TLR signaling controls lethal encephalitis in WNV-infected brain

Abstract

Toll-like receptors (TLRs) are known to be activated in Central Nervous System (CNS) viral infections and are recognized to be a critical component in innate immunity. Several reports state a role for particular TLRs in various CNS viral infections. However, excessive TLR activation was previously reported by us in correlation with a pathogenic, rather than a protective, outcome, in a model of SIV encephalitis. Here we aimed at understanding the impact of TLR-mediated pathways by evaluating the early course of pathogenesis in the total absence of TLR signaling during CNS viral infections. We utilized a mouse model of sublethal West Nile virus (WNV) infection. WNV is an emerging neurotropic flavivirus, and a significant global cause of viral encephalitis. The virus was peripherally injected into animals that simultaneously lacked two key adapter molecules of TLR signaling, MyD88 and TRIF. On day 2 pi (post infection), MyD88/Trif-/- mice showed an increased susceptibility to WNV infection, and revealed an impairment in innate immune cytokines, when compared to wild type mice (WT). By day 6 pi, there was an increase in viral burden and robust expression of inflammatory cytokines as well as higher cell infiltration into the CNS in MyD88/Trif-/-, when compared to infected WT. A drastic increase in microglia activation, astrogliosis, and inflammatory trafficking were also observed on day 6 pi in MyD88/Trif-/-. Our observations show a protective role for TLR signaling pathways in preventing lethal encephalitis at early stages of WNV infection.

Keywords: MyD88; Toll-like receptors (TLRs); Trif; West Nile Virus.

Figure 1

Survival and viral burden analyses of wild-type C57BL/6 and MyD88/Trif−/− mice following WNV infection. (A) Survival curve of WT C57BL/6 (n=26) and MyD88/Trif−/− (n=17) mice infected with 1×10⁷ PFU of WNV Eg101 strain. One hundred percent of MyD88/Trif−/− mice succumbed to the infection by 8 days after infection (p<0.0001, Logrank test). (B) There was a positive correlation between WNV PFU and WNV RNA levels in the brain of WT and MyD88/Trif −/− mice; p<0.01, r= 0.95 (Two-tailed Pearson r correlation test). WNV RNA levels from (C) spleen and (D) brain were determined by quantitative RT-PCR and expressed relative to 18 S at 2, 4, 6 and 8 days after infection. RT-PCR was performed in triplicate at each time point per group. Error bars represent +/− SD from the mean value. *: p<0.05.

Figure 2

Expression of relevant TLRs in the brain of WNV infected WT and MyD88/Trif−/− mice. Quantitative RT-PCR analyses of TLRs mRNA levels in the brain of C57BL/6 WT and MyD88/Trif−/− were measured in uninfected as well as 2 and 6 days after infection with 1×10⁷ PFU of WNV. Total RNA was analyzed for the expression of (A) TLR3, (B) TLR4, (C) TLR7 and (D) TLR9. The data are expressed as log ₂ of ddCT ± SEM of mRNA copies, normalized to the expression of GAPDH. Experiments were performed in 5 animals per group, and in triplicates at each time point. * p<0.05; (ANOVA, Bonferroni test).

Figure 3

Transcriptional levels of cytokines in the brains of WNV infected WT and MyD88/Trif−/− mice in uninfected, or 2 and 6 days after infection. Quantitative RT-PCR analyses of cytokine mRNA levels in the brain of C57BL/6 WT and MyD88/Trif−/− were measured in uninfected as well as 2 and 6 days after infection with 1×10⁷ PFU of WNV. Total RNA was analyzed for the expression of (A) IFNα, (B) TNFα and (C) IL-6. The data are expressed as log 2 of ddCT ± SEM of mRNA copies, normalized to the expression of GAPDH. Experiments were performed in 5 animals per group, and in triplicates at each time point. * p<0.05; (ANOVA, Bonferroni test).

Figure 4

Transcriptional levels of chemokines in the brains of WNV infected WT and MyD88/Trif−/− mice in uninfected, or 2 days and 6 days after infection. Quantitative RT-PCR analyses of chemokine mRNA levels in the brain of WT and MyD88/Trif−/− were measured in uninfected as well as 2 and 6 days after infection with 1×10⁷ PFU of WNV, Eg101 strain. Total RNA was analyzed for the expression of (A) MIP1α, (B) MIP1β, (C) RANTES, (D) CCL2 (MCP1), (E) IP-10, and (F) MIG. The data are expressed as Median ± SEM of chemokine mRNA copies per copy of 18S. Experiments were performed in triplicates at each time point per group. * p<0.05 (ANOVA, Bonferroni test).

Figure 5. Histopathology of the brains of C57BL/6 WT and MyD88/Trif−/− animals at day 6 after WNV infection

Brain sections were immunohistochemically stained for detection of specific cell types and infected cells. (A), (C), (E) and (G) represent brain sections from infected C57BL/6 WT mice and (B), (D), (F) and (H) represent brain sections from infected MyD88/Trif−/− mice. (A) and (B) show WNV-positively stained neurons; (C) and (D) show Iba-1+ activated microglia, (E) and (F) show Mac3+ infiltrating macrophages; and (G) and (H) show GFAP+ astroglia. MyD88/Trif−/−animals show a more robust inflammatory response in correlation with brain viral load at day 6 after infection.

Figure 6

Brain-infiltrating macrophages and myeloid dendritic cells in WNV-infected C57BL/6 WT and MyD88/Trif−/− mice. Uninfected controls and WNV-infected C57BL/6 WT and MyD88/Trif−/−mice at day 6 after infection were perfused and brain-infiltrating innate immune cells were evaluated using flow cytometry. Values represent the average ± SD of 4 uninfected and 7 infected animals in each group. (A) Percentage of CD45LCA+ CD11b+ CD11c- cells represent infiltrating macrophages and CD45LCA+ CD11b+ CD11c+ cells represent myeloid dendritic cells in the brain of controls and WNV-infected C57BL/6 and MyD88/Trif−/− mice. *p<0.05. (B) Class II expression on the surface of infiltrating innate immune cells. Brain-infiltrating macrophages failed to efficiently up-regulate class II expression (IA/IE) in MyD88/Trif−/− mice compared to C57Bl/6 WT mice. (C) Representative histograms showing the expression of class II molecules on uninfected (gray line) and infected (black line) C57BL/6 WT and MyD88/Trif−/− mice.

Figure 7

Survival curve of TLR3−/−, TLR7−/−, TLR4−/− and CpG1 mice infected with WNV. TLR3−/− and TLR7−/− mice are highly susceptible to WNV infection. C57BL/6 WT (n=26), TLR3−/− (n=13), TLR4−/− (n=6), TLR7−/− (n=5), and CpG1 (n=7) mice were infected with 1×10⁷ PFU of WNV Eg101 strain. (A) TLR3−/− (p<0.05) and TLR7−/− (p <0.001) mice are more susceptible to WNV infection compared to C57BL/6 WT mice (Logrank test). (B) The susceptibility of TLR4−/− and CpG1 mice to WNV infection was equivalent compared to what was observed in C57BL/6 WT animals (p>0.05).