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. 2021 Sep 6;12:735643. doi: 10.3389/fimmu.2021.735643

Figure 7.

Figure 7

Mechanism, specificity and effectiveness of IFN-γ-mediated antiviral action in primary human skin cells. (A) Evaluation of IFN-γ effect on HSV immediate early (IE) gene transcription via RNA FISH. In situ detection and visualization of IE gene expression using pooled probes specific to each of the five IE genes. Primary human keratinocytes 6 h.p.i. with K26 (MOI=1) either mock-treated or pretreated with IFN-γ (10 U/ml or 100 U/ml). Scale bar, 50 µm. (B) Quantitative assessment of IFN-γ-mediated reduction of IE gene expression via qRT-PCR. Primary human fibroblasts infected with wildtype strain KOS, MOI=1, 4 h.p.i. Mock treated compared to IFN-γ treated, 100 U/ml. (C) IFN-γ effect on HSV gene transcription (ICP27, ICP8, and glycoprotein B) in the presence or absence of interferon blockers, GIR208 and B18R. Primary human keratinocytes infected with KOS, MOI=1, 4 h.p.i. (D) Time-dependent inhibition by IFN-γ. Percentage of GFP-expressing cells in K26-infected primary human keratinocytes 16 h.p.i. (MOI=1) with IFN-γ treatment initiated at 2, 4, 8, 16, 24, or 48 hours prior to infection. (E, F) Dose-dependent inhibition by IFN-γ. GFP expression (E) and viral DNA replication (F) under increasing dosage of IFN-γ in K26-infected keratinocytes. MOI=1, 16 h.p.i. HSV genome DNA were quantified by qPCR and compared between 2 and 16 h.p.i. (G) Growth kinetics of strain KOS under low and high MOI in primary human fibroblasts with (100 U/ml) or without IFN-γ. (H) Responsiveness to IFN-γ in IFI16 knockout and Cas9 control human fibroblasts. Immunoblot of whole-cell lysate from IFI16 knockout and Cas9 control cells mock-treated or pretreated with IFN-γ (500U/mL) for 20 hours. One of three biological replicates is shown. (I) Viral yield 12 h.p.i. following wildtype HSV-2 strain 186 infection (MOI=0.1) of IFI16 knockout and Cas9 control fibroblasts mock-treated or pretreated with IFN-γ (500U/mL) for 20 hours. Two-way ANOVA with Holm-Sidak’s multiple comparisons test. Values are mean ± S.D. (error bars), from three independent experiments.