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. 2016 Dec 12;213(13):2931–2947. doi: 10.1084/jem.20160303

Figure 3.

Figure 3.

Dysregulation of type I IFN signaling during SVN infection is not the cause of accelerated disease and death in Irf2−/− mice. (A) Serum IFN-α levels in WT and Irf2−/− mice on days 0–7 p.i. were measured by ELISA during SVN infection. Between three and six infected mice per genotype per time point were tested. Because 70% of the Irf2−/− mice succumbed to SVN infection between days 6 and 8 p.i., serum samples from less than three mice were tested on days 6 and 7 p.i. The dotted line indicates the detection limit of the ELISA (concentration of the lowest standard), and error bars represent SD. P-values were determined by the unpaired, two-tailed Student’s t test. *, P = 0.0167; **, P = 0.0073. (B) Protein concentrations of MCP-1 and IL-6 were measured by cytometric bead array. The right halves of the brains from three to four mice per genotype at baseline or on days 1, 3, and 5 p.i. were harvested and homogenized for measurement of inflammatory cytokines. Error bars represent SD. Two-way ANOVA test: MCP-1, P < 0.0001; IL-6, P < 0.05. Bonferroni posttests: ****, P < 0.0001. (C) Mice were infected i.p. with SVN and injected i.p. with Evans blue (EB) dye to measure BBB permeability on day 2, 3, or 5 p.i. Evans blue cannot cross the BBB unless there is a breach. Brains were harvested and homogenized 3 h after dye injection, and fluorescence present in the homogenates was measured spectrophotometrically. Between five and six mice per genotype served as uninfected controls or were infected, injected with the dye at various time points p.i., and harvested in a total of 11 independent experiments. Between two and four SVN-infected mice per genotype were not injected with the dye but instead harvested for measurement of background fluorescence. RU, relative units. (D) mRNA levels of the indicated ISGs in the brains of WT and Irf2−/− mice were measured by RT-qPCR. The right halves of the brains from three to four mice per genotype at baseline or on days 1, 3, and 5 p.i. were harvested and homogenized for RNA extraction. ISG mRNA levels present in the brains of the five WT mice with high CNS viral titers (Fig. 2 D) were also determined. Fold-changes normalized to baseline WT mice for all ISGs tested are shown in the table. ISG mRNA fold-changes with significant differences between the WT and Irf2−/− mice were determined using a two-way ANOVA test (IFITM3, P < 0.05; IFIT1, P < 0.05) and Bonferroni posttests (IFITM3 on day 5 p.i., P < 0.01; IFIT1 on day 5 p.i., P < 0.01; MX1 on day 5 p.i., P < 0.05; protein kinase R [PKR] on day 5 p.i., P < 0.01; IRF1 on day 5 p.i., P < 0.05). pos, positive. (E) Mortality of mice treated with 500 µg or 1 mg IFNAR1-blocking antibody or 500 µg isotype control antibody by i.p. route 1 d before or 2 d after i.p. infection with SVN. A total of 9–11 Irf2−/− and 7–10 WT mice were treated with IFNAR1-blocking antibody or isotype control and infected with SVN, divided among nine independent experiments. The p-values for survival curves were determined by the log-rank test. Only the significant differences are shown (isotype vs. IFNAR1 500 µg day −1 Irf2−/−, P = 0.0044; isotype vs. IFNAR1 500 µg 2 d p.i. Irf2−/−, P = 0.0436; isotype vs. IFNAR1 1 mg day −1 Irf2+/+, P = 0.0479).