Expression of the COVID‐19 receptor ACE2 in the human conjunctiva

Abstract SARS‐CoV‐2 is assumed to use angiotensin‐converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory‐confirmed severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), and there has been speculation that SARS‐CoV‐2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high‐throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS‐CoV‐2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS‐CoV‐2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median: 0.0 transcripts per million [TPM], min: 0.0 TPM, max: 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS‐CoV‐2 via these mediators unlikely.

by inhaling droplets or aerosols released by an infected individual and possibly by a feco-oral transmission route. 1 Recent studies have described "conjunctival congestion" in 9 of 1099 patients (0.8%) with laboratory-confirmed SARS-CoV-2 2 and some clinicians have speculated that the disease can be transmitted through the conjunctiva [3][4][5] highlighting the need for further investigations on transmission pathways.
Similar to other coronaviruses, SARS-CoV-2 uses angiotensinconverting enzyme 2 (ACE2) protein to gain entry into cells. 6,7 Since the outbreak, many studies described ACE2 expression across human tissues, including lung, stomach, ileum, colon, liver, and kidney, 8,9 supporting the clinical observation that SARS-CoV-2 can infect multiple organs. Interestingly, Zou et al 9 reported that alveolar type 2 cells (the proposed main target cell of SARS-CoV-2) in the lung actually expressed rather low levels of ACE2. It was, therefore, speculated that SARS-CoV-2 infection may depend on coreceptors or other auxiliary membrane proteins. 8 For example, Hoffmann et al 6 demonstrated that SARS-CoV-2 uses ACE2 for entry and requires the cellular protease TMPRSS2 for priming. Furthermore, potential candidate coreceptors were suggested to facilitate virus entry including glutamyl aminopeptidase (ENPEP), alanyl aminopeptidase (ANPEP), and dipeptidyl peptidase 4 (DPP4), 8 the latter two being established coreceptors for the human coronavirus (hCov) 229E 10 and hCov-EMC. 11 Currently, it is not clear whether conjunctival epithelia express ACE2 or potential auxiliary proteins and coreceptors such as TMPRSS2, ANPEP, DPP4, and ENPEP.
In view of the lacking data, this study aims to explore the expression levels of ACE2 and its potential coreceptors such as ANPEP, DPP4, ENPEP, and TMPRSS2 in transcriptome data of conjunctival samples. We show that ACE2 and its potential coreceptors are not significantly expressed in the human conjunctiva, which suggests a very low probability of SARS-CoV-2 propagation in the conjunctiva.

| Patients
To obtain information on the transcription of ACE2 and associated molecules required for cell entry by SARS-CoV-2, existing datasets of 38 conjunctival samples from 38 patients were included in this study.
The samples comprised twelve healthy conjunctival tissue specimens from twelve subjects who underwent buckle or 20-gauge vitrectomy surgery for retinal detachment as well as twelve conjunctival melanoma, seven conjunctival squamous cell carcinoma and seven conjunctival papilloma specimens that had been treated at the Eye Centre of the University Freiburg from 1996 to 2017. Another eight healthy conjunctival samples from eight subjects undergoing retinal detachment surgery were included for immunohistochemical staining. All specimens contained conjunctival epithelium and subconjunctival connective tissue. All tissue samples were analyzed in an anonymized manner. Institutional Review Board (IRB)/Ethics Committee approval had been obtained for specimen acquisition, use, and data generation.
2.2 | Tissue processing, library preparation, and sequencing Formalin fixation and paraffin embedding of ocular samples were performed immediately after tissue excision according to routine protocols, as previously described 12,13 Following routine histological staining, each specimen's histological diagnosis was made by two experienced ophthalmic pathologists. Fifteen 4-µm-thick FFPE conjunctival sections were collected and stored in tubes before RNA extraction. RNA isolation from FFPE specimens was carried out as previously described. 12 Briefly, total RNA was extracted from FFPE samples using the Quick-

| Immunohistochemistry
ACE2 immunohistochemistry was performed as previously described 12,21 . In brief, slides were exposed to citrate buffer at 95°C in a steamer for 30 minutes to achieve antigen recovery.

| Patient characteristics
In total, the transcriptome of 38 samples from 38 patients was analyzed in this study. Furthermore, eight healthy conjunctival samples from eight patients were included for immunohistochemistry. A detailed summary of the patient's characteristics is summarized in Table 1.  Figure 1A,B) which represent the targets of hepatitis C viruses for which a transconjunctival transfection has been reported. 21 Immunohistochemical staining of eight healthy conjunctival samples confirmed a negligible ACE2 expression in all analyzed samples ( Figure 2). In contrast to a strong staining in the kidney, none of the analyzed conjunctival samples revealed a significant ACE2 staining. Both primary antibodies used in this study, which bind different ACE2 epitopes, showed a very similar staining pattern thus reinforcing the results. Interestingly, the MAB933 antibody showed an incidental staining of a few goblet cells in four out of eight samples which were less pronounced when using the AMAB91262 antibody.  Figure 3B).

| DISCUSSION
The possible transmission of SARS-CoV-2 through the conjunctiva is controversial and has significant public health implications. Some recent reports have speculated that SARS-CoV-2 can be transmitted via the mucous membranes including the conjunctiva, [3][4][5]   transporting the virus through the nasolacrimal drainage system towards the nasopharynx. In addition to an effective mask that covers the mouth and nose, protective goggles can cover the narrow gap below the top of a mask due to the uneven surface at the base of the nose, thereby reducing the likelihood of pathogen exposure. 29 In addition, potential currently unidentified alternative SARS-CoV-2 receptors may be present in the conjunctiva, which could still allow SARS-CoV-2 infection through the conjunctiva.

ACKNOWLEDGMENTS
We thank Sylvia Zeitler and Brigitte Joos for their excellent technical assistance.