Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

Figures

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  Fig. 1. EV71 infection induces an acute IL-12p40 response in the brainstem. A Cytokine antibody microarray analysis of brainstem of ICR mice infected with MP4 virus. The ICR mice at 7-day of age were orally infected with or without a 50% lethal dose of a mouse-adapted EV71 strain (MP4) virus and the brainstem tissues were harvested for the analysis at 7 days post-infection (dpi). B qRT-PCR analysis of IL-12p40, IL12A, IL23, and IFNγ mRNA levels at 7 dpi in the brainstem of ICR mice infected with or without MP4 virus. Values are represented as mean (n = 4). Significantly different from control group: **P < 0.01 by unpaired t test. C Western blot analysis of IL-12p40 protein levels in the brainstem of hSCARB2-Tg mice infected with EV71 (3 × 104 PFU/mouse). The hSCARB2-Tg (TG) and non-Tg (WT) mice at 7-day of age were injected s.c. with 3 × 104 pfu of 5746 (C2) strain of EV71. The brainstem tissues (n = 3) were harvested at the indicated dpi for western blot analysis. D, Temporal expression of IL-12p40 in the brainstem of hSCARB2-Tg (TG) and non-TG (WT) mice infected with EV71. Time-course qRT-PCR analysis of IL-12p40 at the indicated dpi. Values are represented as mean (n = 4).…

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  Fig. 2. The IL-12p40 response is essential in the pathogenesis of EV71-induced brainstem encephalitis. A Kaplan-Meier survival curve of EV71-infected hSCARB2-Tg mice receiving treatment with anti-IL-12p40 antibody or control IgG. The 7-day-old hSCARB2-Tg mice were injected s.c. with 3 × 104 pfu of EV71 and subsequently intraperitoneally injected with anti-IL12p40 antibody (2 ug/mouse) or IgG (Ctrl) at 2 and 4 dpi. Significantly different from control group at p < 0.01 by Log Rank test. B Representative photomicrographs and quantification of anti-GFAP immunostaining in the brainstem regions of the EV71-infected hSCARB2-Tg mice treated with anti-IL-12p40 antibody or control IgG. Scale bars, 50 μm. The GFAP-positive signals in five consecutive sections per animal was quantified by ImageJ and presented as mean (n = 5, right panel). Significantly different from control group: ***P < 0.01 by unpaired t test. C qRT-PCR analysis of TNFa and IL6 mRNA levels in the brainstem of the EV71-infected hSCARB2-Tg mice treated with anti-IL-12p40 antibody or control IgG. Values are represented as mean (n = 4). Significantly different from control group at *p < 0.05 by unpaired t test. D Progressive limb…

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  Fig. 3. TLR9 is required for EV71 to elicit an IL-12p40 response and include brainstem encephalitis. A Temporal expression pattern of TLR9 in the brainstem of hSCARB2-Tg (TG) and non-TG (WT) mice infected with EV71. qRT-PCR analysis of TLR9 at the indicated dpi. Values are represented as mean (n = 3). Significantly different from control group: *P < 0.05, **P < 0.01 by unpaired t test. B Representative photomicrographs of TLR9 (green) colocalized with IL-12p40 (red) in the brainstem regions of mice infected with EV71 at 4dpi. Scale bars, 40 μm. C qRT-PCR analysis of IL-12p40 expression levels in the brainstem of the EV71-infected hSCARB2-Tg mice with the treatment ODN2088 or control IgG. Values are represented as mean (n = 5). Significantly different from control group: **P < 0.01 by unpaired t test. D EV71-induced limb paralysis at indicated dpi in hSCARB2-Tg mice treated with or without ODN2088. Significantly different from control group: p < 0.001 by unpaired t test. E Representative photomicrographs of anti-GFAP immunostaining in the brainstem regions of the EV71-infected hSCARB2-Tg mice treated with or without ODN2088 at 3 dpi. Scale bars, 50 μm. The GFAP-positive signals in five…

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  Fig. 4. IL-12p40 promotes neuronal damage through activating iNOS/NO response. A Graphical illustration of how different subunits of IL-12 interact with specific cell surface receptors in controlling distinct downstream transcription factors and signaling events. B qRT-PCR analysis of iNOS expression levels in the brainstem of hSCARB2-Tg (TG) or non-TG (WT) mice during the time course of EV71 infection. Values are represented as mean (n = 3). Significantly different from control group: **P < 0.01 by unpaired t test. C qRT-PCR analysis of iNOS expression levels in the EV71-infected hSCARB2-Tg mice treated with or without anti-IL-12p40 antibody (n = 5). D qRT-PCR analysis of iNOS expression levels in the EV71-infected U87-MG cells treated with or without anti-IL-12p40 antibody (n = 3). Effect of NO level on EV71-induced cell viability. SH-SY5Y and primary neuronal cells were pre-treated with or without NO donor SNAP (E, F) or Nitric oxide synthase inhibitor LNNA (G, H) 1 h before infection with EV71 (MOI = 1 for SH-SY5Y and MOI = 3 for primary neuron). WST-1 analysis was conducted at 9 hpi. Values are represented as mean (n = 4). Significantly different from control group at *p < 0.05, **p…

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  Fig. 5. Graphical summary of major findings of this study on the TLR9-IL-12p40-iNOS signaling pathway in the EV71-induced brainstem encephalitis. Severe EV71 infection induces an acute IL-12p40 response through intracellular sensor TLR9 in glia cells of the brainstem, subsequently generating neurotoxic NO causing encephalitis. These findings provide support for the therapeutic value of blocking the TLR9 mediated IL-12p40 response in the treatment of severe EV71 infection-induced brainstem encephalitis.