Journal: Frontiers in Immunology

Article Title: Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

doi: 10.3389/fimmu.2022.985792

Figure Lengend Snippet: Zebrafish and human LGP2s play antithetic roles in regulating IFN response by poly(I:C) in fish cells (A–C) DrLGP2 and HsLGP2 downregulated fish IFN and human IFN promoter activation by poly(I:C) at a high concentration of 1 μg/ml in EPC cells. EPC cells seeded in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (A, B) , or HsIFNβpro-luc (200 ng each) (C) , together with DrLGP2 or HsLGP2 at increasing doses (0, 10, 50, 100, 200 ng). 24 h later, cells were transfected with 1 μg/ml poly(I:C) for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. ** P < 0.01, *P < 0.05. (D–G) DrLGP2 and HsLGP2 switched a first positive role to a following negative one in regulating IFN response by increasing concentrations of poly(I:C) in EPC cells. EPC cells seeded overnight in 24-wells plates were co-transfected with DrIFNφ1pro-luc or CaIFNpro-luc (D, E) , or HsIFNβpro-luc (F, G) , together with DrLGP2 (D, F) or HsLGP2 (E, G) (200 ng each). 24 h later, cells were transfected with poly(I:C) at increasing doses for another 24 h, followed by luciferase assays. P values were calculated using Student’s t-test. ** P <0.01. (H) Agarose electrophoresis showed the molecular weight spanning of MMW poly(I:C) and HMW poly(I:C). (I) RNA pull-down assays verified the binding of poly(I:C) to DrLGP2 and HsLGP2 in fish cells. CO cells seeded in 10 cm dishes were transfected with DrLGP2-HA, HsLGP2-HA or GFP-HA as control. 24 h later, cells were lysed. One-tenth of cell lysates were taken as input, the remaining was incubated with 100 ng biotinylated poly(I:C), followed by western blots with anti-HA antibody.

Article Snippet: The medium molecular weight (MMW) poly(I:C) was purchased from SIGMA (Catalog no. I3036), and the high molecular weight (HMW) poly(I:C) from Enzo Life Sciences (Catalog no. ALX-746-021).

Techniques: Activation Assay, Concentration Assay, Transfection, Luciferase, Electrophoresis, Molecular Weight, Binding Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: Function conservation and disparities of zebrafish and human LGP2 genes in fish and mammalian cells responsive to poly(I:C)

doi: 10.3389/fimmu.2022.985792

Figure Lengend Snippet: Zebrafish and human LGP2s play antithetic roles under low concentrations of poly(I:C) in mammalian cells and do so alone in fish cells. (A–E) Zebrafish and human LGP2s played antithetic roles under low concentrations of poly(I:C) in mammalian cells. HEK293T cells seeded in 24-wells plates were co-transfected with HsIFNβpro-luc (200ng), together with HsLGP2 (A–D) or DrLGP2 (E) (200 ng each). 24h later, cells were transfected again with MMW poly(I:C) [indicated as poly(I:C) in the text or all Figures] at 2 μg/ml (B, E) or at 4 ng/ml (C) , or with HMW poly(I:C) at 4 ng/ml (D) . Renilla vector (pRL-TK, 0.2 ng) was transfected as internal control. Another 24 h later, cells were collected for luciferase assays. P values were calculated using ANOVA. ** P < 0.01. (F) Overexpression of zebrafish or human LGP2s alone revealed antithetic roles in mammalian cells. EPC cells seeded in 24-well plates were transfected with DrLGP2 at the indicated increasing doses for 48 h. Or at 24 h post transfection, cells were transfected again with poly(I:C) at a high concentration of 1 μg/ml for another 24 h, followed by luciferase assays. P values were calculated using ANOVA. ** P < 0.01.

Article Snippet: The medium molecular weight (MMW) poly(I:C) was purchased from SIGMA (Catalog no. I3036), and the high molecular weight (HMW) poly(I:C) from Enzo Life Sciences (Catalog no. ALX-746-021).

Techniques: Transfection, Plasmid Preparation, Luciferase, Over Expression, Concentration Assay

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Simvastatin suppressed poly(I:C)-induced expression of IFN-β and ISGs in mice with HFD-induced hyperlipidemia. a. Schematic summary of the experimental design; b. Body weight changes in LFD-fed (gray circles; n=30) and HFD-fed (black circles; n=30) mice (left panel). Representative photographs of LFD- and HFD-fed mice (right panel); c. Changes to IFN-β and ISG expression in the lungs of mice treated with PBS (white columns), poly(I:C) (pIC; black columns), or poly(I:C) plus simvastatin (pIC+simva; gray columns) (n=10 in each group). Data are expressed as the fold change relative to PBS-treated mice, and shown as the mean ± SD, * p < 0.01, ** p < 0.05; d. IFN-β level in the BAL fluid, as measured by ELISA (n=8-9 in each group). The results are presented as the mean ± SD, * p < 0.01, ** p < 0.05; e. Left panel: Representative images of cells in the BAL fluid (Diff-Quik, x 400). Right panel: The percentage of macrophages to total cells in the BAL fluid of mice treated with PBS (white column), poly(I:C) (pIC; black column), or poly(I:C) plus simvastatin (pIC+simva; gray column) (n=4-5 in each group). The results are presented as the mean ± SD.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Diff-Quik

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Statins attenuated the expression of IFN-β and ISGs in poly(I:C)-treated macrophages. a. Changes to gene expression in J774.1/JA-4 cells after 48 h incubation with vehicle (hatched columns), simvastatin (simva; black columns), or pitavastatin (pitava; gray columns) (100 nM each) followed by treatment with 10 μg/mL of poly(I:C) for 4 h. Data are expressed as the fold change relative to untreated cells (white columns). The results are presented as the mean ± SD from at least three independent experiments, * p < 0.01; b. Quantification of secreted IFN-β protein. J774.1/JA-4 cells (white column) were incubated with vehicle (hatched column), simvastatin (simva; black column), or pitavastatin (pitava; gray column) (100 nM each) for 48 h before they were treated with 10 μg/mL of poly(I:C) for 20 h. The results are presented as the mean ± SD from three independent experiments, * p < 0.01.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Statins impaired the expression of inflammatory cytokines in poly(I:C)-treated macrophages. a. Changes to the expression of inflammatory cytokine genes. J774.1/JA-4 cells were cultured and treated as described in the legend of . Data are shown as the fold change relative to vehicle-treated cells. The results are presented as the mean ± SD from at least three independent experiments, * p < 0.01; b. Quantification of inflammatory cytokine levels. J774.1/JA-4 cells were cultured and treated as described in the legend of . The results are presented as the mean ± SD from three independent experiments, * p < 0.01.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Expressing, Cell Culture

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Statins did not affect the cellular uptake of poly(I:C). J774.1/JA-4 cells were cultured with simvastatin (simva) or pitavastatin (pitava) (100 nM each) for 48 h, then treated with (black columns) or without (white columns) 1 μg/mL of rhodamine-labeled poly(I:C) (red) for 2 h. The cells were also stained with Hoechst 33342 (blue). Fluorescence intensity of rhodamine-labeled poly(I:C) was normalized to that of Hoechst 33342. Scale bar = 10 μm. The results are presented as the mean ± SD from three independent experiments.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Cell Culture, Labeling, Staining, Fluorescence

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: The effects of statins on the expression of TLR3 and involvement of TLR3 in the expression of IFN-β and ISGs in poly(I:C)-treated macrophages. a. Western blot analysis for TLR3. J774.1/JA-4 cells were cultured with simvastatin or pitavastatin (100 nM) for 48 h then subjected to western blot analysis using 25 μg of protein in each lane. β-actin was used as the internal control. The results are representative of three independent experiments; b. Efficiency of TLR3 knockdown. J774.1/JA-4 cells were transfected with TLR3 siRNA (siTLR3; 20 nM, black column) or negative control siRNA (siCTL; 20 nM, white column), and incubated for 24 h. The results are presented as the mean ± SD from at least three independent experiments, * p < 0.01; c. Changes to the expression of IFN-β and ISGs after siRNA treatment. siTLR3-transfected (black columns) and negative control siRNA-transfected (white columns) J774.1/JA-4 cells were treated with 10 μg/mL of poly(I:C) for 4 h. The results are presented as the mean ± SD from at least three independent experiments, * p < 0.01.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Expressing, Western Blot, Cell Culture, Transfection, Negative Control, Incubation

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Statins inhibited IRF3-mediated JAK/STAT signaling in poly(I:C)-treated macrophages. a. Western blot analysis for IRF3 and STAT1. J774.1/JA-4 cells were cultured with simvastatin or pitavastatin (100 nM each) for 48 h, then treated with 10 μg/mL of poly(I:C) for the indicated time periods. An equal amount of protein (15 μg) was applied in each lane. The results are representative of three independent experiments. β-actin was used as the internal control; b. The effect of tofacitinib (JAK inhibitor) on poly(I:C)-induced activation of STAT1. J774.1/JA-4 cells were pre-treated with vehicle or 1 μM of tofacitinib for 1 h, then exposed to 10 μg/mL of poly(I:C) for the indicated time periods. The results are representative of three independent experiments, *non-specific signals that were detected in all samples; c. Changes to gene expression in tofacitinib-treated cells. J774.1/JA-4 cells were pre-treated with vehicle (veh; black columns) or 1 μM tofacitinib (tof; gray columns) for 1 h, then exposed to 10 μg/mL of poly(I:C) for 4 h. The mRNA levels are expressed as the fold change relative to untreated cells (white columns). The results are presented as the mean ± SD of three independent experiments, * p < 0.01, ** p < 0.05.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Western Blot, Cell Culture, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Mevalonate and GGPP reversed the inhibitory effect of statins on IFN-β and ISG expression in poly(I:C)-treated macrophages. a. Schematic representation of the cholesterol and isoprenoid biosynthesis pathway; b. Mevalonate and GGPP reversed the inhibitory effect of simvastatin and pitavastatin on IFN-β and ISG expression. J774.1/JA-4 cells (white columns) were cultured with simvastatin (simva; black columns) or pitavastatin (pitava; gray columns) for 24 h, then incubated with 300 μM of mevalonate (mev), 1 μg/mL of GGPP, or 1 μg/mL of cholesterol (cho) for 24 h. The cells were further treated with 10 μg/mL of poly(I:C) for 4 h. The mRNA levels are expressed as the fold change relative to untreated cells. The results are presented as the mean ± SD from three independent experiments, * p < 0.01; c. Quantification of IFN-β levels. The cells were treated as described in the legend of , and incubated with 10 μg/mL of poly(I:C) for 20 h. The results are presented as the mean ± SD from three independent experiments, * p < 0.01.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Expressing, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Statins attenuate antiviral IFN-β and ISG expression via inhibiting IRF3/JAK/STAT signaling in poly(I:C)-treated hyperlipidemic mice and macrophages

doi: 10.1101/2020.06.21.163873

Figure Lengend Snippet: Mevalonate and GGPP reversed the inhibitory effect of simvastatin on IRF3-mediated JAK/STAT1 signaling in poly(I:C)-treated macrophages. Western blot analysis for IRF3 and STAT1. J774.1/JA-4 cells were cultured with simvastatin (simva), mevalonate (mev), and GGPP as described in the legend of , then treated with 10 μg/mL of poly(I:C) for the indicated time periods. The results are representative of three independent experiments. β-actin was used as the internal control, *non-specific signals that were detected in all samples.

Article Snippet: To observe the cellular uptake of poly(I:C), the cells were treated with 1 μg/mL of poly(I:C) (HMW)-Rhodamine for 2 h at 37 °C in 8-well glass chamber slides (Corning, Corning, NY, USA).

Techniques: Western Blot, Cell Culture