Mortality and pathogenicity of B6 mice subcutaneously infected with JaOArS982 and JaTH160.

<div><p>(A) Mortality and infectivity rates of B6 mice (n=10) following subcutaneous infections with 10⁰, 10², 10⁴ and 10⁶ pfu of JaOArS982 and JaTH160. Mice were recorded for 21 days and no mice died after 21 days. Infectivity was determined by anti-JEV IgG antibody seroconversion for more than 1:1000 of IgG ELISA titer. (B and C) B6 mice were subcutaneously infected with 10⁴ pfu of JaOArS982 (n=30) and JaTH160 (n=15).</p> <p>Survival curves P: Gehan-Breslow-Wilcoxon Test. (C) The averages ratio of weight change of living mice at the time points compared with those of day 0 following subcutaneous infections with 10⁴ pfu of JaOArS982 (n=30) and JaTH160 (n=15). Error bars represent the standard deviations. (D) Viral loads in distinct regions of the CNS following subcutaneous infections with 10⁴ pfu of JaOArS982 (Day 5: n=5, Day 9: n=15) and JaTH160 (Day 5: n=5, Day 9: n=8). P: Mann Whitney test. (E) mRNA levels of TNF-α, IFNγ, IL-2 and IL-10 quantified by real-time PCR in the brain cortex of B6 mice infected with 10⁴ pfu of JaOArS982 (Day 5: n=5, Day 9: n=12), JaTH160 (Day 5: n=5, Day 9: n=5) and mock (n=8). P: Mann Whitney test. (F) Histopathological features of brain cortex in B6 mice infected with 10⁴ pfu of JaOArS982 and JaTH160 at 9 days pi. JEV antigens were detected using E-protein specific JEV antibody (insets). Each experiment represents six and four mice infected with JaOArS982 and JaTH160, respectively. JaOArS982-infected mice showed slight inflammatory infiltration in meninges. In brain cortex, a few degenerated cells were presented (arrow) and were virus antigen-positive cells. In JaTH160-infected mice, severe inflammatory reactions were seen in meninges and perivascular area (asterisks). Many virus antigen-positive cells were detected in degenerated neuronal cells of the cortex (arrows).</p></div

Viral loads and cytokine levels in the brains of severe and mild cases of JaOArS982-infected mice.

<p>(A) Viral loads in the CNS of severe (weight loss: <0.75, n=8) and mild (weight loss: >0.90, n=24) cases of JaOArS982-infected B6 mice at 13 days pi. P: Mann Whitney test. (B) mRNA levels of TNF-α, IL-10, IFNγ, IL-2, IL-4 and IL-5 quantified by real-time PCR in the brain cortex of JaOArS982-infected B6 mice at 13 days pi. Uninfected group (U group, n=8), Severe group: S (n=8), Mild group with high viral load of >10⁶ pfu/g of brain tissue: MH (n=11), Mild group with low viral load of <10⁶ pfu/g of brain tissue: ML (n=13). P: Kruskal-Wallis test, <i>p</i>: Mann Whitney test.</p

Histopathological features of brain cortex in severe and mild cases of JaOArS982-infected B6 mice.

<p>Histopathological features of B6 mice infected with 10⁴ pfu of JaOArS982 at 13 days pi. Each experiment represents five and sixteen mice of severe and mild cases, respectively. JEV antigens were detected using E-protein specific JEV antibody (insets). (A) Severe case (weight ratio: 0.63) showed acute neuronal necrosis in the brain cortex. Virus antigen-positive cells were the necrotic neurons. (B) In a mild case (weight ratio: 0.90), massive inflammatory infiltration was exhibited. Acute necrosis was seen in the area. Some JEV antigen-positive cells were detected. (C) In a mild case (weight ratio: 1.05), focal inflammatory infiltration was seen in the brain cortex (asterisk). JEV antigens were not clearly detected. (D) Mock-infected mice (weight ratio: 1.02).</p

Mortality and pathogenicity of TNF-α and IL-10 KO mice infected with JaOArS982.

<p>(A) Survival curves of IL-10 KO (n=37) and TNF-α KO (n=22) B6 mice following subcutaneous infections with 10⁴ pfu of JaOArS982. WT B6 mice (n=30) show the same data as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071643#pone-0071643-g001" target="_blank">Figure 1B</a>. P: Gehan-Breslow-Wilcoxon Test. (B) Viral loads in the brain cortex of WT (Day 5: n=5, Day 9: n=15, Day 11: n=20), IL-10 KO (Day 5: n=5, Day 9: n=15, Day 11: n=12) and TNF-α KO (Day 5: n=5, Day 9: n=7, Day 11: n=7) mice following subcutaneous infections with 10⁴ pfu of JaOArS982 at 5, 9 and 11 days pi. WT B6 mice at 5 and 9 days pi show the same dataset as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071643#pone-0071643-g001" target="_blank">Figure 1D</a>. (C and D) mRNA levels of cytokines in the brain cortex of WT, TNF-α KO and IL-10 KO mice infected with 10⁴ pfu of JaOArS982 at 9 days pi. The levels of IFNγ, IL-1β, IL-2, IL-4, IL-5 and IL-6D (C) and perforin, granzyme A, granzyme B and FasL (D) were quantified by real-time PCR. WT B6 mice show the same data as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071643#pone.0071643.s001" target="_blank">Figure 1E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071643#pone.0071643.s001" target="_blank">Supplemental Figure 1</a>. P: Kruskal-Wallis test, <i>p</i>: Mann Whitney test.</p

Mortality and pathogenicity of TNF-α and IL-10 KO mice infected with JaTH160.

<p>(A) Survival curves of IL-10 KO (n=15) and TNF-α KO (n=14) B6 mice following subcutaneous infections with 10⁴ pfu of JaOArS982. WT B6 mice (n=15) show the same data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071643#pone-0071643-g001" target="_blank">Figure 1B</a>. P: Gehan-Breslow-Wilcoxon Test. (B) Viral loads in the brain cortex of WT (Day 5: n=5, Day 7: n=9, Day 9: n=8), IL-10 KO (Day 5: n=5, Day 7: n=8, Day 9: n=6) and TNF-α KO (Day 5: n=5, Day 7: n=5) mice following subcutaneous infections with 10⁴ pfu of JaTH160 at 5, 7 and 9 days pi. Data of WT B6 mice at 5 and 9 days pi were same to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071643#pone-0071643-g001" target="_blank">Figure 1D</a>. (C and D) mRNA levels of IFNγ, IL-2, IL-4 and IL-5 in the brain cortex of WT, TNF-α KO and IL-10 KO mice infected with 10⁴ pfu of JaTH160 at 5 (C) and 7 (D) days pi. P: Kruskal-Wallis test, <i>p</i>: Mann Whitney test. (E) Histopathological features of WT, TNF-α KO and IL-10 KO mice infected with 10⁴ pfu of JaTH160 at 9 days pi. JEV antigens were detected using E-protein specific JEV antibody (insets). Each experiment represents four, five and six mice of WT, TNF-α KO and IL-10 KO mice, respectively. The B6 WT and IL-10 KO mice showed severe inflammatory reactions in the brain cortex. On other hand, the TNF-a KO mice exhibited acute necrotic changes with slight inflammatory reactions in the brain cortex.</p

Proposed model of the mechanism of fatal disease following JEV infection in a mouse model.

<p>Proposed model of the mechanism of fatal disease following JEV infection in a mouse model.</p

Neuronal degeneration and inflammation in the brains of TNF-α KO mice infected with JaOArS982.

<p>(A and B) Histopathological features of WT (A) and TNF-α KO (B) mice infected with 10⁴ pfu of JaOArS982 at 10 days pi. JEV antigens were detected using E-protein specific JEV antibody (insets). Each experiment represents six and five mice of WT and TNF-α KO, respectively. Inflammatory reactions and neuronal degeneration were seen in the WT mice. The TNFa KO mice showed acute necrotic changes in the brain cortex. (C) Number of infiltrating leukocytes, T cells (CD3⁺), CD4⁺ T cells, CD8⁺ T cells, B cells (CD19⁺), NK cells (NK1.1⁺) and macrophages including microglia (F4/80⁺) in brains of WT (mock: n=5, Day 10: n=6) and TNF-α KO (mock: n=5, Day 10: n=7). <i>p</i>: Mann Whitney test.</p

TNF-α Acts as an Immunoregulator in the Mouse Brain by Reducing the Incidence of Severe Disease Following Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis