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.

Keywords: ACE2; COVID-19; SARS-CoV-2; TMPRSS2; human conjunctiva.

Figure 1

Box‐Plot showing ACE2, CD81, and LDLR expression values of all analyzed conjunctival samples (n = 38). A, Conjunctival samples do not express the SARS‐CoV‐2 receptor ACE2 but CD81 and LDLR which are common receptors for hepatitis C viruses. B, Subgroup analysis demonstrating that ACE2 expression is similarly low in healthy and in diseased conjunctival samples, while CD81 and LDLR expression is increased among all samples. Each dot represents one sample. ACE2, angiotensin‐converting enzyme 2; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus‐2

Figure 2

Representative immunohistochemical images of an ACE2 staining of conjunctival samples with two different monoclonal antibodies (upper row AMAB91262, lower row MAB933). While the kidney tissue shows a strong ACE2 staining, healthy conjunctival samples (n = 8) show a negligible immunoreactivity. For the negative control the primary antibody was omitted. ACE2, angiotensin‐converting enzyme 2

Figure 3

Boxplots showing TMPRSS2, ANPEP, ENPEP, and DPP4 expression values of all analyzed conjunctival samples (n = 38). A, Conjunctival samples express minor quantities of ANPEP and insignificant numbers of TMPRSS2, ENPEP, and DPP4 RNA. B, Subgroup analysis demonstrating that TMPRSS2, ANPEP, ENPEP, and DPP4 expression is similarly low in healthy and in diseased conjunctival samples. Each dot represents one sample. ANPEP, alanyl aminopeptidase; DPP4, dipeptidyl peptidase 4; ENPEP, glutamyl aminopeptidase