TBK1 and IRF3 are potential therapeutic targets in Enterovirus A71-associated diseases

Figures

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  Fig 1. TBK1 and IRF3 interventions on EV-A71-induced disease severity in a neonatal mouse model. (A) and (E): The experimental design of TBK1 and IRF3 intervention. For TBK1 intervention, EV-A71 infected mice were treated with TBK1/IKKε-IN-2 (TBK1 inhibitor) via i.p. at a dose of 1.5, 2.0 or 2.5 μg per mouse at indicated time points. For IRF3 intervention, EV-A71 infected mice were treated with KIN1148 (IRF3 agonist) via i.p. at a dose of 1.0, 1.5 μg or 2.0 μg per mouse at indicated time points. Survival rates (B, F), body weight (C, G), and clinical scores (D, H) of mice were monitored and recorded daily up to 10 or 15 days after inoculation. Data represent the mean ± SD. TBK1 and IRF3 interventions mice with EV-A71 infection vs EV-A71-infected mice; *P < 0.05; **P < 0.01; *** P < 0.001; **** P < 0.0001; ns, no significance.

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  Fig 2. TBK1 and IRF3 interventions on type I IFN response in the brains and lungs of EV-A71-infected mice. qPCR was conducted to measure the expression levels of IFN-α and IFN-β in the brains (A and B) and lungs (C and D) of EV-A71-infected mice or mock mice with TBK1 and IRF3 interventions at 1 and 5 dpi. Results were normalized by β-actin. Data represent mean ± SD. n = 3 per group; TBK1 and IRF3 interventions alone, EV-A71 vs Mock; TBK1 and IRF3 interventions mice with EV-A71 infection vs EV-A71-infected mice; *P < 0.05; **P < 0.01; *** P < 0.001; ns, no significant. All the experiments were repeated at least three times.

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  Fig 3. TBK1 and IRF3 interventions on the antiviral activity against EV-A71 in mice. The brains (A, F, K, P), spinal cords (B, G, L, Q), muscles (C, H, M, R), hearts (D, I, N, S) and lungs (E, J, O, T) of mice were dissected for immunohistochemical analysis at 5 dpi. The black arrows indicated the positive staining (brown) and pictures were captured using an inverted fluorescence microscope. Bar = 200 μm. n = 4 per group, all experiments were repeated three times.

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  Fig 4. TBK1 and IRF3 interventions on EV-A71 replication. Viral loads in the brains, lungs, hearts, livers, kidneys, spleens and skeletal muscles of mice at 5 dpi were determined by qPCR. Results were expressed as Log10 RNA copies/mg tissue. n = 4 per group; TBK1 and IRF3 interventions in mice with EV-A71 infection vs EV-A71-infected mice; * P < 0.05; **P <0.01; **** P < 0.0001; ns, no significant. All the experiments were repeated at least three times.

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  Fig 5. Pathological examination of mouse tissues. Pathological changes in the brains (A, E, I, M), spinal cords (B, F, J, N), muscles (C, G, K, O) and lungs (D, H, L, P) of mice at 5 dpi were evaluated by H&E staining. Scoring for each section was evaluated on scale of 0 to 4 with increments of 0.5 independently by three observers for pathological changes. n = 4 per group; EV-A71 vs Control; EV-A71-infected mice with TBK1 and IRF3 interventions vs EV-A71-infected mice; *P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. (Q-T) Fresh lung tissues dissected from different experimental groups. Black arrows indicate typical pathological lesions. All experiments were repeated three times.

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  Fig 6. TBK1 and IRF3 interventions on EV-A71-induced proinflammatory cytokines expression in mice. The concentrations of IL-6, TNF-α, IL-1β, MCP-1and IL-10 in the brains (A-E) and lungs (F-J) of mice at 5 dpi were determined using ELISA. The data are shown as means ± SD. n = 3 or 4 per group; EV-A71 vs Control; EV-A71-infected mice with TBK1 and IRF3 interventions vs EV-A71-infected mice. * P < 0.05; **P < 0.01; ns, not significant. All the experiments were repeated at least three times.