Pterostilbene, an active constituent of blueberries, enhances innate immune activation and restricts enterovirus D68 infection

Figures

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  Figure 1. Pterostilbene (Pte) and pinostilbene (Pin) enhance EV-D68-induced innate immune responses. (A) Structure of Pte (molecular formula: C16H16O3). (B) Structure of Pin (molecular formula: C15H14O3). (C–F) Gene expression of IFN-α (C), IFN-β (D), IFIT1 (E), and MX1 (F), respectively, as indicators of virus-stimulated innate immune response. The mRNA was quantified by qRT-PCR at 0, 12, and 24 h post-infection. (G, H) Detection of IFN-α and IFN-β protein concentrations in supernatants by ELISA. (I) Detection of MX1 and IFIT1 in cells by Immunoblotting. Supernatants or cells were collected 24 h post-infection or mock-infection. Error bars indicate the standard deviation (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). EV-D68, Enterovirus D68; IFN, interferon; IFIT1, interferon-induced protein with tetratricopeptide repeats 1; MX1, MX dynamin-like GTPase 1; qRT-PCR, quantitative reverse transcription polymerase chainreaction.

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  Figure 2. Pterostilbene (Pte) and pinostilbene (Pin) relieve CPEs in EV-D68-infected respiratory cells. (A, B) Indicated cells were pre-treated with increasing concentrations of Pte or DMSO vehicle before infection or mock-infection with EV-D68 virus. CPEs were observed 24 h post-infection. (C, D) Indicated cells were pre-treated with increasing concentrations of Pin or DMSO vehicle before infection or mock-infection with EV-D68 virus. CPEs were observed 24 h post-infection. EV-D68, Enterovirus D68; DMSO, dimethyl sulfoxide; CPE, cytopathic effects.

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  Figure 3. Pterostilbene (Pte) and pinostilbene (Pin) inhibit EV-D68 replication. (A–D) Determination of progeny virion production. Supernatants were collected 24 h post-infection and viral titers were determined by standard plaque assay. (E–J) qRT-PCR assessment of EV-D68 RNA replication. (G–L) Cell viability assay. Cellular toxicity was evaluated by CCK-8 assay and is expressed as the percentage relative to DMSO vehicle-treated control cells. Experiments were performed in triplicate. Error bars indicate the standard deviation (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). EV-D68, Enterovirus D68; DMSO, dimethyl sulfoxide; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

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  Figure 4. Pterostilbene (Pte) and pinostilbene (Pin) do not influence EV-D68 attachment or entry into cells. (A–D) Cells were treated with Pte, Pin, or DMSO vehicle in 12-well plates under standard conditions. Cells were incubated at 4°C or 37°C for 30 min before infection and then incubated with EV-D68 viruses for 2 h. The qRT-PCR was performed to quantify viral RNA. EV-D68, Enterovirus D68; DMSO, dimethyl sulfoxide; qRT-PCR, quantitative reverse transcription polymerase chain reaction. Error bars indicate the standard deviation (n.s., no significance, *, P < 0.05; **, P < 0.01).

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  Figure 5. Pterostilbene (Pte) and pinostilbene (Pin) suppress EV-D68 RNA replication and VP1 synthesis post viral entry. A549 or BEAS-2B cells were pre-treated with Pte (25 µM), Pin (25 µM) or DMSO for 2 h and subsequently infected with EV-D68 at an MOI of 0.01. Cell or supernatant samples were collected at 0, 2, 4, 8, and 12 h post-infection. Relative intracellular viral RNA was detected using qRT-PCR (A–D), Viral structural protein VP1 was measured by immunoblotting (E, F). EV-D68, Enterovirus D68; DMSO, dimethyl sulfoxide; qRT-PCR, quantitative reverse transcription polymerase chain reaction; MOI, multiplicity of infection. Error bars indicate the standard deviation (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

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  Figure 6. Pterostilbene (Pte) and pinostilbene (Pin) inhibit isolated circulating strains of EV-D68. (A–H) A549 or BEAS-2B cells were pre-treated with 25 μM Pte, or 25 μM Pin, or DMSO vehicle and then infected with US/MO/14-18947 (MO) or US/KY/14-18953 (KY). Cells were incubated under standard conditions. The titers of progeny virions were determined 12 or 24 h post-infection. The experiment was performed thrice. Error bars indicate the standard deviation (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). EV-D68, Enterovirus D68; DMSO, dimethyl sulfoxide; MO and KY, isolated EV-D68 circulating strains.

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  Figure 7. Pterostilbene (Pte) and pinostilbene (Pin) mediate innate immune activation through IRF3 signaling pathway. (A) IRF3 gene knock-down efficiency validation by Immunoblotting. (B) A549-shctl or A549-shIRF3 cells were pre-treated with Pte (25 µM), Pin (25 µM) or 0.1% DMSO for 2h and then infected with EV-D68 at an MOI of 0.01. Cells were collected 24 h post-infection. Viral protein VP1 was detected using Immunoblotting. (C–F), A549 cells were pre-treated with Pte, Pin or DMSO for 2h, then transfected with Poly I:C (1 µg per well). Cells were collected at 0h and 12h post-transfection. Relative intracellular IFN-α, IFN-β, MX1 and IFIT1 mRNA were detected using qRT-PCR. Error bars indicate the standard deviation (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).