Japanese encephalitis virus neuropenetrance is driven by mast cell chymase

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis.


Supplementary Figure 2. UV-inactivated JEV induces human MC degranulation.
Human ROSA cells exposed to UV-inactivated SA14-14-2 JEV degranulate in a concentration-dependent manner, as assessed by by β-hexosaminidase assay, analyzed using 1 way ANOVA with means compared to uninfected control. Error bars represent the SEM.

Supplementary Figure 3. Similar levels of peripheral JEV infection and serum cytokines in WT and MC-deficient mice.
(a) PCR products showing detection of the JEV negative-strand in the brain at day 5 and spleen at day 1 post i.p. infection of mice confirming JEV replication in vivo. Uninfected tissues were used as controls. Representative images from n=5 animals are shown. P.I.= post infection. (b) Plaque assay demonstrating similar levels of infectious virus in the serum of WT and Sash mice 1 day after i.p. infection with SA-14-14-2 JEV. Representative image of serum plaque assay from n=5. Serum of JEV-infected WT and Sash mice had similar levels of multiple cytokines including (c) IL-2, (d) IL-4, (e) IL-10, and (f) IL-17. (g) Serum of Sash mice had higher levels of IL-6 as compared to WT mice at 6h post-infection and similar levels thereafter. * denotes P <0.05. Error bars represent the SEM. Panels c-g were analyzed by 2-way ANOVA with Bonferroni's post-test, n=5 per group, representative of 3 independent experiments. (h) Representative flow cytometry plots demonstrating the positive and negative controls for the cytokine array assay and (i) a histogram presentation of a representative standard curve for the cytokine IFN-γ are provided.

Supplementary Figure 4. JEV burden in the brains of WT and Sash mice.
A dot-plot alternative representation of the data provided in Figure 2g. Error bars represent the SEM.

Supplementary Figure 5. Kinetics of JEV infection after intracerebralventricular injection (i.c.v.) of virus in WT and MC-deficient mice.
JEV genome copies in the brain were detected by real time qPCR in WT and Sash after i.c.v. infection with SA 14-14-2 JEV (1x10 4 PFU). From days 1-5, no significant differences in viral burden were observed but burden was higher in Sash mice on days 7 and 10 post-infection. These results indicate defects in viral clearance in Sash mice when the brain is the site of inoculation. For all groups n=5 and data were analyzed by two way-ANOVA with Holm-Sidak's multiple comparison test to obtain p-values. Error bars represent the SEM. Data are compiled from two independent experiments. ** denotes P <0.01 and *** denotes P <0.001.

Supplementary Figure 6. Peripheral JEV infection leads to increased BBB leakiness.
Alternative representation of Figure 3a showing a dot plot and mean±SEM. Graph depicts a time course of SA 14-14-2 JEV-induced BBB breakdown, detected by EBD leakage into the brain from 1 to 14 days after infection. EBD concentration was normalized to levels detected in vehicle treated controls. ** denotes P <0.01 as analyzed by two-way ANOVA with Bonferroni's post-test; n=6. Error bars represent the SEM and data are representative of 3 independent experiments.  Figure 5e showed that bEND.3 cells exposed to JEV-activated supernatant demonstrated reduced (a) claudin-5, (b) ZO-1, (c) ZO-2, and (d) occludin levels when compared to media, JEV, and resting MC-stimulated controls. Chymase inhibition with TY-51469 at one or both doses (100uM and 200uM) reduced the breakdown of these TJ proteins. Protein levels were normalized to GAPDH. *denotes P <0.05, **denotes P <0.01, and ***denotes P <0.001, as analyzed by individual groups to controls from 3 independent experiments by Student's unpaired t-test. Error bars represent the SEM.

Supplementary Figure 12.
Chymase inhibitor TY-51469 treatment specifically inhibits chymase activity but not tryptase activity or histamine concentration in WT mice. (a) Serum of WT mice had increased chymase activity as compared to Sash mice. Inhibition of chymase with TY-51469 led to reduced chymase activity to level of Sash mice. Serum of WT mice showed increased (b) tryptase activity and (c) histamine concentration as compared to Sash mice. *denotes P<0.05, **denotes P<0.01, ***denotes P<0.001, as analyzed by individual Student's unpaired t-tests. N=5 mice, representative of 3 independent experiments. Error bars represent the SEM. Treatment with TY-51469 did not alter the activity of tryptase or concentration of histamine.