Fig. 3
From: Japanese encephalitis virus neuropenetrance is driven by mast cell chymase
Mast cells increase JEV-induced breakdown of the BBB. a Time course of SA 14-14-2 JEV-induced BBB breakdown detected by EBD leakage into the brain from 1 day to 14 days post-infection. Differences in EBD leakage between WT and Sash mice were observed starting 5 days but also at 7 days and 10 days after infection. EBD concentration was normalized to levels detected in vehicle-treated controls. * denotes P < 0.01 as analyzed by two-way ANOVA, n = 6, representative of three independent experiments. A dot-plot representation of this data is provided in Supplementary Figure 6. b Representative images showing extent of EBD leakage into the brain from a. c Immunofluorescent images comparing ZO-1 staining (red) in brains of uninfected, JEV-infected WT, and JEV-infected Sash mice. Nucleic acid was stained green. Brains were harvested 5 days after i.p. injection of SA 14-14-2 JEV (4 × 107 PFU). Brain endothelial cells of JEV-infected WT mice showed decreased ZO-1 levels compared to uninfected controls and JEV-infected Sash mice. Scale bar denotes 10 μm. d Comparison of EBD leakage into brains of WT, Sash, Sash reconstituted with BMMCs peripherally (Sash-RP), and Sash reconstituted with BMMCs in the CNS (Sash-RC) at 5 days post-i.p. SA 14-14-2 JEV infection (4 × 107 PFU). EBD concentration was normalized to levels detected in vehicle-treated controls and baseline set as 100%. * denotes P < 0.05, as determined by one-way ANOVA with Holm-Sidak’s post-test; n = 6, representative of three independent experiments. e Representative images showing the extent of EBD leakage into the brain from d. f i.c.v Injection of DT (0.5 mg/kg) into Mcpt5-Cre; iDTRfl/fl mice depleted ~80% of MCs in the brain (refer to Supplementary Figure 8a, b). Injection of 4 × 107 PFU of SA 14-14-2 JEV, i.p., in CNS MC-depleted (DT-injected Mcpt5-Cre; iDTRfl/fl) mice resulted in decreased EBD leakage 5 days post-infection, compared to MC-sufficient control mice. EBD leakage was normalized to uninfected controls. * denotes P < 0.05, as analyzed by Student’s unpaired two-tailed t-test; n = 10, representative of two independent experiments. Error bars represent the SEM. g Representative images showing extent of EBD leakage into the brain from f. Both peripheral and CNS MCs contribute to BBB permeability, yet peripheral MCs are sufficient to induce BBB permeability during JEV infection
