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. 2020 Nov 3;33(5):108339. doi: 10.1016/j.celrep.2020.108339

Figure 3.

Figure 3

The IFN-λ Receptor (IFNλR1) Regulates Antiviral ISG Expression and Restricts HSV-1 Growth in Human Corneal Explants

(A) RNA ISH and histological analysis were performed on human donor corneas to determine IFNLR1 mRNA expression in the corneal epithelium. Panels shown are positive control housekeeping gene (left), negative control probe (middle), and IFNLR1 (right). Scale bar, 10 μm. Data are representative of three independent experiments with unique donor corneas. Black arrows indicate positive staining of cells for IFNLR1.

(B–F) Human donor corneas were treated with IFN-λ or PBS vehicle control, and ISG expression from human corneal explants was quantitated using qRT-PCR. Data represent the mean ± SEM of 12 samples, including 4 cornea segments per donor from 3 donor corneas. Results were pooled from two independent experiments.

(G) ZIKV replication in a representative human donor cornea sample treated with anti-IFNλR1 or isotype control antibody. Infectious units of ZIKV were quantitated by focus forming assay. ZIKV replicated in only 2 of 5 human cornea samples under these conditions. Results were analyzed using two-way ANOVA.

(H) HSV-1 replication in human donor corneas treated with anti-IFNλR1 or isotype control antibody. Infectious units of HSV-1 were quantitated at the 3 h eclipse phase and every 24 h thereafter by plaque assay. Data represent the mean ± SEM of n = 12 replicates, including 4 technical replicates per group from 3 corneas. Results in (H) were pooled from three independent experiments and were analyzed using two-way ANOVA.

(I and J) IFNL1 and IFNL2 mRNA expression levels were quantitated using qRT-PCR 24 h after infection with HSV-1. Data represent the mean ± SEM of mock-infected (n = 3) and infected (n = 3) human donor corneas with 4 cornea segments per donor in two independent experiments.

(K) RNA ISH and histological analysis were performed on human donor corneas to determine IFNL1 mRNA expression in the cornea. Scale bar, 10 μm. Data are representative of 10 samples from seven independent donors. Samples from two donors repeatedly did not stain for positive control (or target) and were excluded. Black arrows indicate positive staining of cells for IFNL1.

(L) SARS-CoV-2 replication in human donor corneas samples treated with anti-IFNλR1 or isotype control antibody. SARS-CoV-2 mRNA expression was measured using qRT-PCR. Data represent the mean ± SEM of n = 7–11 replicates, with two to four technical replicates per group from 3 donor corneas. Results in (L) were pooled from two independent experiments and were analyzed by two-way ANOVA.

Results in (B)–(F), (I), and (J) were pooled from two independent experiments and were analyzed using the Mann-Whitney test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. See also Figure S1.