Conditional reprogrammed human limbal epithelial cell model for anti-SARS-CoV-2 drug screening

Abstract

To minimize the global pandemic COVID-19 spread, understanding the possible transmission routes of SARS-CoV-2 and discovery of novel antiviral drugs are necessary. We describe here that the virus can infect ocular surface limbal epithelial, but not other regions. Limbal supports wild type and mutant SARS-CoV-2 entry and replication depending on ACE2, TMPRSS2 and possibly other receptors, resulting in slight CPE and arising IL-6 secretion, which symbolizes conjunctivitis in clinical symptoms. With this limbal model, we have screened two natural product libraries and discovered several unreported drugs. Our data reveal important commonalities between COVID-19 and ocular infection with SARS-CoV-2, and establish an ideal cell model for drug screening and mechanism research.

Keywords: COVID-19; Cell model; Drug screening; Limbal.

Fig. 1

Expression of ACE2 and TMPRSS2 in primary human epithelial cells isolated from human eyes. A. Three limbal epithelial cells (HNCEC/HL-008, HNCEC/HL01038 and HNCEC/HL01049), two human normal bronchial epithelial cells (HAWEC-02014 and 02032), two lacrimal sac epithelial cells (HNLSEC-1 and 2), one lacrimal gland epithelial cell (HNLGEC-1), one nasolacrimal duct epithelial cell (HNNEC-1), one conjunctiva epithelial cell (HNCoEC-463) and one retina (HNRPE-2) were fixed with 4 % PFA and then strained with antibodies against human ACE2 and TMPRSS2. Scale bar: 100 μm. B. qRT-PCR for ACE2, TMPRSS2 and GAPDH with total RNA extracted from epithelial cells. Significance analysis data was showed in Supplementary Materials. *adjusted p < 0.05, **adjusted p < 0.01, ***adjusted p < 0.001, ****adjusted p < 0.0001 (Student t-test).

Fig. 2

Sensitivity of conditional reprogrammed human limbal epithelial cells and human airway epithelial cells to SARS-CoV-2 infection. A and B. Conditional reprogrammed human limbal epithelial cells and human airway epithelial cells were infected with WIV-04 strain of SARS-CoV-2 for 4 days. The transcript of SARS-CoV-2 spike gene in supernatant and the expression of SARS-CoV-2 nucleocapsid (NP) in cells were measured by RT-qPCR (A) and Immunofluorescence assay (B) respectively. Scale bar: 200 μm. C. Syncytia formation by fusion of SARS-CoV-2 spike expressing HEK293 cells and ACE2-expressing cells. HNCEC/HL-008, HAWEC-02014 and Vero cells were co-cultured with SARS-CoV-2 Spike-GFP expressing HEK293 or GFP expressing HEK293 cells at rate 2:1 for 96 h, and then fixed with 4 % PFA and strained with DAPI for microscope observation Scale bar: 200 μm.

Fig. 3

Susceptibility of SARS-CoV-2 variants infection of HNCEC/HL-008. A. HNCEC/HL-008 infected with mutant strains for 4 days, detected SARS-CoV-2 subgenomic spike transcript in supernatant by RT-qPCR and plotted replication curves. B. Cells in A were immunoblotted with antibody against nucleocapsid of SARS-CoV-2at dpi. 2 and dpi. 3, and photographed by fluorescence microscope at Objective 4x. Scale bar:500 μm. C. Cells in A were immunoblotted with antibody against nucleocapsid of SARS-CoV-2 and strained with DAPI for fluorescence microscope observation at dpi.4 (Objective 10x). The expression of nucleocapsid (SARS-CoV-2) and spike (SARS-CoV-2) were analyzed with Western Blot. The full, non-adjusted images were displayed in the supplementary material 2 (Figure.1). Scale bar: 200 μm.

Fig. 4

SARS-CoV-2 infection of HNCEC/HL-008 induces IL-6 secretion. A and B HNCEC/HL-008 cells were infected with WIV-04 strain or mutant strains of SARS-CoV-2 at different MOI, then detected supernatant IL-6 (A) and IL-8 (B) by ELISA. Significance analysis data was showed in Supplementary Materials. *adjusted p < 0.05, **adjusted p < 0.01(Student t-test).

Fig. 5

Antiviral effect of five reported positive drugs in different cell models. A. Vero (right), HAWEC-02014 (middle)and HNCEC/HL-008 (left) cells were pretreated with diluted drugs for 2 h before viral infection, then incubated with Delta virus for 48 h (Vero) or 96 h (HAWEC-02014 and HNCEC/HL-008). The inhibition rate was analyzed by qRT-PCR of spike transcript in supernatant and graphed with GraphPad Prism 9.0. B. Cells were incubated with drugs for 48 h (Vero) or 96 h (HAWEC-02014 and HNCEC/HL-008), then measured the cell viability by Cell Counting Kit-8 and graphed with GraphPad Prism 9.0. The IC₅₀ and CC₅₀ of five positive drugs in Vero, HAWEC-02014 and HNCEC/HL-008 were shown.

Fig. 6

Inhibition of SARS-CoV-2 infection by IFN-α1 (type Ⅰ), IFN-γ (type Ⅱ) and IFN-λ1 (type Ⅲ) interferons in different cell models. Vero, HAWEC-02014 and HNCEC/HL-008 cells were pretreated with diluted recombinant human IFN-α1,IFN-γ or IFN-λ1 and infected at MOI of 0.05 (Delta strain). The expression of SARS-CoV-2 spike glycoprotein, nucleocapsid, and GAPDH was evaluated by Western Blot at dpi.2 (Vero cells) or dpi.4 (HAWEC-02014 and HNCEC/HL-008 cells). The full, non-adjusted images were displayed in the supplementary material 2 (Figure.2, Figure.3 and 4).

Supplementary Fig. 1

CR-lacrimal gland, lacrimal sac, nasolacrimal duct epithelial cells and retina cells were fixed with 4 % PFA and then strained with antibody against p63 and DAPI. Scale bar: 100 μm.

Supplementary Fig. 2

For tissue specific marker evaluation, Cr-cells were fixed with 4 % PFA and strained with different antibodies for microscope observation. HNLGEC-1 was strained with Aquaporin 5 antibody. HNLSEC-1, 2 and HNNEC-1 were strained with CK19 antibody. HNPRE-2 was strained with RPE-65 antibody.

Supplementary Fig. 3

A. Sensitivity of SARS-CoV-2 infection of human primary epithelial cells isolated from globes. Cells were infected with SARS-CoV-2 WIV-04 strain at MOI = 0.01, 0.05, 0.1 and the transcript of viral Spike gene in supernatant was measured each day by qRT-PCR analysis.B. HNCEC/HL-008 cells infected with SARS-CoV-2 WIV-04 strain at MOI = 0.01, 0.05, 0.1 were immunoprobed with antibody against SARS-CoV-2 nucleocapsid at dpi.2, 3, 4 for Immunofluorescence detection. Bright spotting of dpi.4 were presented at the right panel. Scale bar: 500 μm for fluorescent figure, Scale bar: 200 μm for bright spotting.

Supplementary Fig. 4

Tropism of Delta and Omicron BA2.2 to HNCEC/01038 and HNCEC/01049. HNCEC/01038 and HNCEC/01049 cells were infected with SARS-CoV-2 variants Delta and Omicron BA 2.2 at MOI = 0.01, 0.05, 0.1, and then immunoprobed with antibody against SARS-CoV-2 nucleocapsid and strained with DAPI at dpi.2, 3, 4 for Immunofluorescence detection. Scale bar: 200 μm.