SARS-CoV-2 infects human adult donor eyes and hESC-derived ocular epithelium

Abstract

The SARS-CoV-2 pandemic has caused unparalleled disruption of global behavior and significant loss of life. To minimize SARS-CoV-2 spread, understanding the mechanisms of infection from all possible routes of entry is essential. While aerosol transmission is thought to be the primary route of spread, viral particles have been detected in ocular fluid, suggesting that the eye may be a vulnerable point of viral entry. To this end, we confirmed SARS-CoV-2 entry factor and antigen expression in post-mortem COVID-19 patient ocular surface tissue and observed productive viral replication in cadaver samples and eye organoid cultures, most notably in limbal regions. Transcriptional analysis of ex vivo infected ocular surface cells and hESC-derived eye cultures revealed robust induction of NF-κB in infected cells as well as diminished type I/III interferon signaling. Together these data suggest that the eye can be directly infected by SARS-CoV-2 and implicate limbus as a portal for viral entry.

Keywords: ACE2; COVID-19; NF-κB; SARS-CoV-2; cornea; eye; interferon; limbus; scRNA-seq; stem cells.

Graphical abstract

Figure 1

Limbal region of post-mortem human SARS-CoV-2-positive cadaver donor tissue Post-mortem human ocular surface tissue stained for limbal epithelial marker cytokeratin 15 (K15) and co-staining with antibodies to SARS-CoV-2 (S) and ACE2. Scale bar, 200 μm in tile-scan images. Scale bar, 50 μm in zoomed in images; iso-type controls can be seen in Figure S1. Images are representative from one donor (donor A); images of tissue from two additional donors can be seen in Figure S2 (iso-type controls for Figure S2 can be found in Figures S4A and S4B).

Figure 2

Expression of ACE2, TMPRSS2, and SARS-CoV-2 in primary ocular tissues isolated from adult human eye Post-mortem globes were dissected and cells cultured. (A) Confirmation of cell identity was conducted using antibodies to cell-specific markers keratin 3 (K3) and 12 (K12) for cornea, keratin 15 (K15) and tumor protein p63 (p63) for limbus, STRO-1 for sclera, co-expression of PAX6 and αSMA for iris muscle, co-expression of MITF and OTX2 for RPE, and expression of MITF and negative expression of OTX2 for choroid melanocytes. See also Figure S5A for ISO-type controls. (B) Cells were exposed to SARS-CoV-2 for 24 h, then fixed and stained for antibodies to SARS-CoV-2 (S) and ACE2. See also Figure S5B for ISO-type controls. (C) qRT-PCR for ACE2 on total RNA extracted from healthy ocular tissues; p = 0.0062. (D) Same as (C) for TMPRSS2; p = 0.0018 (Vidal et al., 2015). (E) qRT-PCR for SARS-CoV-2 subgenomic N transcript on total RNA extracted from ocular tissues infected with SARS-CoV-2 as in (B). For (C)–(E), n = 6 for cornea, n = 5 for limbus and sclera, n = 4 for RPE, and n = 3 for iris and choroid. Adjusted p < 0.05 and ^∗∗adjusted p < 0.01. Horizontal bars denote mean ± standard error of the mean.

Figure 3

Whole-transcriptome analysis of human ocular surface cells infected by SARS-CoV-2 (A) Read coverage of the SARS-CoV-2 genome in human donor cornea, limbus, and sclera tissues infected with SARS-CoV-2 (MOI = 1.0, 24 h) and subjected to mRNA sequencing (mRNA-seq). (B) Quantification of reads mapped in (A), indicated as mean reads per million. Error bars indicate SD (n = 3/condition); ^∗∗adjusted p = 0.0024 and ^∗∗∗adjusted p = 0.0001 (one-way ANOVA). (C–E) Differential gene expression of all genes plotted by the log₂ fold change and the statistical significance as −log₁₀ of the adjusted p value the experiment in (A) and (B) in (C) cornea, (D) limbus, and (E) sclera. The ten genes with highest increases in gene expression that also reached statistical significance (adjusted p < 0.05) were named. Horizontal line indicates adjusted p = 0.05, and vertical lines indicate log₂ fold change = ±1.5. (F) Heatmap comparing the differential gene expression of genes that showed increased gene expression upon infection (log₂ fold change > 1.5, adjusted p < 0.05) in at least one of the tissues as in (C)–(E). (G) Venn diagram showing the overlap of genes with increased expression (log₂ fold change > 1.5, adjusted p < 0.05) in response to SARS-CoV-2 infection as in (C)–(F). (H) Relative expression of subgenomic SARS-CoV-2 N gene RNA in infected human donor limbus tissue (MOI = 1.0, 24 h) pre-treated with TPCK or vehicle control (DMSO) quantified by qRT-PCR. Relative expression = 1 is the average expression in the vehicle control (n = 3/condition). See also Table S1.

Figure 4

Zone 3 in the SEAM whole-eye colonies is composed of cells expressing ocular surface ectoderm gene profile (A) Schematic of SEAM culture differentiation and lineages of each SEAM zone. hESC-derived SEAM organoids were differentiated for 55 days, then processed for immunohistochemical analysis single-cell RNA-seq (n = 1 biological replicate). Unbiased clustering of SEAM culture was conducted using the Seurat package (Satija et al., 2015) in R. (B) Cells possessing ocular surface ectoderm annotation were further clustered, presented as UMAP and principal-component analysis (PCA). (C) Heatmap of genes distinguishing each cluster by relative expression. (D) Expression pattern of genes associated with ocular surface ectoderm clusters. (E) Violin plots of known markers of corneal, limbal, conjunctival, and epidermal lineages. See also Figures S6 and S7.

Figure 5

Presumptive corneal cell clusters from SEAM whole-eye colonies express ACE2 and TMPRSS2 (A) Relative expression of ACE2 in corneal clusters from SEAM colonies presented as UMAP and violin plot. (B) ACE2-positive cells evaluated by Jensen TISSUES, Mouse Gene Atlas, and Gene Ontology analyses. (C) Relative expression of TMPRSS2 in corneal clusters from SEAM colonies presented as UMAP and violin plot. (D) TMPRSS2-positive cells evaluated by Jensen TISSUES, Mouse Gene Atlas, and Gene Ontology analyses. (E) Table showing total cell number and percentage of the corneal cells from SEAM colonies expressing potential SARS-CoV-2 targets. (F) Bar graph illustrating the percentage of ACE2, TMPRSS2, and ACE2+TMPRSS2-positive cells quantified by immunofluorescence (IF) staining of zone 3 in SEAM colonies (SEAM differentiated for 31 days). Error bars indicate standard error of mean (SEM) from six images counted, with an average total number of cells of per image of 916.67 ± 91.68 (mean ± SEM). (G) Immunohistochemistry of antibodies targeting ACE2 and TMPRSS2 in SEAM colonies. White arrows indicate double-positive cells. Blue is DAPI staining illustrating nuclei in merged image, and ACE2 and TMPRSS2 are shown in green and red, respectively. Scale bars, 50 μm.

Figure 6

SARS-CoV-2 infection of SEAM whole-eye colonies (A) Read coverage of the SARS-CoV-2 genome from next-generation sequencing of RNA from three different infected SEAM cultures (MOI = 1.0, 24 h). (B) Virus growth in SEAM cultures shown as plaque-forming units per milliliter of supernatant at the time points indicated. Error bars show SD (n = 3). (C) UMAP based on unbiased clustering of integrated scRNA-seq data from a non-infected SEAM culture (healthy [SEAM differentiated for 31 days]) and a SEAM infected with SARS-CoV-2 (MOI = 1.0, 24 h) (infected [SEAM differentiated for 54 days]), resolution 0.75. (D) UMAP of integrated scRNA-seq data colored by origin of data (healthy [blue] versus infected [red]). (E) Feature plot showing the location of cells expressing the SARS-CoV-2 spike gene (S) in red. (F) Zoom in of the cornea and limbal clusters made up of four sub-clusters. (G) Violin plots showing some differences in genes expression between the corneal and limbal sub-clusters used for sub-annotation of cell-type identity. (H) Dot plots showing differential expression of inflammation related genes between the corneal and limbal sub-clusters split by data origin, healthy (blue) versus infected (red). Top: SARS-CoV-2 genes and TNF-α/NF-κB pathway-related gens. Bottom: SARS-CoV-2-stimulated genes.

Figure 7

Genes downregulated in limbus cells after infection with Sars-CoV-2 (A) Venn diagram of genes downregulated after infection with SARS-CoV-2 in primary adult limbus cells (brown) and limbus cell cluster from scRNA-seq of infected SEAM (purple). (B) Log₂ fold change of overlapping downregulated genes after infection with SARS-CoV-2 at 1 MOI for 24 h in limbus cells isolated from human cadaver donors (bulk sequencing, DEseq2) and limbus clusters from SEAM organoid (scRNA-seq, Wilcoxon p < 0.05). (C) Volcano plot showing log₂ fold change of gene expression after infection on the basis of Wilcoxon test between limbus clusters (infected cluster and nearest neighbor cluster). The inset shows the full volcano plot. Genes with log₂ fold change < −1 were labeled. (D) Gene expression analysis on overlapping downregulated genes from BioPlanet2019, GO Biological Processes 2017b, COVID-19 Regulated Gene Sets and MSigDB Hallmark 2020. All databases accessed through Enrichr.