Elevated proinflammatory cytokine levels in the brain of Irf3R278Q/R278Qmice upon HSV-1 infection. (A–F) Expression of Ifnb, Mx1, Tnfa, Il1b, Il6, and Ccl2 in brain stems from WT or Irf3R278Q/R278Q mice on days 1–4 after infection, measured by RT-qPCR. UI, uninfected control. WT, n = 5–7; Irf3R278Q/R278Q, n = 5–12. (G–J) Mesoscale analysis of cytokine levels (TNFα, CCL2, and IFNγ) (G–I) and HSV-1 titer in brain stem homogenates on day 5 after infection quantified by plaque assay (J) (WT UI, n = 4, WT, n = 9; Irf3R278Q/R278Q UI, n = 4; Irf3R278Q/R278Q, n = 8). (K) Expression of Tnfa in brain stems from WT (n = 7), Irf3−/−(n = 5), Irf3R278Q/R278Q (n = 9), and Irf3WT/R278Q (n = 6) mice on day 5 after HSV-1 infection, measured by RT-qPCR. (L–N)Ifnb, Tnfa, and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with 0.5 μM PLX5622 (PLX) for microglia depletion. (O and P)Tnfa and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with NF-κB inhibitors BMS-345541 (2 μM), PDTC (25 μM), or saline. UI controls were included. (Q and S)Ifnb and Tnfa mRNA levels in murine microglia from WT, Irf3WT/R278Q, and Irf3R278Q/R278Q mice 24 h after HSV-1 infection at increasing virus MOI as indicated. (R and T)IFNB1 and TNFA mRNA levels in iPSC-derived microglia from the IRF3WT/R285Q HSE patient and controls 24 h after HSV-1 infection at increasing virus MOI as indicated. Expression data were normalized to β-actin and shown as fold change compared with the UI control. All in vitro experiments were performed in triplicates and independently repeated at least three times. Cytokine expression kinetics were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype) (A–F), error bars; SEM. Statistical analyses of cytokine and virus levels in brain stem homogenates (G–J and L–T) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. CNS cell experiments (K) were analyzed with a two-tailed one-way ANOVA for difference of means followed by an unpaired t test of means. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. UT, untreated; MOI, multiplicity of infection.
Figure 3.

Elevated proinflammatory cytokine levels in the brain of Irf3 R278Q/R278Q mice upon HSV-1 infection. (A–F) Expression of Ifnb, Mx1, Tnfa, Il1b, Il6, and Ccl2 in brain stems from WT or Irf3R278Q/R278Q mice on days 1–4 after infection, measured by RT-qPCR. UI, uninfected control. WT, n = 5–7; Irf3R278Q/R278Q, n = 5–12. (G–J) Mesoscale analysis of cytokine levels (TNFα, CCL2, and IFNγ) (G–I) and HSV-1 titer in brain stem homogenates on day 5 after infection quantified by plaque assay (J) (WT UI, n = 4, WT, n = 9; Irf3R278Q/R278Q UI, n = 4; Irf3R278Q/R278Q, n = 8). (K) Expression of Tnfa in brain stems from WT (n = 7), Irf3−/−(n = 5), Irf3R278Q/R278Q (n = 9), and Irf3WT/R278Q (n = 6) mice on day 5 after HSV-1 infection, measured by RT-qPCR. (L–N)Ifnb, Tnfa, and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with 0.5 μM PLX5622 (PLX) for microglia depletion. (O and P)Tnfa and Il6 response 16 h after infection with 1.0 MOI HSV-1 in MBCs treated with NF-κB inhibitors BMS-345541 (2 μM), PDTC (25 μM), or saline. UI controls were included. (Q and S)Ifnb and Tnfa mRNA levels in murine microglia from WT, Irf3WT/R278Q, and Irf3R278Q/R278Q mice 24 h after HSV-1 infection at increasing virus MOI as indicated. (R and T)IFNB1 and TNFA mRNA levels in iPSC-derived microglia from the IRF3WT/R285Q HSE patient and controls 24 h after HSV-1 infection at increasing virus MOI as indicated. Expression data were normalized to β-actin and shown as fold change compared with the UI control. All in vitro experiments were performed in triplicates and independently repeated at least three times. Cytokine expression kinetics were compared between the groups using a mixed-effects analysis with Geisser-Greenhouse correction for multiple interacting variables (time and genotype) (A–F), error bars; SEM. Statistical analyses of cytokine and virus levels in brain stem homogenates (G–J and L–T) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. CNS cell experiments (K) were analyzed with a two-tailed one-way ANOVA for difference of means followed by an unpaired t test of means. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. UT, untreated; MOI, multiplicity of infection.

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