Regulation of SARS-CoV2 viral entry-related proteins in human ocular surface epithelium

Purpose : Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). While the ocular surface is considered one of the major SARS-CoV2 transmission routes, SARS-CoV2 specific tropism in ocular epithelial cells is not fully understood. In the current study, we evaluated the expression of two SARS-CoV2 viral entry proteins, ACE2 and TMPRSS2, in human ocular epithelial cells. Additionally, we investigated the role of an eye-specific transcription factor PAX6 in regulating the expression of ACE2 and TMPRSS2. Methods : Human donor corneas were obtained from the Saving Sight eye bank, Kansas City, MO. Flow cytometry was performed to detect ACE2 and TMPRSS2 in ABCB5-positive LSCs and ABCB5-negative limbal epithelial cells. RNAseq data of conjunctival swab samples were obtained from GSE135455 Gene Expression Omnibus datasets, and Spearman's rank correlation coefficients between PAX6, ACE2 and TMPRSS2 were calculated using R version 4.0.3. RNAseq analyses of PAX6 overexpressing oral mucosal epithelial cells were obtained from the DNA Data Bank of Japan Sequence Read Archive (DRA002960). PAX6 knockdown (KD) was performed in cultured immortalized conjunctival epithelial cells using five distinct siRNAs. PAX6, ACE2, and TMPRSS2 expression was analyzed by qRT-PCR and Western blotting. Results : Flow cytometry analyses revealed enhanced expression of ACE2 and TMPRSS2 in PAX6 low-expressing ABCB5-positive LSCs compared to PAX6 high-expressing ABCB5- negative limbal epithelial cells. Intriguingly, TMPRSS2 mRNA expression negatively correlated with PAX6 in conjunctival swab samples (R=-0.48, p<0.0001). Mechanistically, PAX6 overexpression in oral mucosal epithelial cells decreased TMPRSS2 mRNA levels by 76.8%, while PAX6 KD in immortalized conjunctival epithelial cells resulted in induction of TMPRSS2 RNA and protein expression suggesting a possible functional role of PAX6 in regulating TMPRSS2. Conclusions : High expression of ACE2 and TMPRSS2 in LSCs raises the possibility of specific COVID-19 cellular tropism to this cell population. Based on the here reported novel role of PAX6 as a negative regulator of TMPRSS2 expression, lower levels of PAX6 might contribute to the induction of TMPRSS2 in LSCs. Our study points to the importance of COVID-19 testing of donor corneas before clinical transplantation to patients with limbal stem cell deficiency.