated with virus receptor activity (GO category) revealed lower ACE2 gene expression in S. epidermidis-inoculated NHNE cells (0.51-fold decrease) relative to the control (Fig. 2c). We also analyzed signi cant gene expression associated with serine-type peptidase activity (GO category) and found that TMPRSS2 expression was decreased in S. epidermidis-inoculated NHNE cells (0.59-fold decrease) relative to the control (Fig. 2d). Interestingly, of the scatter plot data of genes associated with GO category, serine-type peptidase inhibitor activity showed that both SERPINE1 (17.2-fold increase) and SERPINE2 (40.8-fold increase) expression was signi cantly elevated in S. epidermidis-inoculated NHNE cells (Fig. 2e).
Next, we analyzed the signi cant change of ACE2, TMPRSS2, SERPINE1, and SERPINE2 expression in NHNE cell subsets of basal cells, secretory-like cells, unde ned intermediate cells, suprabasal cells, and multiciliated cells in the presence or absence of S. epidermidis. Heatmap data depicting genes classi ed into virus receptor activity revealed that decrease in ACE2 transcription in response to S. epidermidis inoculation was most pronounced in basal cells and secretory-like NHNE cells. A decrease of ACE2 gene expression was observed in suprabasal and unde ned intermediate cells of S. epidermidis-inoculated NHNE cells. ACE2 gene expression was not signi cantly altered in multiciliated cells (Fig. 2f).
Based on dot plot data, normalized TMRPSS2 expression was signi cantly higher in multiciliated cultured NHNE cells, and a larger proportion of SERPINE1 and SERPINE2 gene expression was found in basal NHNE cells (Fig. 2g). The heatmap of serine-type peptidase transcript activity also showed that TMPRSS2 transcription was signi cantly reduced in multiciliated S. epidermidis-inoculated NHNE cells (Fig. 2h). In contrast, a signi cant increase of serine-type peptidase inhibitor SERPINE1 and SERPINE2 transcripts was determined in all NHNE basal, secretory-like, suprabasal, and unde ned intermediate cell subsets with S. epidermidis inoculation. The baseline transcript levels of SERPINE1 and SERPINE2 were minimal in multiciliated cells, but the expression of both was highly induced after S. epidermidis inoculation (Additional le 1: Figure S3). Based on these ndings, we suggest that human nasal commensal S. epidermidis reduced gene expression associated with host entry of SARS-CoV-2, including ACE2 and TMPRSS2, depending on NHNE cell subset. Contrary to these ndings, S. epidermidis inoculation enhanced the gene expression of serine-type protease inhibitors SERPINE1 and SERPINE2, which might be involved in reduction of serine protease activity, including TMPRSS2, in NHNE cells.
To better determine the in uence of S. epidermidis on SARS-CoV-2 host entry factors in the nasal epithelium, NHNE cells from ve healthy subjects were inoculated with human nasal S. epidermidis at an MOI of 0.25, and transcriptional changes of ACE2, TMPRSS2, SERPINE1, and SERPINE2 were evaluated. S. epidermidis mRNA level in the cell lysate and the colony count of S. epidermidis in the supernatant were assessed until 1-day post infection (dpi). Real-time PCR data revealed that the mean mRNA level of S. epidermidis femA increased signi cantly starting from 8 h post infection (0.8x10 9 ), and that the highest levels were observed at 1 dpi (4.2´10 9 ; Fig. 3a). The mean colony forming unit (CFU) of S. epidermidis was signi cantly increased in the supernatant of S. epidermidis-inoculated NHNE cells until 1 day (2.4x10 4 CFU/ml) after S. epidermidis inoculation (Fig. 3b). Subsequently, we tested whether S. epidermidis-inoculated NHNE cells exhibited the decrease of ACE2 and TMPRSS2 as shown in scRNA-seq data. Real-time PCR and immunohistochemistry results showed that ACE2 mRNA and protein levels were reduced signi cantly at 1 day after S. epidermidis inoculation (Fig. 3c, 3d). In addition, TMRPSS2 mRNA level decreased signi cantly in the cell lysates of S. epidermidis-inoculated NHNE cells until 1 day after inoculation. A gradual increase of SERPINE1 and SERPINE2 gene expression was seen in NHNE cells in response to S. epidermidis, with the highest expression observed at 24 h after inoculation (Fig. 3e).
Considering the in vitro effect of the nasal commensal S. epidermidis on entry factors of SARS-CoV-2 in the nasal epithelium, we investigated the relationship between S. epidermidis abundance and mRNA levels of ACE2 and TMPRSS2 in human nasal mucosa. Nasal mucus and middle turbinate mucosa of 20 healthy subjects was collected, and the number of S. epidermidis CFUs from nasal mucus and ACE2 or TMPRSS2 mRNA levels from the human nasal mucosa were compared. Interestingly, S. epidermidis CFUs from healthy human nasal mucus was inversely correlated with ACE2 (Spearman r = -0.7469) and TMPRSS2 (Spearman r = -0.6581) mRNA levels in the nasal mucosa (Fig. 3f, 3g). These data indicate that subjects who have more number of S. epidermidis in their nasal mucus show relatively lower levels of ACE2 and TMPRSS2 gene expression, and that subjects with decreased number of S. epidermidis in the nasal mucus have higher transient expression of ACE2 and TMPRSS2.

Discussions
Altogether, our ndings indicate that the most abundant human nasal commensal, S. epidermidis, restricts host entry of SARS-CoV-2 into the nasal epithelium through reduction of host virus receptors and a principal host protease that are necessary for cellular transmission. In addition, S. epidermidis can increase expression of the serine-type protease inhibitors SERPINE1 and SERPINE2 in nasal epithelial cells.
Host protection against viral infections can be conferred by the nasal microbiome via a specialized immune mechanism and recent work has highlighted S. epidermidis is capable of combating invasion by respiratory viruses [13]. Growing evidence shows that the entry factors for SARS-CoV-2, including ACE2 and TMPRSS2, are dominantly found in the nasal epithelium, and nasal epithelial cells have been determined as a potential cellular target of SARS-CoV-2 infection [17][18][19][20]. Thus, we characterized the contribution of the nasal commensal S. epidermidis to the defense mechanisms against SARS-CoV-2 infection, which mainly targets nasal epithelial cells.
Our scRNA-seq ndings indicate that primary nasal epithelial cells support entry of SARS-CoV-2 leading to spread to the respiratory tract, and the human nasal commensal S. epidermidis downregulated cellular entry factors in the nasal epithelium. Both ACE2 and TMPRSS2 transcription in the nasal epithelium was signi cantly reduced after inoculation with S. epidermidis in suprabasal, secretory-like, and multiciliated NHNE cells. This result is underscored by the inverse correlation between ACE2 and TMPRSS2 of the nasal mucosa and S. epidermidis colony number in human nasal mucus. Thus, a greater abundance of S. epidermidis in the nasal mucus results in lower ACE2 and TMPRSS2 expression in the nasal mucosa of healthy subjects.

Conclusions
The present study suggests that the nasal commensal S. epidermidis-regulated transcription of ACE2, TMPRSS2, SERPINE1, and SERPINE2 in nasal epithelium, even in a cellular environment free from SARS-CoV-2 infection and S. epidermidis, can restrict cellular entry factors of SARS-CoV-2 in nasal epithelial cells to impede SARS-CoV-2 invasion into the human respiratory tract. Our work highlights the importance of host-bacterial commensalism in shaping the cellular environment of the nasal epithelium, resulting in decreased SARS-CoV-2 invasion into epithelial cells through modulation of host entry factors.

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 xed in a xative solution and transported immediately to the laboratory for identi cation 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 identi ed using GS-FLX 454 pyrosequencing and 16S rRNA gene ampli cation. 13 Four S. epidermidis strains were isolated from four individuals.

Cell culture
Normal human nasal epithelial (NHNE) cells were cultured as described previously [21]. Briefly, passage-2 NHNE cells (1 x 10 5 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 ltering 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 les using the Cell Ranger pipeline. We followed the 10x Genomics standard sequence protocol by trimming the barcode and unique molecular identi er (UMI) end to 26 bp and the mRNA end to 98 bp. Then, the FASTQ les were aligned to the human reference genome (GRCh38). Subsequently, we applied Cell Ranger for preliminary data analysis and generated a le 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).

Real-time PCR
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). Ampli cation was performed using the TaqMan Universal PCR master mix (PE Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Brie y, 12 μl ampli cation reactions contained 2 μl of cDNA (reverse transcription mixture), oligonucleotide primers ( nal concentration of 800 nM), and TaqMan hybridization probe (200 nM). Realtime PCR probes were labeled at the 5' end with carboxy uorescein (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 quanti ed using target-speci c 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´10 6 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