key: cord-279518-z3k7zaw4
authors: Xue, Xiaotong; Mi, Zihao; Wang, Zhenzhen; Pang, Zheng; Liu, Hong; Zhang, Furen
title: High expression of ACE2 on the keratinocytes reveals skin as a potential target for SARS-CoV-2
date: 2020-05-23
journal: J Invest Dermatol
DOI: 10.1016/j.jid.2020.05.087
sha: 
doc_id: 279518
cord_uid: z3k7zaw4

nan

To the Editor:

Since December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has emerged in Wuhan, Hubei, China, as an etiological agent causing a new infectious respiratory disease (Corona virus disease 2019, , which manifests as fever, severe respiratory illness and pneumonia (Huang et al., 2020) . With significant morbidity and mortality, COVID-19 has now evolved into a global pandemic (Park, 2020) . To date, more than three million COVID-19 cases have been reported worldwide, including 252,336 deaths (World Health Organization, WHO).

The entry of coronavirus into target cells mainly depends on the binding of the viral spike (S) proteins to cellular receptors (Hoffmann et al., 2020) . Angiotensin-converting enzyme 2 (ACE2) has been identified as a crucial functional receptor of SARS-CoV-2 . The receptor-binding domain (RBD) domain of the SARS-CoV-2 S protein were reported to have strong interaction with human ACE2 molecules, which was confirmed in molecular structure (Xu et al., 2020b; Wrapp et al., 2020) . These studies suggested that the ACE2-expressing cells were vulnerable to SARS-CoV-2 infection.

The RNA and protein expressions of ACE2 have been widely investigated by bulk samples from the heart, lung, kidney, and other organs (Hamming et al., 2004) . Recently, single-cell RNA sequencing (scRNA-seq) was applied to analyze the ACE2 mRNA expression in different cell subtypes. High ACE2 expression was identified in the type II alveolar cells (AT2), bronchial transient secretory cells, small intestinal epithelium cells and the oral epithelial cells in accordance with respiratory clinical manifestations and rare clinical manifestations such as gastrointestinal symptoms, suggesting the respiratory droplet, digestive ２ and fecal-oral transmission routes of SARS-CoV-2 (Lukassen et al., 2020; Liang et al., 2020; Xu et al., 2020a) . Therefore, we hypothesized that the expression and distribution of ACE2 in human organs and tissues could reflect the potential infection routes of SARS-CoV-2.

However, scRNA-seq has not yet been applied to examine the ACE2 expression in the cells of skin tissues, and the transmission of this virus by percutaneous routes remains unclear.

To investigate whether skin was a potential target for SARS-CoV-2 infection, we firstly analyzed the ACE2 mRNA expression and the ACE2-positive cell composition in skin tissues based on the public databases (GEPIA2 and ARCHS4), and found ACE2 was expressed in skin tissues in addition to testis, kidney, colon, lung, and so on. Especially, the expression of ACE2 was significantly higher in keratinocytes than other cell types in skin tissues, such as fibroblasts and melanocytes (Figure 1a Human skin as a functional physical and immune barrier could prevent the invasion of foreign pathogens, including bacteria, fungi and viruses. Once the skin barrier was disrupted, humans had an increased susceptibility to microbial colonization and infections (Boguniewicz and Leung, 2011) . Recently, the cutaneous manifestations of SARS-CoV-2 infection were reported in 20.4% (18 / 88) patients, and were found to be similar with other viral skin infections (Recalcati, et al., 2020) . In this study, we systematically analyzed the ACE2 expression and the ACE2-positive cell composition in skin tissues and found the high ４ expression of ACE2 in keratinocytes, especially in differentiating keratinocytes and basal cells, suggesting that skin might be a potential target of SARS-CoV-2. Eczematoid dermatitis was induced by long-term wearing protective clothing and contacting disinfectant, which might be a crucial factor to cause percutaneous infection in COVID-19 patients and medical personnel (Yan et al., 2020) . The healthcare workers, especially those worked at first-line, and the dermatosis patients with skin barrier dysfunction may be the risk populations for percutaneous infection. In addition, keratinocytes maybe become infected through hematogenous viral spreading following inoculation of the upper airways .

Our study provided a viewpoint to the routes of SARS-CoV-2 transmission, which were not reported previously.

In conclusion, the high expression of ACE2 on keratinocytes in human skin indicated that percutaneous transmission might be a potential risk route for SARS-CoV-2 infection, especially in condition of skin barrier dysfunction. Also, keratinocytes are the potential target cells for the viral infection when a patient is in a state of viraemia. Currently, SARS-CoV-2 has been pandemic worldwide. The potential risk routes, by which SARS-CoV-2 infects keratinocytes, and cutaneous manifestations of SARS-CoV-2 infection should be brought to our attention as well.

The raw sequence data reported in this paper has been deposited in the GSA (Genome 

The authors state no conflict of interest. 

The public RNA-seq data of ACE2 expression in various normal tissues were obtained from Gene Expression Profiling Interactive Analysis 2 (GEPIA2, http://gepia2.cancer-pku.cn/ #index).

The public RNA-seq data of ACE2 expression in various cell types of different tissues were obtained from All RNA-seq and CHIP-seq Sample Search Space (ARCHS4, https://amp.pharm.mssm.edu/archs4/data.html), a web source providing the majority of published RNA-seq data from human and mouse at the gene and transcript levels (Lachmann et al., 2018) . 

Fresh skin samples were cut into approximately 3 mm 3 pieces and digested using Dispase II (Gibco) to separate dermis and epidermis. Epidermis was minced finely with dissection scissors in 5 ml 1 x PBS (Gibco), and was digested in 1 mL 0.25% Trypsin-EDTA (Gibco) for 30 min.

Subsequently, 6 ml PBS containing 10% FBS was added to terminate the digestive reaction.

Dermis was minced finely using dissection scissors, and was digested in 5 mL digestion buffer (1 mg/mL Collagenase P, 100 μg/mL DNase I) (Sigma-Aldrich) for 50 min. After that, 5ml PBS containing 10% FBS was added to terminate the digestive reaction. The cell suspension was subsequently passed through a 70 μm cell strainer and centrifuged at 500 x g for 7 min. After washing twice with 1 x PBS, the cell pellets were resuspended in 100 μl 1 x PBS, and the cells were counted under the microscope using cell counting chamber.

The was used for dimensionality reduction and clustering the cells. The cell types for the analysis were derived from the Human Primary Cell Atlas. UMAP plots and violin plots were generated with Seurat in R.

Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instruction, and reverse transcribed into cDNA (ABI). The primers were designed by Primer-BLAST (NCBI). Quantitative real-time PCR assays were conducted in triplicate using the SYBR green method (CWBIO) on a StepOnePlus Real-Time PCR Systems (ABI) according to manufacturer's instructions. The sequence of primers for ACE2 genes were as follows: forward primer, 5'-CGAGTGGCTAATTTGAAACCAAGAA-3'; reverse primer, 5'-ATTGATACGG CTCCGGGACA-3' (Zhang et al., 2019) .

The tissue slides were deparaffinized and rehydrated, and water-bath heating method was used to perform antigen retrieval. After washing with 1 x PBS, the endogenous peroxidase was blocked by 0.3% H 2 O 2 for 15 min, and nonspecific binding was blocked by 5 x BSA for 25 min at 37 o C. The slides were incubated with rabbit anti-human ACE2 polyclonal antibody (Proteintech) or rabbit polyclonal IgG isotype control at 1:200 dilution overnight at 4°C. Next day, the slides were washed with 1 x PBS, and incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Abcam) for 30min. Sections were mounted by neutral resins and examined under microscopy (EVOS TM FL Auto 2 Imaging System, Thermofisher).

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The work was supported by the Academic promotion programme of Shandong First