key: cord-0957432-8xdzeztp
authors: li, s.; li, d.; fang, j.; liu, q.; sun, x.; xu, g.; cao, w.
title: COVID-19 receptor ACE2 is expressed in human conjunctival tissue, expecially in diseased conjunctival tissue
date: 2020-05-23
journal: nan
DOI: 10.1101/2020.05.21.20109652
sha: d994ad8573eb0126a0cfbcceb574eab9691ee496
doc_id: 957432
cord_uid: 8xdzeztp

COVID-19 virus has currently caused major outbreaks worldwide. ACE2 is a major cellular-entry receptor for the COVID-19 virus. Although ACE2 is known to be expressed in many organs, whether it is expressed by the conjunctival tissue is largely unknown. Human conjunctival tissues from 68 subjects were obtained, which included 10 subjects with conjunctival nevi, 20 subjects with conjunctivitis, 9 subjects with conjunctival papilloma, 16 subjects with conjunctival cyst, 7 subjects with conjunctival polyps, and 6 ocular traumas as normal subjects. Expression of ACE2 was evaluated by immunohistochemistry, immunofluorescence, reverse transcriptase-quantitative polymerase chain reaction, and western blot assay. We observed the expression of ACE2 by conjunctival tissues, expecially in conjunctival epithelial cells. ACE2 was significantly (p<0.001) overexpressed in conjunctival cells obtained from subjects with conjunctivitis, conjunctival nevi, conjunctival papilloma, conjunctival cyst, and conjunctival polyps epithelial cells when compared to that in conjunctival epithelial cells obtained from control subjects. Collectively, clinical features of reported COVID-19 patients combined with our results indicate that COVID-19 is likely to be transmitted through the conjunctiva.

In December 2019, coronavirus disease 2019 (COVID- 19) was first reported in people in Wuhan, Hubei province, China. [1] Since then, the COVID-19 virus has rapidly spread throughout the world. [2, 3] As of May 16, 2020, 4425485 laboratory-confirmed cases were reported. Furthermore, the World Health Organization reported 302059 fatalities.

[4] The typical signs and symptoms of COVID-19 are fever, dry cough, and fatigue. The virus is mainly transmitted through respiratory droplets and by close contact with infected individuals, as reported by the Health commission, PRC. Furthermore, about 0.8% of COVID-19 patients showed symptoms of conjunctival congestion. [5] Thus, the ocular surface may as a possible site of virus entry and also as a source of contagious infection [6] . However, whether COVID-19 can be transmitted through the conjunctiva is still unclear.

Studies provide powerful evidence that angiotensin-converting enzyme 2 (ACE2) is a major cellular-entry receptor for the COVID-19 virus. [7, 8] Lukassen et al. [9] and Hamming I et al. [10] have shown the expression of ACE2 in lung tissue, which may explain why most COVID-19 patients show increased sputum production and experience shortness of breath. [11] Using single-cell transcriptome analysis, Haoyan Chen et al. [12] and Hao Zhang et al. [13] have shown that ACE2 is expressed in the epithelial cells of the colon and digestive system, which may explain why 3.8% of COVID-19 patients experience nausea or vomiting, and diarrhoea. [1] Moreover, previous studies have shown that oral, nasal, and nasopharyngeal epithelia do not express ACE; [10] these results are consistent with the lack of any obvious upper . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint respiratory distress symptoms in COVID-19 patients. [1, 11] These studies, along with the typical symptoms exhibited by COVID-19 patients, may provide evidence that the expression of ACE2 gene may be a factor involved in the mode of transmission of the virus.

Currently, it is still unknown whether the COVID-19 virus is transmitted through the conjunctiva, and whether the conjunctival tissue can express ACE2. [14] In order to understand the relationship between the expression levels of ACE2 and the likelihood of COVID-19 transmission through the conjunctiva, we studied the mRNA and protein level of ACE2 in conjunctival tissue.

This study was conducted at the Department of Pathology, Renji Hospital, Shanghai Jiao Tong University, Shanghai, China. The ethics committee of the Renji Hospital of Shanghai Jiao Tong University approved this study, and it adhered to the principles of the Declaration of Helsinki. Informed consent was obtained from all participating subjects. All subjects were recruited from Renji Hospital of Shanghai Jiao Tong University.

Human conjunctival tissues were obtained from patients undergoing biopsy for diagnoses or prior to surgery. Diseased conjunctival tissues were obtained from 62 different subjects with the following conditions: ten patients with conjunctival nevi, 20 patients with conjunctivitis, nine patients with conjunctival papilloma, 16 patients . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint with conjunctival cyst, and seven patients with conjunctival polyps; six normal conjunctival tissues were obtained from patients with ocular trauma, who needed ophthalmectomy.

Total RNA was extracted using GenEluteTM FFPE RNA Purification Kit (RNB400, Sigma-Aldrich) as per the manufacturer's instructions. The quality and integrity of the acquired total RNA were evaluated using a NanoDrop™ 2000c (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). For reverse transcription-quantitative polymerase chain reaction (RT-qPCR), 1,000 ng of total RNA was reverse-transcribed with 2 μL of 5X OneStep RT Mix. The RT-qPCR reaction was performed using 1 μL of RT products, 0.2 μL of 10 μM forward primer, 0.2 μL of 10 μM pmol reverse primer, and 5 μL of 2X SYBR Green I qPCR mix and completed to 10 μL with nuclease-free water. The primers used were as follows: ACE2-forward, 5′-AAAGGAACAGTCCACACTTGCCC-3′, and ACE2-reverse, 5′-TGAAGACCCATTTTGCTGAAGAGCC-3′.

Total protein was extracted from conjunctival tissue using ice-cold RIPA lysis buffer together with PMSF mixture. For gel electrophoresis, 80 µg total extracted proteins of each sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% non-fat milk for ~2 h at room temperature. Then the membranes were incubated with primary . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint antibodies against GAPDH (1:10000; ab181602, Abcam, Cambridge, MA, USA) and ACE2 (1:1000; ab15348, Abcam, Cambridge, MA, USA). After being washed three times with TBST, the membranes were further incubated with specific horseradish peroxidase conjugated secondary antibodies (1:5,000; ab6721; Abcam, Cambridge, MA, USA). Immuno-stained bands were detected by chemiluminescent method.

Image software (Imagepro plus 6.0, Media Cybernetics, Inc., USA) were used to quantify band intensities.

Paraffin-embedded sections (4 μm) of conjunctival tissue collected from the patients were prepared on slides. The antigen was retrieved by microwaving the samples at a temperature over 90°C after they were dewaxed and rehydrated. Slides were then blocked with 5% bovine serum albumin (BSA) for 1 h to reduce nonspecific binding.

Two specimens were taken from each sample. One specimen was incubated with . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint Image processing software (Imagepro Plus, Media Cybernetics, Inc., USA) was used to quantify the degree of immunohistochemical staining. The area (sum) and integrated optical density (IOD) (sum) were counted in four different fields using a computer-automated method (Imagepro plus 6.0, Media Cybernetics, Inc., USA).

Area of interest (AOI) = IOD (sum) / area (sum); the results were averaged.

Briefly, 4μm FFPE sections of human conjunctiva were dewaxed and hydrated. Tissue is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint value of immunofluorescence staining. The results were averaged.

All analyses were performed using the Statistical Package for the Social Sciences software, version 13.0 (SPSS Inc., Chicago, IL, USA). The figures were created using GraphPad Prism 6 software (La Jolla, CA, USA). The results were analysed using the two-tailed Mann-Whitney test. A value of p<0.05 was considered statistically significant.

Sixty-eight subjects with a mean age of 47.42 ±16.19 years [(32 males (47.06%); 36 females (52.94%)] were recruited in the study. A total of 68 conjunctival tissues were collected from subjects with the following conditions: conjunctival nevi (n=10), conjunctivitis (n=20), conjunctival papilloma (n=9), conjunctival cyst (n=16), conjunctival polyps (n=7), and normal conjunctiva tissues (n=6). The demographic data of the study subjects are summarised in Table 1 .

RT-qPCR was used to examine the expression level of ACE2. A significant difference in mRNA level of ACE2 between normal conjunctival tissues and other diseased conjunctival tissues was observed (p<0.001; Figure 1A ). The ACE2 mRNA levels was significantly overexpressed in the conjunctival tissues of inflamed conjunctiva, conjunctival nevi, conjunctival cyst, conjunctival papilloma, and conjunctival polyps . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint as compared to that in normal conjunctival tissues (p<0.001; Figure 1A ). A significant difference in ACE2 mRNA level between conjunctivitis tissues and other diseased conjunctival tissues was observed (p<0.001; Figure 1A ). Conjunctival tissues from conjunctival cyst, conjunctival papilloma, and conjunctival polyps exhibited a higher ACE2 expression than that suffering from conjunctivitis. Moreover, there was no significant difference in ACE2 expression among diseased conjunctival tissues expect conjunctivitis ( Figure 1A , p=0.08).

As presented in Figure 1B , the protein level of ACE2 was detected by western blot assay. The protein level of ACE2 was significantly (p<0.001 for both) higher in the conjunctival tissues of inflamed conjunctiva, conjunctival nevi, conjunctival cyst, conjunctival papilloma, and conjunctival polyps as compared to that in normal conjunctival tissues ( Figure 1C ). Furthermore, in diseased conjunctival tissues, the protein level of ACE2 was significantly (p<0.001) lower in the conjunctival tissues of inflamed conjunctiva as compared to other diseased conjunctival tissues ( Figure 1C ).

Two different antibodies [polyclonal rabbit anti-ACE2 antibody (Figure 2, 4) and monoclonal mouse anti-ACE2 antibody (Figure 3 Additionally, ACE2 was also expressed in the conjunctival lamina propria lymphocytes and vascular endothelial cells ( Figure 4A, Figure 5A ). Strong staining was seen in the vascular endothelium and lymphocytes in the subepithelial tissues of the conjunctival polyps ( Figure 4B, Figure 5B ), conjunctival papilloma ( Figure 4C , Figure 5C ), conjunctival cyst ( Figure 4D , Figure 5D ), and conjunctivitis ( Figure 4F , Figure 5F ). In the conjunctival tissue exhibiting conjunctival nevi ( Figure 4E , Figure   5E ), strong staining was seen in the melanocytes and eccrine sweat-gland cells.

We also observed the presence of ACE2 in conjunctival epithelial cells by immunofluorescence ( Figure 6 

A summary of the quantitative data is presented in Figure 7A -C. A significant difference in ACE2 expression between normal conjunctival epithelial cells and other diseased conjunctival epithelial cells was observed (p<0.05). The ACE2 was . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint significantly (p<0.05 for both) overexpressed in the epithelial cells of inflamed conjunctiva, conjunctival nevi, conjunctival papilloma, conjunctival cyst, and conjunctival polyps as compared to that in normal conjunctival epithelial cells.

Furthermore, in diseased conjunctival tissues, the level of ACE2 was significantly (p<0.001) lower in the conjunctival epithelial cells of inflamed conjunctiva as compared to other diseased conjunctival tissues.

COVID-19 is currently a global pandemic; however, the possibility of its transmission through the conjunctiva is still unclear. In this study, we report the level of ACE2, the major cellular-entry receptor of the COVID-19 virus, in human conjunctival tissue. It is evident that several respiratory viruses are capable of using the eye as both a site for replication as well as a port of entry, which could result in a productive respiratory infection. [15] Athanasiu P et al. [16] reported human parainfluenza viruses in conjunctival cells. Warren D et al. [17] reported these viruses in children presenting with conjunctivitis and upper respiratory tract illnesses. Further, Peiris JS et al. [18] reported that SARS-CoV can be transmitted either through direct or indirect contact . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05. 21.20109652 doi: medRxiv preprint with the mucous membranes of the eyes. Collectively, it is evident that the virus, in the form of droplets, can be transmitted through the human conjunctival epithelium. [19] Lu CW et al. [20] have proposed that the COVID-19 virus transmission through the ocular surface must not be ignored, indicating the possibility of its transmission via the eye. They also reported that Guangfa Wang, a member of the national expert panel on pneumonia, was infected by the COVID-19 virus during inspection in Wuhan.

Wang complained of redness of the eyes, which indicates that he was infected through the ocular surface. [20, 21] Furthermore, Xia J et al. [22] performed a prospective interventional case study series to assess the presence of novel coronavirus in tears and conjunctival secretions of COVID-19-infected patients. They report that conjunctival swab samples collected from patients with conjunctivitis yielded positive RT-PCR results, whereas negative RT-PCR results were obtained for the tear fluid and conjunctival secretion samples in patients without conjunctivitis. In our study, a marked immunostaining revealed that ACE2 was abundantly expressed in epithelial cells in patients with conjunctivitis as compared to that in the epithelial cells from normal conjunctivae. This could explain why some studies reported positive RT-PCR results only in patients with conjunctivitis.

Moreover, our study also showed that ACE2 was significantly overexpressed in conjunctivitis, conjunctival nevi, conjunctival papilloma, conjunctival cyst, and conjunctival polyps epithelial cells as compared to that in normal conjunctival epithelial cells. Our results were highly consistent with the anatomical features that . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint viruses enter the host via the conjunctival route. Conjunctiva exposed to the environment is easily overlooked. Thus, subjects with diseased conjunctivae may be more susceptible to COVID-19 infection via conjunctival epithelial cells; therefore, individuals with diseased conjunctivae should use protective eyewear. Since medical staffs are in contact with patients at a close-range during examination, protective goggles are required to be worn, especially by ophthalmologists.

We conclude that ACE2 is present in human conjunctival tissue, especially in conjunctival epithelial cells, which might provide a possible route of entry for the COVID-19 virus. Abundant ACE2 is expressed in diseased conjunctival tissue; therefore, subjects with diseased conjunctivae may be more susceptible to COVID-19.

Collectively, clinical features of reported COVID-19 patients combined with our results indicate that COVID-19 is likely to be transmitted through the conjunctiva.

Future studies have to elucidate whether COVID-19 was bind to a co-receptor in addition to ACE2.

(COVID-19).

February [16] [17] [18] [19] [20] [21] [22] [23] [24] 2020 . https://www.who.int/docs/ default-source/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf.

[6] Jun ISY, Hui KKO, Songbo PZ. Perspectives on Coronavirus Disease 2019

Control Measures for Ophthalmology Clinics Based on a Singapore Center . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint

Clinical Characteristics of Coronavirus Disease 2019 in China

Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore

Health security capacities in the context of COVID-19 outbreak: an analysis of International Health Regulations annual report data from 182 countries

Report of the WHO-China Joint Mission on Coronavirus Disease

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus

SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet

Profiling ACE2 expression in colon tissue of healthy adults and colorectal cancer patients by single-cell transcriptome analysis

The digestive system is a potential route of 2019-nCov infection: a bioinformatics analysis based on single-cell transcriptomes

Epub ahead of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted

Ocular tropism of respiratory viruses

Rapid detection by immunofluorescence of multiple viral infections in patients with keratitis

A large outbreak of epidemic keratoconjunctivitis: problems in controlling nosocomial spread

The severe acute respiratory syndrome

Avian influenza and sialic acid receptors: more than meets the eye? Lancet Infect Dis

2019-nCoV transmission through the ocular surface must not be ignored

Peking University Hospital Wang Guangfa disclosed treatment status on Weibo and suspected infection without wearing goggles

Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection

The authors declare that they have no competing interests.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. In the conjunctival nevi (E), strong staining can be seen in the melanocytes (arrow-head) and eccrine sweat gland cells (double arrow). Results are representative of specimens. The specimens were incubated with polyclonal rabbit anti-ACE2 primary antibody.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 23, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted May 23, 2020. . https://doi.org/10.1101/2020.05.21.20109652 doi: medRxiv preprint