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Objective: Although the neurological and olfactory symptoms of coronavirus disease 2019 have been identified, the neurotropic properties of the causative virus, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), remain unknown. We sought to identify the susceptible cell types and potential routes of SARS-CoV-2 entry into the central nervous system, olfactory system, and respiratory system. Methods: We collected single-cell RNA data from normal brain and nasal epithelium specimens, along with bronchial, tracheal, and lung specimens in public datasets. The susceptible cell types that express SARS-CoV-2 entry genes were identified using single-cell RNA sequencing and the expression of the key genes at protein levels was verified by immunohistochemistry. We compared the coexpression patterns of the entry receptor angiotensin-converting enzyme 2 (ACE2) and the spike protein priming enzyme transmembrane serine protease (TMPRSS)/cathepsin L among the specimens. Results: The SARS-CoV-2 entry receptor ACE2 and the spike protein priming enzyme TMPRSS/cathepsin L were coexpressed by pericytes in brain tissue; this coexpression was confirmed by immunohistochemistry. In the nasal epithelium, ciliated cells and sustentacular cells exhibited strong coexpression of ACE2 and TMPRSS. Neurons and glia in the brain and nasal epithelium did not exhibit coexpression of ACE2 and TMPRSS. However, coexpression was present in ciliated cells, vascular smooth muscle cells, and fibroblasts in tracheal tissue; ciliated cells and goblet cells in bronchial tissue; and alveolar epithelium type 1 cells, AT2 cells, and ciliated cells in lung tissue. Conclusion: Neurological symptoms in patients with coronavirus disease 2019 could be associated with SARS-CoV-2 invasion across the blood–brain barrier via pericytes. Additionally, SARS-CoV-2–induced olfactory disorders could be the result of localized cell damage in the nasal epithelium. Keywords: ACE2, coronavirus disease 2019, SARS-CoV-2, single-cell RNA sequencing, TMPRSS2 ZK, JW, and TM contributed equally to the article. Introduction Department of Rheumatology and Immunology, Changzheng Hospital, The main clinical manifestations of coronavirus disease 2019 Department of Neurosurgery, Changhai Hospital, Second Military Medical (COVID-19) resulting from severe acute respiratory syndrome University, Division of Spine, Department of Orthopedics, Shanghai General coronavirus 2 (SARS-CoV-2) infection are fever, cough, and dys- Hospital, School of Medicine, Shanghai Jiaotong University, Tongji University [1–5] e pnea. However, numerous neurological abnormalities have Cancer Center, School of Medicine, Tongji University, Department of Orthopaedic been described in patients with COVID-19; these abnormalities Oncology, Changzheng Hospital, Department of Urology, The Third Affiliated Hospital, Second Military Medical University, Department of Pathology, Shanghai include olfactory and gustatory disorders (anosmia, hyposmia, Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China, ageusia, and dysgeusia), headache, epilepsy, encephalitis, and Zeenat Qureshi Stroke Institutes and University of Missouri, Columbia, MO, [4–8] acute myelitis. SARS-CoV-2 RNA has also been identified in i j USA, Peking-Tsinghua Center for Life Sciences, School of Medicine, Tsinghua [9,10] cerebrospinal fluid via genome sequencing. These findings University, Beijing, China suggest that SARS-CoV-2 can directly or indirectly enter the *Corresponding authors: Huji Xu, Department of Rheumatology and Immunology, central nervous system (CNS). However, the underlying mecha- Changzheng Hospital, Second Military Medical University, Shanghai 200003, nism remains unknown. China. E-mail: firstname.lastname@example.org; Jianmin Liu, Department of Neurosurgery, Changhai Hospital, Second Military Medical University, Shanghai 200003, China. The CNS can be invaded through the hematogenous route or  E-mail: email@example.com by axonal transport ; these routes involve infections of endo- thelial cells in the blood–brain barrier (BBB) or epithelial cells Copyright © 2023 The Chinese Medical Association, Published by Wolters Kluwer Health, Inc. This is an open-access article distributed under the terms of in the blood–cerebrospinal fluid barrier, as well as infections  the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 of the olfactory bulb through the olfactory nerve. The tro- (CCBY-NC-ND), where it is permissible to download and share the work provided pism of coronaviruses is primarily dependent on the presence of it is properly cited. The work cannot be changed in any way or used commercially attachment and entry receptors, along with protease availability without permission from the journal. and the activities of internalization-related and trafficking-re- Journal of Bio-X Research (2023) 6:23–36 [13,14] lated pathways. SARS-CoV-2 uses angiotensin-convert- [15,16] Received: 4 May 2022; Accepted: 7 December 2022 ing enzyme 2 (ACE2) as an entry receptor. Other entry receptor molecules associated with SARS-CoV-2 may include http://dx.doi.org/10.1097/JBR.0000000000000137 23 ReseaRch aR ticle Journal of Bio-X Research [17,18] CD147 and CD209. Upon receptor binding, spike protein Cell type identification and gene expression analysis priming enzymes (eg, cell surface transmembrane serine pro- Clusters were annotated based on the expression patterns of tease [TMPRSS] and endosomal cathepsins) are necessary for known cellular markers; the clustering information was visu- [19,20] some coronaviruses to achieve membrane fusion. Thus, the alized by uniform manifold approximation and projection detection of coexpression involving key genes in the viral entry (UMAP) plots. Then, lost values in the gene expression matrix process may help to identify cells that are susceptible to SARS- were imputed by the “RunALRA” function. The imputed gene CoV-2 infection; this will contribute to further investigations of expression values are displayed in feature and violin plots. the mechanisms by which SARS-CoV-2 enters the CNS. The mean expression levels of gene sets are displayed in box In this study, we used single-cell RNA sequencing (scRNA- plots. For comparisons of expression levels among datasets, seq) of normal brain and nasal epithelium specimens, as well as the quantile method was used to correct expression data; this tracheal, bronchial, and lung specimens, to identify susceptible [29,30] global normalization method is widely used. To calculate cell types and potential routes of SARS-CoV-2 entry into the the endocytosis signaling score, we obtained endocytosis-as- CNS, olfactory system, and respiratory system. We sought to sociated genes from “GOBP_ENDOCYTOSIS” gen set in the clarify how SARS-CoV-2 invades various tissues in the human MigDB dataset (https://www.gsea-msigdb.org). The average body, with a particular interest in elucidating the olfactory and expression level of all the genes in this gene set was considered CNS symptoms in patients with COVID-19. as the expression level of the endocytosis signaling. Materials and methods Immunohistochemical staining Data sources Human brain tissue specimens were obtained from five patients We analyzed single-cell transcriptomes of the brain, nasal olfac- who had undergone surgery for treatment of traumatic brain tory epithelium (OE), nasal respiratory epithelium (RE), lung, injury, including three males and two females. Human lung tis- tracheal epithelium, and bronchial epithelium (Additional Table sue specimens were obtained during routine surgical intervention 1, http://links.lww.com/JR9/A46). Nasal RE/OE and trachea from three lung cancer patients, including two males and one transcriptome data were obtained from the Gene Expression female. Ethical approval was granted by the Ethics Committee Omnibus (GEO; https://www.ncbi.nlm.nih.gov/), using acces- of Shanghai Changhai Hospital (Approval No. CHEC2020-   sion codes GSE139522 and GSE134355. Brain transcrip- 164). All patients were fully informed of the research and signed tome data were obtained from the Sequence Read Archive the informed consent forms. Formalin-fixed paraffin-embedded (SRA; https://www.ncbi.nlm.nih.gov/sra), using accession code brain tissue sections on glass slides were incubated at 37°C over-  SRA667466. Lung transcriptome data were obtained from night, deparaffinized in xylene, and rehydrated in a descending Cell Atlas Data Portal (https://data.humancellatlas.org), using ethanol gradient. Then, tissue sections were incubated in antigen  accession code PRJEB31843. Bronchial transcriptome data retrieval solution at 95°C for 30 minutes. Next, tissue sections  were obtained from research data published by Lukassen et al. were incubated in 3% hydrogen peroxide for 10 minutes, fol- The single-cell data within each dataset had similar sequencing lowed by serum-free protein blocking solution (Cat# 9048-46- depth to ensure the quality of consensus sequence. Because the 8, Sigma, St. Louis, MO, USA) for 20 minutes; they were then data were obtained from public datasets, approval for our study incubated with rabbit anti-human ACE2 primary antibody (Cat# by the ethics committee and patient consent were unnecessary. ab108252, Abcam, Cambridge, UK) at a dilution ratio of 1:200 overnight at 4°C temperature and goat anti-rabbit IgG second- ary antibody (Cat# ab6721, Abcam) at a dilution ratio of 1:1000 Quality control for 1 hour at room temperature. After incubation with primary Barcodes with outlier gene counts may be indicative of dying and secondary antibodies, color development was performed cells, cells with broken membranes, or doublets. A high frac- using 3-amino-9-ethylcarbazole as the chromogenic substrate tion of mitochondria is indicative of leaky cytoplasmic mem- and tissue was counterstained with hematoxylin as described   branes allowing mRNA to escape. We removed low-quality previously. The microscopic observation was performed by data from cells with <200 or >5000 expressed genes, as well as the optical microscope (Eclipse 80i, Nikon, Tokyo, Japan). data from cells in which >10% of unique molecular identifiers mapped to mitochondrial genes. Results Single-cell maps of mouse brain, healthy human Data integration, dimension reduction, and cell olfactory system, and healthy human respiratory system clustering   To identify susceptible cell types in the brain, olfactory system, The R package Seurat and R package Harmony were and respiratory system, we collected scRNA-seq data for six used to process scRNA-seq data. Unique molecular identifier types of tissue from published papers (Additional Table 1, http:// counts were normalized using the “NormalizeData” function. The top 1000 highly variable genes were identified using the links.lww.com/JR9/A46). In the brain, 11 cell types were identi- “FindVariableGenes” function. Principal component analy- fied based on the analysis of 29,678 cells, after the application sis was performed on single-cell expression matrices using the of quality control criteria (Fig. 1A). The main cell types were oli- “RunPCA” function. Subsequently, “Runharmony” was used for godendrocytes, interneurons, astrocytes, microglia, and neurons batch-effect correction in each dataset. Cell clustering analysis 1 and 2. The other identified cell types included ependymal cells, was performed after the construction of a K-nearest-neighbor endothelial cells, pericytes, dividing cells, and myeloid cells. graph based on the adjusted principal component analysis The nasal epithelium was divided into two types: OE and RE. embeddings by harmony algorithm. In total, 19,903 cells and 14 cell types were identified in the OE 24 Journal of Bio-X Research ReseaRch aR ticle Figure 1. Single-cell maps of healthy human respiratory system and mouse brain tissue. UMAP plots showing the distributions of brain cells (A), nasal olfactory epithelial cells (B), nasal respiratory epithelial cells (C), tracheal epithelial cells (D), bronchial epithelial cells (E), and lung cells (F). Single-cell map of respiratory system (G). All the single-cell data in the human respiratory system were combined for another round of reduction and clustering analysis. UMAP plots show the distributions of the cell clusters in the human respiratory system. DCs=dendritic cells, HBCs=horizontal basal cells, NK=natural killer, UMAP=uniform manifold approximation and projection, VSMC=vascular smooth muscle cells. (Fig. 1B); 10,657 cells and 12 cell types were identified in the RE as well as chondrocytes, VSMCs, endothelial cells, fibroblasts, (Fig. 1C). Epithelial cell types in the RE were horizontal basal and immune cells (ie, myeloid and B cells). Additionally, 17,228 cells (HBCs) and Bowman’s gland cells, as well as sustentac- cells and seven cell types were identified in bronchial specimens ular, ciliated, and endodermal progenitor cells. Non-epithelial after the application of quality control criteria (Fig. 1E). The cell types in the RE were microvillar and endothelial cells, as main cell types identified were ciliated, basal, and goblet cells. well as fibroblasts and immune cells (ie, mast, myeloid, T, and B The other identified cell types consisted of brush cells, fibro- cells). Epithelial cell types in the OE were HBCs, pericytes, and blasts, dividing cells, and ionocytes. Bowman’s gland cells, as well as gland progenitor, sustentacular, After the application of quality control criteria, 24,710 cells and ciliated cells. Non-epithelial cell types in the OE were neu- and 14 cell types were identified in lung specimens (Fig. 1F). rons, olfactory ensheathing glia cells (OECs), fibroblasts, vascu- The cell types identified were alveolar epithelium type 1 (AT1), lar smooth muscle cells (VSMCs), and immune cells (ie, mast, alveolar epithelium type 2 (AT2), and ciliated cells, as well as myeloid, T, and B cells). fibroblasts, lymph vessel cells, VSMCs, and endothelial cells. After the application of quality control criteria, 9614 cells and The following immune cell types were identified: mast, T, B, and 10 cell types were identified in tracheal specimens (Fig. 1D). The natural killer (NK) cells, along with monocytes, macrophages, cell types were goblet, brush, ciliated, basal, and granular cells, and dendritic cells. 25 ReseaRch aR ticle Journal of Bio-X Research Because of the similar cells present among tissues in the respi- progenitor, ciliated, and sustentacular cells, as well as neu- ratory system, we constructed a map of the respiratory system rons; its expression was minimal in fibroblasts, HBCs, and based on olfactory epithelum data, nasal respiratory epithelium Bowman’s gland cells (Fig. 3C and D). CTSL was expressed data, trachea data, bronchus data, and lung data from 83,126 in all non-immune subclusters, but it was only expressed in cells. In this map, we identified 17 subclusters: HBCs, endo- myeloid cells among the immune subclusters (Fig. 3C and D). thelial cells, Bowman’s gland cells, sustentacular cells, alveolar Additionally, endocytosis signaling was strongly expressed cells, ciliated cells, neurons, OECs, fibroblasts, microvillar cells, in non-immune subclusters and weakly expressed in immune VSMCs, chondrocytes, basal cells, mast cells, myeloid cells, B subclusters (Fig. 3C and D). PIKFYVE and TPCN2 were cells, and T/NK cells (Fig. 1G). expressed in most cell types (Fig. 3D). The viral RNA repli- cation target gene MTHFD1 was detected in all subclusters in the OE, except T and B cells (Fig. 3D). Overall, the data Coexpression patterns of key entry genes in mouse brain indicated that multiple cell types in the OE could serve as tar- tissue get cells for SARS-CoV-2. The gland progenitor, ciliated, and To identify susceptible cell types in brain tissue and elucidate sustentacular cells may constitute the most susceptible cells potential routes of SARS-CoV-2 entry, we collected scRNA-seq in the OE because they exhibited the strongest expression of data from mouse brains. In total, 11 subclusters were identified ACE2 and TMPRSS2 (Fig. 3C and D). Thus, we speculate that in mouse brain cells (Fig. 2A); marker genes for each subclus- non-neuronal cell types, rather than sensory or bulb neurons, ter are shown in Figure 2B. ACE2 was strongly expressed in are responsible for anosmia and associated odor perception pericytes and weakly expressed in other subclusters (Fig. 2C). deficits in patients with COVID-19. To confirm the expression of ACE2 at the protein level, we performed immunohistochemical analysis of normal human Coexpression patterns of key entry genes in healthy brain tissues; the immunohistochemistry results were consistent human nasal respiratory epithelial tissue with the scRNA-seq findings. ACE2 was strongly expressed in vascular pericytes, as well as endothelial cells (Fig. 2D). Lung RE is a major component of the nasal epithelium. We identified alveolar cells also exhibited strong expression of ACE2 at the 12 subclusters (Fig. 4A) and the marker genes for each sub- protein level (Fig. 2E); they are regarded as the main target of cluster are shown in Figure 4B. ACE2 was strongly expressed [32,33] SARS-CoV-2 lung tissue. Therefore, pericytes may be the in sustentacular, ciliated, and microvillar cells; it was weakly main target of SARS-CoV-2 in brain tissue; entry through these expressed in endodermal progenitor cells (Fig. 4C and D). cells might be involved in indirect damage to neurons. CD147, CD147 was expressed in almost all subclusters, whereas a potential entry receptor, was expressed in almost all sub- CD209 was expressed in myeloid cells (Fig. 4D). TMPRSS clusters (Fig. 2F). The spike protein priming enzyme TMPRSS and CTSL had similar expression patterns among subclusters was nearly absent from all subclusters in brain tissue, whereas in the RE; they did not exhibit robust expression in immune cathepsin L (CTSL) was expressed in all subclusters (Fig. 2C cells (Fig. 4C and D). Thus, multiple cell types (eg, microvillar, and F); these findings suggested that spike protein hydrolysis ciliated, and sustentacular) expressed ACE2, TMPRSS/CTSL, in brain tissue is mediated by CTSL. Because endocytosis is an endocytosis signaling, PIKFYVE, TPCN2, and MTHFD1; important aspect of coronavirus infection, we examined the these cells were identified as potential targets of SARS-CoV-2 expression of the endocytosis signaling molecules phospho- (Fig. 4D). inositide kinase, FYVE-type zinc finger containing (PIKFYVE) and two pore segment channel 2 (TPCN2). We found that Coexpression patterns of key entry genes in healthy PIKFYVE was strongly expressed in ependymal cells, peri- human tracheal and bronchial tissues cytes, and neurons 1 and 2 in brain tissue (Fig. 2F), whereas the expression of TPCN2 was minimal and primarily observed Among the 11 cell types identified in tracheal tissue (Fig. 5A in myeloid cells. Furthermore, methylenetetrahydrofolate dehy- and B), ACE2 was expressed in fibroblasts, VSMCs, and cili- drogenase 1 (MTHFD1), which is an essential gene for RNA ated cells (Fig. 5C and D). CD147 was expressed in all sub-  virus replication, was expressed in all subclusters in brain clusters, whereas CD209 was only expressed in myeloid cells tissue (Fig. 2F). These results suggested that neurons do not (Fig. 5D). TMPRSS was mainly expressed in goblet, granule, constitute a primary site of infection; however, pericytes are the brush, basal, and ciliated cells; in contrast, CTSL was expressed most susceptible cells in brain tissue because they exhibit strong in chondrocytes, endothelial cells, VSMCs, fibroblasts, myeloid expression of ACE2 and coexpression of CTSL, which facilitate cells, and B cells (Fig. 5C and D). Endocytosis signaling, viral entry and replication. PIKFYVE, TPCN2, and MTHFD1 were strongly expressed in all cell types in tracheal tissue (Fig. 5D). Among the seven sub- clusters in bronchial tissue (Fig. 6A and B), ACE2 was mainly Coexpression patterns of key entry genes in healthy expressed in goblet and ciliated cells, whereas CD147 was human nasal olfactory epithelial tissue expressed in almost all subclusters (Fig. 6C). TMPRSS, CTSL, Next, we explored whether SARS-CoV-2 could enter the CNS endocytosis signaling, PIKFYVE, TPCN2, and MTHFD1 were through the olfactory system. We identified 14 subclusters in expressed in all cell types in bronchial tissue (Fig. 6C and D). the OE (Fig. 3A); the marker genes for each subcluster are Taken altogether, the results indicated that ciliated cells are shown in Figure 3B. ACE2 was strongly expressed in suste- the most susceptible cells in both tracheal and bronchial tissue ntacular, ciliated, and gland progenitor cells; its expression because they generally exhibit strong expression of ACE2 and was weaker in HBCs and Bowman’s gland cells (Fig. 3C and TMPRSS (Figs. 5D and 6C). Goblet cells also demonstrated D). CD147 was expressed in almost all subclusters, whereas susceptibility to SARS-CoV-2 through its coexpression of CD209 was only expressed in myeloid cells (Fig. 3D). TMPRSS ACE2, TMPRSS/CTSL, endocytosis signaling, and MTHFD1 was expressed in many subclusters in the OE, including gland (Figs. 5D and 6C). 26 Journal of Bio-X Research ReseaRch aR ticle Figure 2. Single-cell analysis of mouse brain cells. UMAP plot showing the distribution of mouse brain cells (A). Dot plot showing the marker genes for brain clusters (B). Dot scale indicates the fractions of expressing cells and dot color indicates the expression level of the marker genes. UMAP plots show- ing the expression patterns of ACE2, TMPRSS, and CTSL (C). Red indicates high expression of ACE2 and blue indicates high expression of TMPRSSs or CTSL. Representative immunohistochemical staining of ACE2 in cerebral vascular pericytes (D) and lung tissue of humans (E). Arrows indicate ACE2-postive cells. Violin and box plots for the expression levels of ACE2, CD147, TMPRSS, CTSL, endocytosis-related genes, PIKFYVE, TPCN2, and MTHFD1 across clusters (F). Gene expression levels are measured as log2 (TP10K+1) values and the expression levels of gene sets are measured as mean log2 (TP10K+1) values. ACE2=angiotensin-converting enzyme 2, CTSL=cathepsin L, TMPRSS=transmembrane serine protease, UMAP=uniform manifold approximation and projection. 27 ReseaRch aR ticle Journal of Bio-X Research Figure 3. Single-cell analysis of healthy human nasal olfactory epithelial cells. UMAP plot showing the distribution of nasal olfactory epithelial cells (A). Dot plot showing the marker genes for nasal olfactory epithelial clusters (B). Dot scale indicates the fractions of expressing cells and dot color indicates the expression level of the marker genes. UMAP plots showing the expression patterns of ACE2, TMPRSS, and CTSL (C). Red indicates high expression of ACE2 and blue indi- cates high expression of TMPRSS or CTSL. Violin and box plots for the expression levels of ACE2, CD147, CD209, TMPRSS, CTSL, endocytosis-related genes, PIKFYVE, TPCN2, and MTHFD1 across clusters (D). Gene expression levels are shown as log2 (TP10K+1) values, and the expression levels of gene sets are shown as mean log2 (TP10K+1) values. ACE2=angiotensin-converting enzyme 2, CTSL=cathepsin L, HBCs=horizontal basal cells, OEC=olfactory ensheathing glia cells, TMPRSS=transmembrane serine protease, UMAP=uniform manifold approximation and projection, VSMC=vascular smooth muscle cells. Coexpression patterns of key entry genes in healthy PIKFYVE, TPCN2, and MTHFD1 were strongly expressed in lung cells (Fig. 7D). human lung tissue In total, 14 subclusters (Fig. 7A) in lung tissue were identi- fied on the basis of cell type-specific marker genes (Fig. 7B). Expression patterns of SARS-CoV-2 and neurotropic ACE2 was mainly expressed in AT2 cells; it was also expressed virus receptor genes in healthy human respiratory and in AT1 and ciliated cells (Fig. 7C and D). CD209 was mainly mouse brain tissues expressed in monocytes and macrophages, whereas CLEC4M For comprehensive analysis of susceptible cell types in the respi- was expressed in endothelial cells. CD147 was expressed in ratory system and comparison of similarities among cell types, all subclusters in lung tissue (Fig. 7D). TMPRSS and CTSL we constructed a map of the respiratory system and identified 17 were expressed in most cell types; they were not expressed in subclusters; the main subclusters comprised myeloid, alveolar, mast, NK, T, or B cells (Fig. 7C and D). Endocytosis signaling, 28 Journal of Bio-X Research ReseaRch aR ticle Figure 4. Single-cell analysis of healthy human nasal respiratory epithelial cells. UMAP plot showing the distribution of nasal respiratory epithelial cells (A). Dot plot showing the marker genes for nasal respiratory epithelial clusters (B). Dot scale indicates the fractions of expressing cells and dot color indicates the expression level of the marker genes. UMAP plots showing the expression patterns of ACE2, TMPRSS, and CTSL (C). Red indicates high expression of ACE2 and blue indicates high expression of TMPRSSs or CTSL. (D) Violin and box plots for the expression levels of ACE2, CD147, CD209, TMPRSS, CTSL, endocytosis-related genes, PIKFYVE, TPCN2, and MTHFD1 across clusters. Gene expression levels are shown as log2 (TP10K+1) values, and the expression levels of gene sets are shown as mean log2 (TP10K+1) values. ACE2=angiotensin-converting enzyme 2, CTSL=cathepsin L, HBCs=horizontal basal cells, TMPRSS=transmembrane serine protease, UMAP=uniform manifold approximation and projection. T/NK, endothelial, ciliated, neuronal, and B cells (Additional types in the respiratory system (Fig. 8A). The endocytosis-re- Fig. 1A and B, http://links.lww.com/JR9/A45). We found that lated gene PIKFYVE was expressed mainly in myeloid cells and ACE2 was strongly expressed in ciliated cells in the OE, RE, tra- fibroblasts in the RE, as well as endothelial cells in lung tissue cheal tissue, and bronchial tissue; sustentacular cells in the OE (Fig. 8A). These results showed that factors involved in SARS- and RE; gland progenitor cells in the RE; and AT2 cells in lung CoV-2 entry are strongly expressed in the OE and RE, highlight- tissue (Fig. 8A). CD209 was strongly expressed in myeloid cells ing the potential role of such cells to facilitate viral infection in the OE, RE, and tracheal tissue. TMPRSS, CTSL, endocytosis and spread. The most susceptible cells in the respiratory system signaling, TPCN2, and MTHFD1 were expressed in most cell were ciliated, gland progenitor, and sustentacular cells, as well 29 ReseaRch aR ticle Journal of Bio-X Research Figure 5. Single-cell analysis of healthy human trachea cells. UMAP plot showing the distribution of tracheal cells (A). Dot plot showing the marker genes for tracheal clusters (B). Dot scale indicates the fractions of expressing cells and dot color indicates the expression level of the marker genes. UMAP plots showing the expression patterns of ACE2, TMPRSS, and CTSL (C). Red indicates high expression of ACE2 and blue indicates high expression of TMPRSSs or CTSL. Violin and box plots for the expression levels of ACE2, CD147, CD209, TMPRSS, CTSL, endocytosis-related genes, PIKFYVE, TPCN2, and MTHFD1 across clusters (D). Gene expression levels are shown as log2 (TP10K+1) values, and the expression levels of gene sets are shown as mean log2 (TP10K+1) values. ACE2=angiotensin-converting enzyme 2, CTSL=cathepsin L, TMPRSS=transmembrane serine protease, UMAP=uniform manifold approximation and projec- tion, VSMC=vascular smooth muscle cells. as HBCs; AT2 cells are the known vulnerable cells for SARS- A45). Notably, many viral receptors (eg, AXL, ISG15, ITGA4, [32,33] COV2, their susceptibility could be greater than the suscep- and PECAM1) are expressed in pericytes, suggesting that peri- tibility of AT2 cells, indicating that these cells could serve as the cytes mediate CNS infection (Additional Fig. 3, http://links. portal of entry for SARS-CoV-2. lww.com/JR9/A45). Furthermore, because exosome biogenesis To explore the potential mechanism of SARS-CoV-2 infec- and release are also associated with viral transport, we explored tion in the CNS, we compared the expression patterns of entry the brain-specific and OE-specific expression patterns of genes receptors for various neurotropic viruses between maps of the involved in exosome biogenesis and release. The results showed respiratory system and brain. We found that some neurotropic that neurons, ciliated cells, and OECs had strong capacities for exosome biogenesis and release, which may serve as alterna- virus entry receptors were not expressed in neurons, indicating tive pathways for CNS infection (Fig. 8B). Thus, we speculated that other mechanisms (eg, other cells) were involved in CNS the potential mechanisms involved in CNS infection (Fig. 8C). infection (Additional Figs. 2 and 3, http://links.lww.com/JR9/ 30 Journal of Bio-X Research ReseaRch aR ticle Figure 6. Single-cell analysis of healthy human bronchial epithelial cells. UMAP plot showing the distribution of bronchial epithelial cells (A). Dot plot showing the marker genes for bronchial epithelial clusters (B). Dot scale indicates the fractions of expressing cells and dot color indicates the expression level of the marker genes. UMAP plots showing the expression patterns of ACE2, TMPRSS, and CTSL (C). Red indicates high expression of ACE2 and blue indicates high expression of TMPRSSs or CTSL. Violin and box plots for the expression levels of ACE2 CD147, TMPRSS, CTSL, endocytosis-related genes, PIKFYVE, TPCN2, and MTHFD1 across clusters (D). Gene expression levels are shown as log2 (TP10K+1) values, and the expression levels of gene sets are shown as mean log2 (TP10K+1) values. ACE2=angiotensin-converting enzyme 2, CTSL=cathepsin L, TMPRSS=transmembrane serine protease, UMAP=uniform mani- fold approximation and projection. [33,34] Coexpression of entry receptor ACE2 and spike protein prim- coexpression patterns of viral entry genes. Although most ing enzymes in BBB pericytes enables SARS-CoV-2 entry into analyses are based on transcriptional levels, there is clear con- the cerebrospinal fluid and brain. SARS-CoV-2 may also infect sistency at the cellular level: the expression patterns of entry the telencephalon through the olfactory bulb. genes at the transcriptional level in a particular cell type can accurately predict the protein expression levels of entry recep-  tors. Our results were consistent with the coexpression Discussion patterns (ACE2 and TMPTSS2) previously identified among  scRNA-seq is a powerful technique that can be used to iden- susceptible cell types in multiple tissues. Furthermore, we tify susceptible cells through single-cell-level analyses of the obtained novel information concerning tissues such as the OE, 31 ReseaRch aR ticle Journal of Bio-X Research Figure 7. Single-cell analysis of healthy human lung cells. UMAP plot showing the distribution of lung cells (A). Dot plot showing the marker genes for lung clusters (B). Dot scale indicates the fractions of expressing cells and dot color indicates the expression level of the marker genes. UMAP plots showing the expression patterns of ACE2, TMPRSS, and CTSL (C). Red indicates high expression of ACE2 and blue indicates high expression of TMPRSSs or CTSL. Violin and box plots for the expression levels of ACE2, CD147, CD209, CLEC4M, TMPRSS, CTSL, endocytosis-related genes, PIKFYVE, TPCN2, and MTHFD1 across clusters (D). Gene expression levels are shown as log2 (TP10K+1) values, and the expression levels of gene sets are shown as mean log2 (TP10K+1) values. CTSL=cathepsin L, DCs=dendritic cells, NK=natural killer, TMPRSS=transmembrane serine protease, UMAP=uniform manifold approximation and projection, VSMC=vascular smooth muscle cells. RE, and brain; we also identified coexpression patterns involv- The coronavirus infection process begins with viral bind- ing TMPRSS2 and viral replication genes. These results sug- ing to the cellular membrane through heparan sulfate pro- gest that neurological symptoms and CNS infection in patients teoglycans, which facilitate the interaction between the spike [16,36] with COVID-19 are secondary to SARS-CoV-2 invasion of the protein and the entry receptor (ie, ACE2). Recently, BBB via pericytes. Additionally, SARS-CoV-2–induced olfac- CD147 and CD209 were also identified as potential recep- tory disorders could be the result of localized damage to suste- tors for SARS-CoV-2; interactions with these proteins [17,18] ntacular cells and ciliated cells in the nasal RE and OE. also facilitate viral entry into host cells. In our study, 32 Journal of Bio-X Research ReseaRch aR ticle Figure 8. Expression levels of SARS-CoV-2 binding receptors, spike protein hydrolysis, endocytosis-related genes, and virus RNA replication genes across tissues. Violin and box plots for the expression levels of SARS-CoV-2 binding receptors, spike protein hydrolysis, endocytosis-related genes, and virus RNA replication genes across tissues (A). Gene expression levels are shown as log2 (TP10K+1) values, and the expression levels of gene sets are shown as mean log2 (TP10K+1) values. Box plots showing the expression levels of exosome biogenesis and release-related genes in the brain and olfactory epithelium (B). The expression levels of gene sets are shown as mean log2 (TP10K+1) values. Schematic depicting potential mechanisms involved in CNS infection (C). Coexpression of entry receptor ACE2 and spike protein priming enzyme cathepsin L in BBB pericytes enables SARS-CoV-2 entry into the cerebrospinal fluid and brain. SARS- CoV-2 may also infect the telencephalon through the olfactory bulb. ACE2=angiotensin-converting enzyme 2, BBB=blood–brain barrier, CNS=central nervous system, HBCs=horizontal basal cells, OEC=olfactory ensheathing glia cells, SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. CD147 was expressed in most cell types; however, these in various cells. Thus, cells that include both the entry receptor cells were not consistent with published reports of infected (ACE2) and the spike protein priming enzyme (TMPRSS and/  cells. Thus, the role of CD147 in SARS-CoV-2 pathoge- or CTSL) may be most susceptible to infection by SARS-CoV-2. nicity requires further investigation. In contrast, CD209 was Headache, impaired consciousness, epilepsy, encephalitis, mainly expressed in macrophages and monocytes in most and acute myelitis have been reported in patients with COVID-  tissues. The specific location of CD209 expression implies 19. Notably, a case of SARS-CoV-2 encephalitis was reported a role in SARS-CoV-2 entry into macrophages, which were by physicians in Beijing Ditan Hospital; genomic sequencing  also the target cells for SARS-CoV-2 infection, with the revealed the presence of SARS-CoV-2 in the patient’s cerebrospi- potential for initiation of a cytokine storm. nal fluid. Subsequently, a similar case was reported by physicians  Successful conformational changes and activation of spike in Japan. These reports indicated that SARS-CoV-2 is able to proteins result in membrane fusion; these processes require infect the human CNS in some instances. Intracranial infections  receptor binding, as well as appropriate protease activation. occur after damage to the BBB, which allows viral invasion. Endocytosis and cathepsin-mediated endosomal acidification In the present study, we found that the entry receptor ACE2 are also important processes during viral fusion with a target and spike protein priming enzyme CTSL were coexpressed in [37,38]  cell. In the present study, we found that TMPRSS and CTSL pericytes, which are specialized endothelial cells in the brain. were strongly expressed and exhibited a broad distribution. Furthermore, endothelial cells generally expressed lower levels Either TMPRSS or CTSL was expressed in ACE2-expressing of ACE2, which suggests that they can serve as an alternative cells, consistent with previous reports that ACE2 constitutes a route of infection. Notably, some vascular pericytes (eg, in  limiting factor for initial viral entry. Genes that encode pro- the OE and RE) did not express ACE2, indicating that brain teins involved in endocytosis and viral replication are expressed pericytes have a distinct role in blood pressure regulation and 33 ReseaRch aR ticle Journal of Bio-X Research  BBB maintenance. Endothelial cells in the brain are bound which were higher than the levels in AT2 cells. This finding together by tight junctions to ensure the integrity of the BBB. indicates that such ciliated cells may be the “portal” for SARS- The use of endothelial cells and pericytes as host cells could CoV-2 entry because of their anatomical location; they may also allow viral proliferation; the eventual cell death would destroy serve as a viral reservoir for persistent SARS-CoV-2 infection. the host cells and BBB, thereby releasing viral particles into the These results have important implications for understanding brain. Considering the extensive similarities described above in the symptoms of anosmia and potential mechanisms involved in terms of expression patterns for ACE2, TMPRSS2, and CTSL SARS-CoV-2 infection of the CNS. in mice and humans, neurons presumably do not constitute a primary site of infection; however, vascular pericytes may be Limitations sensitive to SARS-CoV-2, thus allowing entry through the BBB and subsequent CNS infection. In this study, the coexpression pattern of virus entry genes only Respiratory viruses may enter the CNS through neurons in indicates the susceptibility of the cell types. It does not really the olfactory and trigeminal nerves (within the nasal cavity), represent the cell types the SARS-CoV-2 infects. Furthermore, or through neurons in the vagus nerve (within the trachea and we analyzed susceptible cell types based on only normal sam-  lungs). Human coronavirus OC43 can induce CNS neu- ples. It would be more convincing if we used samples from ropathology through interneuron propagation and axonal COVID-19 patients to study the viral load in different cell types.  transport. We found that the entry receptor for human coro- So, the results were limited. navirus OC43 (HLA class I molecule) was expressed in neural cells, which enables the virus to infect neurons (Additional Fig. Conclusion 2, http://links.lww.com/JR9/A45). However, the entry receptor ACE2 and the spike protein priming enzymes were not coex- We identified cells susceptible to SARS-CoV-2 infection in the pressed in neural cells within the CNS, olfactory system, or CNS, olfactory system, and respiratory system. Our findings respiratory system. An earlier epidemic strain of coronavirus, provide insights into the mechanisms that underlie neurological SARS-CoV, also binds ACE2; SARS-CoV particles have been symptoms and CNS infection in patients with COVID-19. detected in brain tissue, where they were located almost exclu-  sively in neurons. Considering the strong capacities for exo- Acknowledgments some biogenesis and release in neurons, ciliated cells, and OECs, None. exosome biogenesis and release could be involved in SARS- CoV-2 infection of the CNS. However, this hypothesis requires additional investigation. Author contributions The OE serves as the main odor detection and conduction HX, JL: study design. ZK, JW, HZ, DX, HG, and WZ: data tissue; it includes ciliated olfactory sensory neurons, glia-like  analysis. ZK, DX, and HG: data collection and generation. HZ, sustentacular cells, and apical microvilli. Olfactory sensory JW, ZL, XC, and JX: data interpretation. TM, ZK, AQ, and HX: neurons are surrounded by sustentacular cells and the axons of manuscript drafting. AQ, HZ, TM, HX, and JL: overall super- OECs. Sustentacular cells serve as structural support for sensory vising and organizing the study. All authors approved the final neurons, whereas OECs guide olfactory sensory neuron axons version for publication. that cross the cribriform plate at the skull base and terminate in [46–48] the olfactory bulb. Additionally, olfactory sensory neurons  express odor receptors on dendritic cilia. Thus, SARS-CoV-2 Financial support invasion into ciliated cells and sustentacular cells may lead to This work was supported by the National Natural Ascience olfactory disorders. Foundation of China (No. 31821003 to HX) and the China SARS-CoV-2 primarily affects the respiratory system, with Ministry of Science and Technology (No. 2018AAA0100300 to  serious effects on the lungs. In this study, we observed coex- HX). The funders had no role in manuscript design, data collec- pression of ACE2 and TMPRSS/CTSL in the AT1, AT2, and cil- tion and analysis, preparation of the manuscript, or decision to iated cells in lung tissue. In lung alveoli, AT1 cells (ie, epithelial publish. cells) are responsible for gas exchange; AT2 cells (ie, alveolar stem-like cells) are responsible for surfactant biosynthesis and  Institutional review board statement immunoregulation. Thus, SARS-CoV-2 infection may cause damage to alveolar infrastructure; the resulting increase in There is no direct involvement of human subjects in this project. alveolar surface tension can induce dyspnea and hypoxia. The All the data use existing biological samples and data from prior  involvement of AT2 cells can lead to extensive inflammation. studies. Therefore, ethical oversight and patient consent were The host immune response produces a proinflammatory cyto- not handled in this study. kine storm, which manifests as acute lung injury and acute [53,54] respiratory distress syndrome. We found that CD209 was Data availability statement mainly expressed in macrophages and monocytes in lung tissue. Notably, CD209 may be responsible for viral antigen activation The single cell data are available at the Gene Expression Omnibus of specific T cells, as well as the secretion of perforin, interferon, under the accession code GSE139522 and GSE134355, Sequence and granzyme B; these processes can cause further injury to the ReadArchive dataset (SRA https://www.ncbi.nlm.nih.gov/sra) at  lungs and remote organs, such as the brain. accession code SRA667466 and Atlas Data Protal (https://data. Finally, we compared the expression levels of viral entry fac- humancellatlas.org) at accession code PRJEB31843. 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