Fig 1.
Wnt3a increases progeny influenza virus production.
(A, B) Mouse lung epithelial E10 cells were pretreated with different concentrations of recombinant human Wnt3a protein for 24 h and infected with A/PR/8/34 at an MOI of 0.1 for 18 h. The media were collected, and viral titers were determined by TCID50 assay. The cells were fixed, and immunostained with anti-NP antibodies. The results of 3 independent experiments are displayed as the mean ± SE. *p<0.05 vs. control (0 ng/ml). (C) E10 cells were pretreated with blank control (DMEM only), 50% Wnt3a_CM or Con_CM for 24 h and infected with influenza virus A/PR/8/34 at an MOI of 1 for 18 h. (D) Mice were instilled with 10x Wnt3a_CM or Con_CM, and the lungs were collected on day 5 post-infection. Relative mRNA expression levels of viral genes (HA, NP and MP) were measured by real-time PCR and normalized to GAPDH. The data are normalized to the blank control and are represented as the mean ± SE (n = 4). *p<0.05 vs. Con_CM. (E) HEK 293 cells were co-transfected with a TOPflash reporter plasmid, and a pRL-TK normalization vector. Twenty four h post transfection, the cells were infected with A/PR/8/34 or A/WSN/33 at a MOI of 0.1 for various times. TOPFlash firefly luciferase activity was normalized to pRL-TK Renilla luciferase activity. The results of 4 independent experiments are displayed as the mean ± SE. ##p<0.01 vs. control cells without infection (0 time); *p<0.05 vs. control cells without infection; **p<0.01 vs. control cells without infection, ***p<0.001 vs. control cells without infection (Student’s t-test).
Fig 2.
iCRT14 reduces progeny influenza virus production.
(A) A549 cells were co-transfected with a TOPflash reporter vector and a pRL-TK normalization vector. Twenty four h post transfection, the cells were treated with Wnt3a_CM or Con_CM in the presence or absence of iCRT14 (12.5 μM) for 24 h. TOPFlash firefly luciferase activity was normalized to pRL-TK Renilla luciferase activity. **p<0.01. n = 3 (one-way ANOVA, followed by Tukey's test). (B, C) A549 cells were treated with iCRT14 (12.5 μM) or vehicle control for 12 h and then infected with influenza virus A/PR/8/34 (B) or A/WSN/33 (C) at an MOI of 1. The cell culture medium was collected at various times post-infection, and viral titers were determined by TCID50 assay. (D) A549 cells were treated with iCRT14 (12.5 μM) or vehicle control for various times, and cell viability was measured using the CellTiter Glo kit (Promega). The results of 3 independent experiments are displayed as the mean ± SE. *p<0.05 vs. vehicle control, **p<0.01 vs. vehicle control at the corresponding time points (Two-way ANOVA, post hoc Tukey). (E) A549 cells were cotrasnfected with an influenza virus sensor reporter (NP-UTR-Luc) and pRL-TK vector. Twenty-four h post-transfection, the cells were infected with A/PR/8/34, pdm/Ok/09 and H3N2 A/Wisconsin/67/05 at an MOI of 0.1 for 48 h. Firefly luciferase activity was normalized to Renilla luciferase activity. *p<0.05 vs. vehicle control, **p<0.01 vs. vehicle control, ***p<0.001 vs. vehicle control (Student’s t-test).
Fig 3.
Knockdown of β-catenin reduced viral replication.
Rat lung epihtelail L2 cells were treated with adenoviral shRNA targeting β-catenin (shβCat) or a control vector (shCon) at an MOI of 100 for 48 h and then infected with A/PR/8/34 (MOI 0.01) for 48 h. (A) GFP signal after 48 h adenoviral infection. (B) Representaive western blots of β-catenin and viral proteins, NS1 and NP. (C) Quantiation of wesetern blots. (D) Virus titer as determined by plaque assay. **p<0.01 vs. shCon, ***p<0.001 vs. shCon, n = 3 (Student’s t-test).
Fig 4.
iCRT14 acts during the early stage of the influenza virus life cycle.
(A) A549 cells were infected with A/PR/8/34 at an MOI of 1. The cells were treated with iCRT14 (12.5 μM) or vehicle control at various times before or after infection. The culture medium was collected 12 h post-infection, and titers were determined by TCID50 assay. The results of 3 independent experiments are displayed as the mean±SE. *p<0.05 vs. vehicle control, **p<0.01 vs. vehicle control, ***p<0.001 vs. vehicle control at the corresponding time points (Two-way ANOVA, post hoc Tukey). (B-G) iCRT14 reduces influenza virus RNA synthesis. A549 cells were infected with A/PR/8/34 at an MOI of 5. The cells were treated with iCRT14 at 12.5 μM (B, C, D) or at the indicated concentrations (E, F, G) 1 h prior to infection. RNA was extracted at 5 h post-infection. The mRNA, cRNA, and vRNA levels of viral genes were determined by real-time PCR and normalized to GAPDH. In B-D, data was expressed as relative expression to GAPDH and in E-G, data was expressed as a percentage of control (without iCRT14). The results of 3 independent experiments are displayed as the mean ± SE. *p<0.05 vs. vehicle control, **p<0.01 vs. vehicle control, ***p<0.001 vs. vehicle control, $p<0.05 vs. vehicle control, $ $p<0.01 vs. vehicle control, $ $ $p<0.001 vs. vehicle control, #p<0.05 vs. iCRT14, ##p<0.01 vs. iCRT14, ###p<0.001 vs. iCRT14 (One-way ANOVA, post hoc Tukey).
Fig 5.
Effects of iCRT14 on influenza virus-induced IFN response and iCRT14-mediated inhibition of influenza virus RNA synthesis in Vero cells.
(A, B) A549 cells were infected with A/PR/8/34 at an MOI of 1 for various times. The cells were treated with iCRT14 (12.5 μM) or vehicle control 1 h prior to infection. IFN α1 and β1 mRNA levels were determined by real-time PCR and normalized to GAPDH. The data are expressed as fold change relative to mock infection. (C) Vero cells were infected with A/PR/8/34 at an MOI of 5. The cells were treated with iCRT14 (12.5 μM) or vehicle control 1 h prior to infection. The cRNA, vRNA and mRNA levels of the NP gene were determined at 5 h post-infection using real-time PCR and normalized to GAPDH. The data are expressed as relative expression. The results of 3 independent experiments are presented as the mean ± SE. *p<0.05 vs. vehicle control, *p<0.01 vs. vehicle control (Student’s t-test).
Fig 6.
iCRT14 reduces influenza virus infection in primary mouse alveolar epithelial cells (AECs).
Mouse AECs were isolated and cultured for 6 days. (A) AECs were immuno-stained with the AEC type I marker T1α and the nuclear dye DAPI. Scale bar = 50 μm. (B-E) AECs were infected with influenza virus A/PR/8/34 or A/WSN/33 at an MOI of 1 for 24 h. The cells were treated with iCRT14 (12.5 μM) or vehicle control 1 h prior to infection. Viral titers in the cell culture medium were determined using the TCID50 assay (B, C). NA mRNA levels in cells were determined by real-time PCR and normalized to GAPDH. The results of 3 independent experiments are displayed as the mean ± SE. *p<0.05 vs. vehicle control at the corresponding time points (Student’s t-test).
Fig 7.
Effects of iCRT14 on weight loss and survival rate in mice challenged with a lethal dose of influenza virus A/PR/8/34.
C57BL/6J mice were treated with iCRT14 (50 mg/kg) or vehicle control daily from one day prior to infection until day 6 post-infection. The mice were challenged with influenza virus A/PR/8/34 (1,000 pfu/mouse). (A) Percent of body weight loss. Body weight loss is presented as the mean ± SE. *p < 0.05 vs. vehicle control at day 8 (Two-way ANOVA followed by post-hoc sidak test). (B, C) Kaplan–Meier survival curves and mean day of survival of mice in the iCRT14 and vehicle control groups. The Mantel-Cox χ2 test and Student’s t-test were used for survival curves and mean day of survival, respectively. *p< 0.05 vs. control group; **p<0.01 v.s control group, n = 12–13 animals per group.
Fig 8.
Effect of iCRT14 on lung injury and inflammation of mice challenged with a sublethal dose of influenza virus A/PR/8/34.
C57BL/6J mice were infected with A/PR/8/34 (100 pfu/mouse). Mice euthanized on day 0 of the study were used as a control. Mice received either iCRT14 (50 mg/kg) or vehicle control daily beginning one day prior to infection until day 2 or day 5 after infection. (A) Virus titers in total lung homogenate (n = 6). (B) Average clinical scores; 0 = normal, 1 = ruffled fur, 2 = inactive, 3 = hunched back (n = 10–20). (C) Lung water content as measured by wet-to-dry ratio (n = 4). (D) Total protein in BAL fluid (n = 4). (E) LDH activity in BAL fluid (n = 4). (F) Inflammatory cells in BAL (n = 3–4). The data are presented as the mean ± SE. *p < 0.05 vs. vehicle control on the corresponding days, **p < 0.01 vs. vehicle control on the corresponding days. For (A) and (C), Two-way ANOVA, post hoc sidak was used and 0 time group was not included in analysis due to lack of equal groups. For (B), Student’s t-test was used duo to unequal animal numbers in different groups. (G) Representative H&E photomicrographs from 6 mice are shown. A, B = day 2 vehicle control mice; C, D = day 2 iCRT14 mice; E, F = day 5 vehicle control mice; G, H = day 5 iCRT14 mice. The dark arrowheads in Panels A, C, E, G represent alveolar histiocytic and neutrophil infiltrations with associated hemorrhage. The arrowheads in Panels B, D, F, H represent major airway degeneration/necrosis, histiocytic and neutrophil infiltration. Scale bar = 50 μm.
Table 1.
Histopathology of the lungs of mice on days 2 and 5 after infection with a sublethal dose of influenza virus.
Table 2.
Real-time PCR primers.