The hIRF3 R285Q/mIRF3 R278Q mutation impairs type I IFN responses in microglia and confers susceptibility to HSE-like disease. (A) Human iPSC-derived microglia, neurons, and astrocytes were generated from patient fibroblasts or control iPSC. Created with BioRender. (B–D)IFNB1 expression in (B) microglia, (C) astrocytes, and (D) cortical neurons 24 h after infection with HSV-1 at MOI 1.0. HSE pt., HSE patient. (E) Alignment of human and murine IRF3 around the region harboring R285 in WT human IRF3. (F–I)Ifnb1 and Isg15 expression after HSV-1 infection in murine microglia and neurons from WT and transgenic mice carrying the IRF3 R278Q mutation. Microglia (F and G) and neurons (H and I) 24 h after infection with HSV-1 at MOI 1.0. All in vitro experiments were performed in triplicates and independently repeated at least three times. Expression data were normalized to β-actin and shown as fold change compared with the UI control. (J–O) Mice were infected in the cornea with HSV-1 McKrea (2 × 106 PFU/eye), and HSE-like disease development was followed over time until reaching humane endpoint or recovering 100% of starting weight. (J) % weight change. (K) Symptom score. (L) Survival curve (UI, n = 7; WT, n = 15; Irf3WT/R278Q, n = 15; Irf3R278Q/R278Q, n = 16; Irf3−/−n = 10). Dead animals were censored in the graphs and thus represented in the graphs with weight and symptom score at time of death. (M) Representative MR images performed on day 5 after infection. Red dotted line and white arrows indicate lesions. (N) Lesion volumes quantified blinded. (O and P) BBB disruption/integrity was assessed visibly by Evans blue perfusion of mice 5 days after HSV-1 infection. Representative microscope images of Evans blue dye leakage in brain stems from UI and HSV-1–infected WT and IRF3R278Q/R278Q mice were obtained from (O) uncut ventral position (2× objective) and (P) coronal slides cut in 5 mm thickness (3.2× objective). Red circles indicate area of Evans blue passive diffusion into lesion sites. n = 3–7 mice per group. In vivo survival experiments were independently repeated three times, and MR-imaging experiment was repeated two times. Statistical analyses of cell culture experiments (B–D and F–I) were analyzed by two-tailed two-way ANOVA for difference of means, followed by two-tailed unpaired t test of means, error bars; SD. Disease development (weight change and symptom score) were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Survival was analyzed using log-rank Mantel–Cox test (L). Error bars; SEM. Lesion volumes (N) were analyzed by two-tailed one-way ANOVA followed by unpaired t test, error bars; SD. P values <0.05 were considered statistically significant, **P < 0.01, and ***P < 0.001.
Figure 1.

The hIRF3 R285Q/mIRF3 R278Q mutation impairs type I IFN responses in microglia and confers susceptibility to HSE-like disease. (A) Human iPSC-derived microglia, neurons, and astrocytes were generated from patient fibroblasts or control iPSC. Created with BioRender. (B–D)IFNB1 expression in (B) microglia, (C) astrocytes, and (D) cortical neurons 24 h after infection with HSV-1 at MOI 1.0. HSE pt., HSE patient. (E) Alignment of human and murine IRF3 around the region harboring R285 in WT human IRF3. (F–I)Ifnb1 and Isg15 expression after HSV-1 infection in murine microglia and neurons from WT and transgenic mice carrying the IRF3 R278Q mutation. Microglia (F and G) and neurons (H and I) 24 h after infection with HSV-1 at MOI 1.0. All in vitro experiments were performed in triplicates and independently repeated at least three times. Expression data were normalized to β-actin and shown as fold change compared with the UI control. (J–O) Mice were infected in the cornea with HSV-1 McKrea (2 × 106 PFU/eye), and HSE-like disease development was followed over time until reaching humane endpoint or recovering 100% of starting weight. (J) % weight change. (K) Symptom score. (L) Survival curve (UI, n = 7; WT, n = 15; Irf3WT/R278Q, n = 15; Irf3R278Q/R278Q, n = 16; Irf3−/−n = 10). Dead animals were censored in the graphs and thus represented in the graphs with weight and symptom score at time of death. (M) Representative MR images performed on day 5 after infection. Red dotted line and white arrows indicate lesions. (N) Lesion volumes quantified blinded. (O and P) BBB disruption/integrity was assessed visibly by Evans blue perfusion of mice 5 days after HSV-1 infection. Representative microscope images of Evans blue dye leakage in brain stems from UI and HSV-1–infected WT and IRF3R278Q/R278Q mice were obtained from (O) uncut ventral position (2× objective) and (P) coronal slides cut in 5 mm thickness (3.2× objective). Red circles indicate area of Evans blue passive diffusion into lesion sites. n = 3–7 mice per group. In vivo survival experiments were independently repeated three times, and MR-imaging experiment was repeated two times. Statistical analyses of cell culture experiments (B–D and F–I) were analyzed by two-tailed two-way ANOVA for difference of means, followed by two-tailed unpaired t test of means, error bars; SD. Disease development (weight change and symptom score) were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype). Survival was analyzed using log-rank Mantel–Cox test (L). Error bars; SEM. Lesion volumes (N) were analyzed by two-tailed one-way ANOVA followed by unpaired t test, error bars; SD. P values <0.05 were considered statistically significant, **P < 0.01, and ***P < 0.001.

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