Antiviral Research 110 (2014) 124–131
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
Alleviation of respiratory syncytial virus replication and inflammation by fungal immunomodulatory protein FIP-fve from Flammulina velutipes Yu-Chi Chang a, Yen-Hung Chow b, Hai-Lun Sun c, Yu-Fan Liu d, Yu-Tzu Lee a, Ko-Huang Lue c,e,⇑, Jiunn-Liang Ko a,c,⇑ a
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan Vaccine Research and Development Center, Miaoli County, National Health Research Institutes, Miaoli, Taiwan Division of Allergy, Department of Pediatrics, Chung Shan Medical University Hospital, Taichung, Taiwan d Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan e School of Medicine, Chung Shan Medical University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 6 May 2014 Revised 23 July 2014 Accepted 6 August 2014 Available online 14 August 2014 Keywords: Immunomodulatory protein FIP-fve Respiratory syncytial virus Inflammatory NF-jB Antiviral agent
a b s t r a c t Respiratory syncytial virus (RSV) causes bronchiolitis in children followed by inflammation and asthmalike symptoms. The development of preventive therapy for this virus continues to pose a challenge. Fungal immunomodulatory proteins (FIPs) exhibit anti-inflammatory function. FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes. To determine whether FIP-fve affects the infection or consequence of immunity of RSV, we investigated viral titers of RSV and inflammatory cytokine levels in vivo and in vitro. Oral FIP-fve decreased RSV-induced airway hyperresponsiveness (AHR), airway inflammation, and IL-6 expression in bronchoalveolar lavage fluid (BALF) of BALB/c mice. RSV replication and interleukin 6 (IL-6) levels in RSV-infected HEp-2 cells were compared before and after FIP-fve treatment. FIP-fve inhibited viral titers on plaque assay and Western blot, as well as inhibited RSV-stimulated expression of IL-6 on ELISA and RT-PCR. The results of this study suggested that FIP-fve decreases RSV replication, RSV-induced inflammation and respiratory pathogenesis. FIP-fve is a widely used, natural compound from F. velutipes that may be a safe agent for viral prevention and even therapy. Ó 2014 Published by Elsevier B.V.
1. Introduction Respiratory syncytial virus (RSV) is the most common cause of severe acute lower respiratory illness in children, in some cases leading to infant hospitalization and even death (Nair et al., 2010; Swanson et al., 2011). In addition, RSV has been associated with long term complications, such as recurrent wheezing and asthma. Recently, prevention of RSV disease has become possible. However, prophylaxis with potent neutralizing monoclonal antibody, Palizivumab, is only available to the highest risk infants (DeVincenzo et al., 2010). The development of an accessible preventive therapy for RSV infection has so far been unsuccessful due to problems associated with stability, purity, reproducibility, tolerability, and potency (Wright et al., 2007). RSV infection has been thought to result in the viral triggering of an exaggerated Th2 immune response and exacerbated pulmonary ⇑ Corresponding authors at: Room 704, No. 110, Sec. 1, Chung Shan Medical University, Institute of Medicine, Chien-Kuo N. Road, Taichung 40203, Taiwan. Tel.: +886 4 24730022 11694; fax: +886 4 24751101. E-mail addresses: [email protected] (K.-H. Lue), [email protected] (J.-L. Ko). http://dx.doi.org/10.1016/j.antiviral.2014.08.006 0166-3542/Ó 2014 Published by Elsevier B.V.
disease. The correlation between viral load and proinflammatory cytokines IL-6 and IL-8 suggests that if viral load is reduced by a robust antiviral, disease severity decreases even if the disease is mediated by these proinflammatory cytokines (DeVincenzo et al., 2010). In the presence of decreased NF-kB reporter activity in lung, liver, and spleen tissues challenged with lipopolysaccharides, there is attenuation of inflammatory gene expressions (cyclooxygenase2, inducible nitric-oxide synthase, and TLR4) and inflammatory cytokine secretion (Karpurapu et al., 2011). The role of NF-jB in mediating inflammation has been established using genetic approaches and chemical inhibitors (Ghosh and Hayden, 2008). Epithelial cells of infected tissue or tissue-resident haematopoietic cells, such as mast cells or dendritic cells (DCs), initiate the inflammatory response by triggering pro-inflammatory pathways through NF-jB in response to inflammatory stimuli (Hayden et al., 2006). RSV-triggered IKK activation, p65 nuclear accumulation, and DNA binding are characteristic of the classical pathway of NF-jB activation (Fink et al., 2008; Harris and Werling, 2003). Phosphorylation at Ser536 is essential for RSV-induced p65-mediated promoter transactivation in airway epithelial cells (Fink et al., 2008).
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
Fungal immunomodulatory protein (FIP-fve) has been isolated and purified from the edible golden needle mushroom (Flammulina velutipes) (Ko et al., 1995). It is comprised of 114 amino acid residues and characterized by a molecular mass of 13 kDa, a high degree of amino acid sequence homology with LZ-8 and 70 invariant amino acid residues (Ko et al., 1995). FIP-fve has been found to stimulate the production of IFN-c in PBMCs (Wang et al., 2004). Th1 cells produce IFN-c, which is essential for the eradication of intracellular pathogens (Teixeira et al., 2005). It has been demonstrated that glial cells are derived from neural progenitor cells with neurotropic JHM strain of mouse hepatitis virus infection and that treatment of infected cells with IFN-c inhibits viral replication in a dosedependent manner (Whitman et al., 2009). Strategies aimed at balancing neonatal Th1 and Th2 responses can facilitate the treatment of allergies (Dubois et al., 2010). Therefore, the aims of this study were to assess the ability of FIP-fve to disrupt RSV infection or the following inflammatory responses and to investigate the effects of FIP-fve on the viral load of RSV and RSV-induced inflammation. 2. Materials and methods 2.1. Intranasal immunization and RSV challenge Female BALB/c mice (6 weeks old) were purchased from the National Laboratory Animal Center, Taiwan, and animal experiments were approved by the Animal Use Committee of Chung Shan Medical University. The mice were divided into three groups of four mice each, as follows: (1) normal control group with oral and intranasal administration of PBS; (2) RSV group with oral administration of PBS and intranasal administration of 2 105 plaque-forming units (PFU) of purified RSV (in endotoxin-free PBS) for 6 days as previously described (Han et al., 2010); (3) FIP-fve/RSV group with oral administration of 10 ml/kg FIP-fve once a day from 2 days before RSV infection and intranasal administration of 2 105 PFU of purified RSV (in endotoxin-free PBS) for 6 days. The effects of FIP-fve on RSV-induced airway hyperresponsiveness (AHR) were measured using whole-body plethysmograph (Buxco, Troy, NY) with increasing doses of inhaled methacholine (Sigma– Aldrich, St. Louis, MO) as previously described (Stark et al., 2005). Immediately after measurement of AHR, lungs were lavaged through the trachea with 1 ml of normal saline to collect bronchoalveolar lavage fluid (BALF). BALF was aspirated and stored at – 70 °C until assay. The animals were sacrificed and the lungs were fixed in formalin and embedded in paraffin. Lung tissue sections were cut from the paraffin blocks and stained with hematoxylin and eosin or specific anti-RSV antibodies (Millipore Cat. No. 5006). 2.2. Measurement of cytokines The levels of IL-6 and IFN-r in BALF were assayed according to the manufacturer’s protocol (Mouse Interleukin-6, eBioscience catalog number 88-7064-88 and Mouse IFN-gamma DuoSet, R&D system catalog number DY485-05, respectively). The levels of IL-6 in medium of HEp-2 cells were assayed according to the manufacturer’s instructions (Human Interleukin-6, eBioscience Catalog Number 88-7066-88). 2.3. H Score The intensity of immunoreactivity was assessed by integrated optic density in a fixed field with a total pixel area of 150 150 on a scale of 0–3 using the Pro Plus 6.1 image analysis program (Media Cybernetic, Inc., Silver Spring, MD). The proportion of positive cells was calculated for each field. Three images of representative areas were acquired and at least 100 cells per tissue section were manually counted on a computer screen. The proportion of
125
positive cells was scored as follows: none, 0; less than one tenth, 0.1; less than one half, 0.5; and greater than one half, 1. Finally, a semi-quantitative H score of RSV immunoreactivity was determined by multiplying the four-tiered intensity score by the fourtiered proportion score of positive cells in a section. Minimum H score was 0 and maximum H score was 3. 2.4. Plaque-forming unit assay Plaque-forming unit assay for detecting abortive RSV replication was performed as previously described. Briefly, HEp-2 cells were seeded onto 12-well tissue culture plates at 3 105 cells/ well. The plates were incubated in a humidified incubator until the cell monolayer achieved confluence. The media were aspirated from each well and washed twice with PBS. Pretreatment with 300 ll FIP-fve per well at various doses (0, 7.5, 30 lM) was carried out for 2 h. Then, 300 ll diluted virus were added to each well, followed by mixing and incubation of the plates at 37 °C, 5% CO2 for 1 h. After the incubation period, the remaining media were aspirated from each well. This was followed by overlaying of 2.5 ml of 0.75% methyl cellulose in 2% FBS-medium in each well. The plates were further incubated at 37 °C in a humidified 5% CO2 incubator for 6 days. After 6 days, the plates were fixed and stained with crystal violet-methanol at room temperature for 20 min. RSV specific immunostaining of RSV plaques was performed on EnVision + Dual Link System-HRP (Dako North American, Inc.). 2.5. RT-PCR and real-time PCR The detailed protocols for RNA isolation and RT-PCR have been previously described (Wang et al., 2011). cDNA was reversetranscribed from 3 lg total cellular RNA using random hexamer primers and murine leukemia virus reverse transcriptase (Promega). The primer sequences for PCR amplification were: RSV sense 50 -AGCAAAGTCAAGTTGAATGATAC-30 and antisense 50 -GGCTGTAAGACCAGATCTATC-30 ; human IL-6 sense 50 -ATGAAGTTTCTCTCCGCAAGAGACTTCCAGCCAG-30 and antisense 50 CTAGGTTTGCCGAGTAGACCTCATAGTGACC-30 ; human b-actin sense 50 -CAGGGAGTGATGGTGGGCA-30 and antisense 50 -CAAACATCATCTGGT CATCTTCTC-30 . Two microliters of cDNA were amplified in a reaction volume of 50 lL containing 0.5 units of Taq polymerase (Promega), 400 lM dNTPS, 10 mM Tris–HCl (pH 8.0), 1.5 mM MgCl2, 50 mM KCl and 10 pmol of each primer. Using ABI PRISM 7000 real-time PCR system and Smart Quant Green Master Mix (Protech, PT-GL-SQGR), real-time PCR was performed in triplicate in a 10 lL reaction volume. The primer sequences for real-time PCR amplification were: RSV sense 50 -AGGATTGTTTATGAATGCCTATGGT-30 and antisense 50 -GCTTTTGGGTTGTTCAATATATGGTAG30 ; human IL-6 sense 50 -GCACTGGCAGAAAACAACCT-30 and antisense 50 -CAGGGGTGGTTATTGCATCT-30 ; human b-actin sense 50 -TCATCACCATTGGCAATGAG-30 and antisense 50 -CACTGTGTTGGCGTACAGGT-30 ; mouse b-actin sense 50 -CCACACCCGCCACCAGTTCG-30 and antisense 50 -CCCATTCCCACCATCACACC-30 . 2.6. Statistical analysis Statistical analysis was performed using ANOVA. All values are presented as means ± standard deviation. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Effects of FIP-fve treatment on RSV infection in BALB/c mice First, we purified FIP-fve from F. velutipes. Purity was confirmed on SDS–PAGE analysis with Coomassie blue staining (Supplemen-
126
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
tary Fig. 1A). To assess the cytotoxic effects of FIP-fve, HEp-2 cells were treated with various concentrations of FIP-fve for 24 h. The results showed no significant cellular toxicity (Supplementary Fig. 1B). To determine whether FIP-fve administered during RSV infection affects the consequences of infection in mice, daily FIPfve was administered orally from 2 days before RSV infection to 6 days after RSV infection, followed by inhalation of methacholine. A decrease in AHR was observed when compared with RSV group (Fig. 1A). Airway inflammation was assessed by cytokines in BALF and evaluated on ELISA. The levels of IL-6 (Fig. 1B) and IFN-c (Fig. 1C) in BALF increased significantly in the RSV group when compared with the untreated control group. The FIP-fve/RSV group demonstrated lower levels of IL-6 (NC, 470.0 ± 21.5 pg/ml; RSV, 1157.3 ± 187.6 pg/ml; FIP, 854.3 ± 19.3 pg/ml; P < 0.05) and higher levels of IFN-c (NC, non-detected; RSV, 69.7 ± 13.6 pg/ml; FIP, 251.2 ± 96.6 pg/ml; P < 0.05) in BALF. Levels of IL-4, IL-10 and TGF-b did not differ among the FIP-fve/RSV group and the remaining two study groups (below the level of detection; data not shown). We also detected RSV content in lungs of mice on real time quantitative PCR. The whole lungs of mice were homogenized followed by extraction of mRNA. The mRNA level of RSV in lung decreased to 0.6-fold in FIP-fve/RSV group when compared with RSV group (as 1.0). However, this difference did not reach statistical significance (Fig. 1D). Tissue sections were fixed in formalin and histopathological analyses of the lungs were performed. Infection of mice with RSV resulted in the development of cell infiltration of peribronchial and perivascular airway tissue. FIP-fve administered during infection reduced this goblet cell metaplasia (Fig. 2A–C). Immunohistochemistry of paraffin-embedded lungs was performed using RSV-specific antibody. Uninfected mice did
not demonstrate positive staining (Fig. 2D). RSV rate was reduced in lung tissues of FIP-fve/RSV group when compared with RSV group (Fig. 2E and F). The H scores of RSV immunostaining were significantly higher in the lung tissues of RSV group (H score = 1.2) when compared with the lung tissues of normal group (H score = 1.2) and significantly lower in the lung tissues of FIP-fve/ RSV group when compared with the lung tissues of normal group (H score = 0.2) (Fig. 2G). 3.2. Effects of FIP-fve on plaque formation We examined the efficacy of FIP-fve as an antiviral agent against RSV in vitro. Pre-treated HEp-2 cells were incubated with FIP-fve at 0, 7.5, or 30 lM for 2 h, then infected with 0.0002 MOI RSV for 1 h. Post-treated HEp-2 cells were infected with 0.0002 MOI RSV, followed by incubation with various concentrations of FIP-fve. Pre- and post-treated HEp-2 cells were overlaid with 0.75% methylcellulose-DMEM medium with 0, 7.5, or 30 lM FIPfve after aspiration of the content. The plates were incubated for 6 days then stained with crystal violet. In the pre-treated group, the plaque count was 65.7 ± 2.1 for the control group and decreased to 25.7 ± 1.2 and 10.3 ± 1.2 in 7.5 lM and 30 lM FIPfve treated cells, respectively (IC50 = 6.2 lM). In the post-treated group, the plaque count was 68.0 ± 4.0 for the control group and decreased to 51.3 ± 3.5 and 33.7 ± 2.5 in 7.5 lM and 30 lM FIPfve treated cells, respectively (IC50 = 29.7 lM) (Fig. 3A and B). We also detected RSV plaques with RSV specific immunostaining. Monolayers of HEp-2 cells were pretreated with FIP-fve for 2 h and infected with 0.0003 MOI RSV for 1 h, followed by removal of the contents. The untreated and treated cultures were overlaid
Fig. 1. Effects of FIP-fve on AHR and cytokines in mouse BALF. BALB/c mice (6–8 weeks old) were inoculated with 2 105 PFU of RSV. FIP-fve at 10 ml/kg was administered for 2 days before RSV infection and for 6 days after infection. (A) Airway responsiveness to inhaled methacholine at 0, 5, 10, 20, and 40 mg/ml was measured. IL-6 (B) and IFNc (C) levels in BALF were assessed on day 6 after infection. (D) RSV content in mouse lung on real time quantitative PCR. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
127
Fig. 2. Effects of FIP-fve treatment on airway responsiveness in RSV-infected mice. BALB/c mice (6–8 weeks old) were inoculated with 2 105 PFU of RSV. FIP-fve at 10 kg/ml was administered 2 days before RSV infection and for 6 days after infection. BALB/c mice lungs were harvested for histologic analyses and revealed histopathological changes such as infiltration induced by RSV. Compared with sham-inoculated control mice (A), RSV-infected mice (B) and RSV-infected mice with FIP-fve administration (C) developed characteristic peribronchial and perivascular airway tissue infiltration. On immunohistochemical staining (IHC) with RSV-specific antibody at 1/100 dilution alveolar epithelium was negative in control group (D). There was visible staining in RSV group (E) and FIP-fve/RSV group (F). (G) H scores of RSV immunostaining. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
with 0.75% methylcellulose-DMEM medium and incubated for 6 days after infection. The plates were fixed followed by staining with RSV specific antibody. The plaque count was 111.0 ± 7.0 for the control group and decreased to 17.3 ± 4.7 and 0 in 7.5 lM and 30 lM FIP-fve treated cells, respectively (Fig. 3C and D).
3.3. FIP-fve reduces the replication of RSV and RSV-induced IL-6 We examined the effects of FIP-fve on RSV and IL-6 in HEp-2 cells. Following pre-treatment or post-treatment with FIP-fve and infection with RSV at 0.1, 0.5 or 3 MOI for 24 h, RSV G protein
128
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
Fig. 3. Effects of FIP-fve treatment on RSV replication in HEp-2 cells. Pre-treated HEp-2 cell monolayers (3 105 cells/well) were incubated with FIP-fve at 0, 7.5, or 30 lM for 2 h before or after RSV for 1 h. After RSV treatment, pre- and post-treated HEp-2 cells were overlaid with 0.75% methylcellulose-DMEM medium with 0, 7.5, or 30 lM FIP-fve after aspiration of the content. The plates were incubated in 5% CO2 at 37 °C for 6 days. (A and B) Fixing and staining with 0.1% crystal violet. (C and D) Fixing and staining with RSV specific antibody. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
expression in HEp-2 cells was detected on Western blot. The data showed that pre-treatment with FIP-fve inhibits RSV G protein expression. For the pre-treated group, the IC50 value was 7.5 lM at 3 MOI RSV infection (Fig. 4A). This value was not reduced in the post-treated group. For the post-treated group, the IC50 value was >30 lM at 3 MOI RSV infection (Fig. 4B). Pre-treatment with FIP-fve inhibited RSV replication after 24 h infection. We next treated HEp-2 cells with FIP-fve for 2 h, followed by infection with RSV at 0.1 and 0.5 MOI for 24 h, 48 h and 72 h, respectively. FIP-fve decreased the expression of RSV G protein at 48 h and 72 h but not at 24 h (Fig. 4C). We analyzed RSV infection via confocal microscopy. HEp-2 cells were pre-treated with FIP-fve for 2 h then infected with 3 MOI RSV for 1 h, followed by fixing and staining with RSV specific antibody. The data showed that FIP-fve does not affect RSV infection (Fig. 4D). The IL-6 level in the cell culture supernatant was induced to 58684.9 qg/ml following RSV infection. After 7.5 lM and 30 lM FIP-fve treatment, IL-6 levels decreased to 41342.8 pg/ml and 16276.0 pg/ml, respectively (Fig. 4E). On RT-PCR, the mRNA levels of RSV decreased to 0.9-fold at 7.5 lM and 0.4-fold at 30 lM FIP-fve treatment. The mRNA levels of IL-6 in HEp-2 decreased to 0.6-fold at 7.5 lM and 0.3-fold at 30 lM FIP-fve treatment when compared with RSV-infected HEp-2 cells, respectively (Fig. 4F). On real-time quantitative PCR, the mRNA levels of RSV were 1.2-fold at 7.5 lM and 0.6-fold at 30 lM FIP-fve treatment. The mRNA levels of IL-6 decreased to
0.4-fold at 7.5 lM and 0.1-fold at 30 lM FIP-fve treatment when compared with RSV-infected HEp-2 cells, respectively (Fig. 4G). 3.4. FIP-fve inhibits RSV-induced NF-jB translocation in A549 cells RSV induces the inflammation of respiratory system. The HEp-2 cells are located on upper respiratory tract. Our previous data showed that FIP-fve inhibited RSV-induced inflammation in HEp2 cells. It has been reported that RSV induced lung inflammation (Ishioka et al., 2011). We further investigated whether FIP-fve inhibited RSV-induced lung cell inflammation by pulmonary alveolar epithelial cells (A549 cell line). To investigate whether FIP-fve inhibits RSV-induced inflammation through NF-jB pathway, laser excited confocal microscope fluorescence method was used to achieve p65 translocation. A549 cells were seeded onto 24 wells with coverslip overnight followed by FIP-fve pre-treatment for 2 h and RSV infection for 8 h. Treatment of A549 cells with RSV induced a 5.6-fold increase in the mean fluorescence intensity of NF-jB/p65 in the nucleus when compared with control cells and significant decreases in FIP-fve content (2.4-fold at 7.5 lM and 1.8-fold at 30 lM) (Fig. 5A and B). We also investigated TNF-ainduced NF-jB translocation as controls. A549 cells were pre-treated with FIP-fve for 8 h followed by TNF-a (50 gg/ml) for 20 min. FIP-fve showed inhibitory ability in TNF-a-induced NF-jB translocation (Fig. 5C and D).
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
129
Fig. 4. FIP-fve inhibits expression of RSV and IL-6 in HEp-2 cells. HEp-2 cells (2 105 cells/well) were (A) pre-treated with FIP-fve (0, 7.5 30 lM) for 2 h then infected or not infected with RSV (MOI = 3) for 1 h. (B) Post-treatment with FIP-fve with RSV infection (MOI = 3) or without RSV infection for 1 h. Then, 0.5 ml 10% FBS-DMEM were added at 1 h after infection for 24 h. Western blot was performed to analyze RSV protein expression. (C) HEp-2 cells (2 105 cells/well) were infected or not infected with RSV (MOI = 0.1, 0.5) for one hour followed by post-treatment with FIP-fve (0, 7.5 30 lM) for 2 h. After infection for one hour, 0.5 ml 10% FBS-DMEM were added. The cells were incubated for 24, 48 and 72 h. Western blot was performed to analyze RSV protein expression. (D) HEp-2 cells (3 104 cells/well) were seeded onto 24 wells with coverslip. These HEp-2 cells were pretreated with FIP-fve (0, 7.5, 30 lM) for 2 h then inoculated with RSV at MOI of 3 for 1 h. This was followed by staining with RSV specific antibody and FITC-conjugated anti-mouse antibody (Green). (E) Conditioned media were subjected to ELISA to measure amounts of secreted IL-6 (mean value from three independent experiments). Total cellular RNA from HEp-2 cells was analyzed using (F) RT-PCR and (G) real-time PCR for RSV, IL-6 and b-actin expressions. The experiments were performed in triplicate. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
4. Discussion We have shown that FIP-fve suppresses AHR and IL-6 secretion in BALF of RSV-infected BALB/c mice. We have also demonstrated that FIP-fve inhibits plaque formation and IL-6 expression induced by RSV infection. Moreover, FIP-fve down regulates the nuclear translocation of NF-jB. We suggest that FIP-fve inhibits RSV replication and RSV-induced inflammation via reduction in NF-jB translocation.
Py-Im polyamide 1 binds to the jB sites within the IL-6 and IL-8 promoters. Incubation of A549 cells with Py-Im polyamide 1, followed by TNF-a stimulation, results in a significant reduction in both IL-6 and IL-8 expressions (Raskatov et al., 2012). In our study, FIP-fve exhibited similar inhibition of TNF-a-induced NF-jB translocation in HEp-2 cells (data not shown). TLR4 with small interfering RNA attenuates high glucose-induced IjB/NF-jB activation and inhibits downstream synthesis of IL-6 and CCL-2. RSV induces
130
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
Fig. 5. Effects of FIP-fve treatment on NF-jB translocation in RSV-infected cells. (A and B) A549 cells (1 104 cells/well) were seeded onto 24 wells with coverslip. A549 cells were pretreated with FIP-fve (0, 7.5, 30 lM) for 2 h then inoculated with RSV at MOI of 3. A549 cells were stained with Texas RedÒ-X phalloidin conjugate after 8 h of infection to detect NF-jB. (C and D) A549 cells (1 104 cells/well) were seeded onto 24 wells with coverslip. A549 cells were pretreated with FIP-fve (0, 7.5, 30 lM) for 8 h then inoculated with TNF-a (50 gg/ml) for 20 min. Cells with NF-jB translocation are indicated by the white arrows. Cells without NF-jB translocation are indicated by the yellow arrows. The experiments were performed in triplicate. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
p65Ser536 phosphorylation resulting from the recognition of replicative viral RNA by RIG-I and TRAF6 functions downstream of RIG-I to trigger NF-jB pathway (Yoboua et al., 2010). In our study, FIP-fve inhibited RSV-induced IL-6 secretion by blocking the NF-jB pathway. In a recent study, the expression and barrier function of tight junction molecules claudin-4 and occludin were induced in addition to IL-8 and TNF-a in human nasal epithelial cells after RSV infection. The increase in tight junction molecules caused budding and replication of RSV, which was regulated through a protein kinase C d/hypoxia-inducible factor-1a/NF-jB pathway (Masaki et al., 2011). The results of the present study suggested that FIP-fve reduces RSV replication by downregulating NF-jB translocation. FIP-fve directly interacts with the target substrate by binding to polysaccharides near the substrates, possibly the TLR4 receptor. LZ-8 is an immunomodulatory protein from Ganoderma lucidum with 63% protein sequence identity with FIP-fve (Liu et al., 2012). It has been reported that anti-TLR4 mAb treatment of human dendritic cells significantly blocks rLZ-8-induced IL-12 p40 and IL-10 production. However, these effects are abolished by anti-TLR1, TLR2, and -TLR3 mAb (Lin et al., 2009). It has also been shown that rLZ-8 primes macrophages via TLR4-independent modus. Moreover, rLZ-8 binding to the surface of a TLR4-deficient macrophage has been demonstrated using fluorescence (Yeh et al., 2010). We speculated that FIP-fve inhibits IL-6 production partly via TLR-4/ MD2 pathway. It has been reported that TLR-4 enhances IFN-c production (Kim and Chung, 2012), and increased IFN signaling attenuates viral replication (Collins and Graham, 2008). The NS1 and NS2 proteins of RSV mediate resistance to the antiviral action of IFN-a/b (Valarcher et al., 2003). These are possible reasons for the pronounced effects of FIP-fve when administered prior to
infection. Moreover, FIP-fve is a stable protein that can be taken orally. FIP-fve has been shown to be resistant to digestive enzymes in simulated gastric fluid and simulated intestinal fluid (Ou et al., 2009). In summary, our findings provide clear evidence that FIP-fve is a potent suppressor of not only RSV infection but also subsequent inflammation. We propose that FIP-fve, a natural compound, is a safe and stable antiviral agent.
Conflict of interest statement The authors have no financial conflicts of interest. Acknowledgments We would like to thank Dr. Yen-Hung Chow for providing A2 strain RSV and HEp-2 cell line. Confocal microscopy was performed at the Instrument Center of Chung Shan Medical University. This study was funded by grants from the National Science Council, Ministry of Education, and Chung Shan Medical University Hospital (CSH-2011-D-001, CSH-2012-D-002). This manuscript was copy edited by Cheryl Robbins, a professional native English speaking editor.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral. 2014.08.006.
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
References Collins, P.L., Graham, B.S., 2008. Viral and host factors in human respiratory syncytial virus pathogenesis. J. Virol. 82, 2040–2055. DeVincenzo, J.P., Wilkinson, T., Vaishnaw, A., Cehelsky, J., Meyers, R., Nochur, S., Harrison, L., Meeking, P., Mann, A., Moane, E., Oxford, J., Pareek, R., Moore, R., Walsh, E., Studholme, R., Dorsett, P., Alvarez, R., Lambkin-Williams, R., 2010. Viral load drives disease in humans experimentally infected with respiratory syncytial virus. Am. J. Respir. Crit. Care Med. 182, 1305–1314. Dubois, A., Deruytter, N., Adams, B., Kanda, A., Delbauve, S., Fleury, S., Torres, D., Francois, A., Petein, M., Goldman, M., Dombrowicz, D., Flamand, V., 2010. Regulation of Th2 responses and allergic inflammation through bystander activation of CD8+ T lymphocytes in early life. J. Immunol. 185, 884–891. Fink, K., Duval, A., Martel, A., Soucy-Faulkner, A., Grandvaux, N., 2008. Dual role of NOX2 in respiratory syncytial virus- and sendai virus-induced activation of NFkappaB in airway epithelial cells. J. Immunol. 180, 6911–6922. Ghosh, S., Hayden, M.S., 2008. New regulators of NF-kappaB in inflammation. Nat. Rev. Immunol. 8, 837–848. Han, J., Jia, Y., Takeda, K., Shiraishi, Y., Okamoto, M., Dakhama, A., Gelfand, E.W., 2010. Montelukast during primary infection prevents airway hyperresponsiveness and inflammation after reinfection with respiratory syncytial virus. Am. J. Respir. Crit. Care Med. 182, 455–463. Harris, J., Werling, D., 2003. Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response. Cell. Microbiol. 5, 671– 680. Hayden, M.S., West, A.P., Ghosh, S., 2006. NF-kappaB and the immune response. Oncogene 25, 6758–6780. Ishioka, T., Kimura, H., Kita, H., Obuchi, M., Hoshino, H., Noda, M., Nishina, A., Kozawa, K., Kato, M., 2011. Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549). Cell Biol. Int. 35, 467–474. Karpurapu, M., Wang, X., Deng, J., Park, H., Xiao, L., Sadikot, R.T., Frey, R.S., Maus, U.A., Park, G.Y., Scott, E.W., Christman, J.W., 2011. Functional PU.1 in macrophages has a pivotal role in NF-kappaB activation and neutrophilic lung inflammation during endotoxemia. Blood 118, 5255–5266. Kim, H.S., Chung, D.H., 2012. TLR4-mediated IL-12 production enhances IFN-gamma and IL-1beta production, which inhibits TGF-beta production and promotes antibody-induced joint inflammation. Arthritis Res. Ther. 14, R210. Ko, J.L., Hsu, C.I., Lin, R.H., Kao, C.L., Lin, J.Y., 1995. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur. J. Biochem. 228, 244–249. Lin, Y.L., Liang, Y.C., Tseng, Y.S., Huang, H.Y., Chou, S.Y., Hseu, R.S., Huang, C.T., Chiang, B.L., 2009. An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and MAPK pathways. J. Leukoc. Biol. 86, 877–889. Liu, Y.F., Chang, S.H., Sun, H.L., Chang, Y.C., Hsin, I.L., Lue, K.H., Ko, J.L., 2012. IFNgamma induction on carbohydrate binding module of fungal immunomodulatory protein in human peripheral mononuclear cells. J. Agric. Food Chem. 60, 4914–4922. Masaki, T., Kojima, T., Okabayashi, T., Ogasawara, N., Ohkuni, T., Obata, K., Takasawa, A., Murata, M., Tanaka, S., Hirakawa, S., Fuchimoto, J., Ninomiya, T., Fujii, N.,
131
Tsutsumi, Himi, T., Sawada, N., 2011. A nuclear factor-kappaB signaling pathway via protein kinase C delta regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells. Mol. Biol. Cell 22, 2144–2156. Nair, H., Nokes, D.J., Gessner, B.D., Dherani, M., Madhi, S.A., Singleton, R.J., O’Brien, K.L., Roca, A., Wright, P.F., Bruce, N., Chandran, A., Theodoratou, E., Sutanto, A., Sedyaningsih, E.R., Ngama, M., Munywoki, P.K., Kartasasmita, C., Simoes, E.A., Rudan, I., Weber, M.W., Campbell, H., 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375, 1545–1555. Ou, C.-C., Hsiao, Y.-M., Wang, W.-H., Ko, J.-L., Lin, M.-Y., 2009. Stability of fungal immunomodulatory protein, FIP-gts and FIP-fve, in IFN-c production. Food Agric. Immunol. 20, 319–332. Raskatov, J.A., Meier, J.L., Puckett, J.W., Yang, F., Ramakrishnan, P., Dervan, P.B., 2012. Modulation of NF-kappaB-dependent gene transcription using programmable DNA minor groove binders. Proc. Natl. Acad. Sci. U.S.A. 109, 1023–1028. Stark, J.M., Khan, A.M., Chiappetta, C.L., Xue, H., Alcorn, J.L., Colasurdo, G.N., 2005. Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection. J. Infect. Dis. 191, 387–395. Swanson, K.A., Settembre, E.C., Shaw, C.A., Dey, A.K., Rappuoli, R., Mandl, C.W., Dormitzer, P.R., Carfi, A., 2011. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl. Acad. Sci. U.S.A. 108, 9619–9624. Teixeira, L.K., Fonseca, B.P., Vieira-de-Abreu, A., Barboza, B.A., Robbs, B.K., Bozza, P.T., Viola, J.P., 2005. IFN-gamma production by CD8+ T cells depends on NFAT1 transcription factor and regulates Th differentiation. J. Immunol. 175, 5931– 5939. Valarcher, J.F., Furze, J., Wyld, S., Cook, R., Conzelmann, K.K., Taylor, G., 2003. Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins. J. Virol. 77, 8426–8439. Wang, P.H., Hsu, C.I., Tang, S.C., Huang, Y.L., Lin, J.Y., Ko, J.L., 2004. Fungal immunomodulatory protein from Flammulina velutipes induces interferongamma production through p38 mitogen-activated protein kinase signaling pathway. J. Agric. Food Chem. 52, 2721–2725. Wang, P.H., Ko, J.L., Yang, S.F., Lin, L.Y., 2011. Implication of human nonmetastatic clone 23 Type 1 and its downstream gene lipocalin 2 in metastasis and patient’s survival of cancer of uterine cervix. Int. J. Cancer 129, 2380–2389. Whitman, L., Zhou, H., Perlman, S., Lane, T.E., 2009. IFN-gamma-mediated suppression of coronavirus replication in glial-committed progenitor cells. Virology 384, 209–215. Wright, P.F., Karron, R.A., Belshe, R.B., Shi, J.R., Randolph, V.B., Collins, P.L., O’Shea, A.F., Gruber, W.C., Murphy, B.R., 2007. The absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines. Vaccine 25, 7372–7378. Yeh, C.H., Chen, H.C., Yang, J.J., Chuang, W.I., Sheu, F., 2010. Polysaccharides PS-G and protein LZ-8 from Reishi (Ganoderma lucidum) exhibit diverse functions in regulating murine macrophages and T lymphocytes. J. Agric. Food Chem. 58, 8535–8544. Yoboua, F., Martel, A., Duval, A., Mukawera, E., Grandvaux, N., 2010. Respiratory syncytial virus-mediated NF-kappa B p65 phosphorylation at serine 536 is dependent on RIG-I, TRAF6, and IKK beta. J. Virol. 84, 7267–7277.
Contents lists available at ScienceDirect
Antiviral Research journal homepage: www.elsevier.com/locate/antiviral
Alleviation of respiratory syncytial virus replication and inflammation by fungal immunomodulatory protein FIP-fve from Flammulina velutipes Yu-Chi Chang a, Yen-Hung Chow b, Hai-Lun Sun c, Yu-Fan Liu d, Yu-Tzu Lee a, Ko-Huang Lue c,e,⇑, Jiunn-Liang Ko a,c,⇑ a
Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan Vaccine Research and Development Center, Miaoli County, National Health Research Institutes, Miaoli, Taiwan Division of Allergy, Department of Pediatrics, Chung Shan Medical University Hospital, Taichung, Taiwan d Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan e School of Medicine, Chung Shan Medical University, Taichung, Taiwan b c
a r t i c l e
i n f o
Article history: Received 6 May 2014 Revised 23 July 2014 Accepted 6 August 2014 Available online 14 August 2014 Keywords: Immunomodulatory protein FIP-fve Respiratory syncytial virus Inflammatory NF-jB Antiviral agent
a b s t r a c t Respiratory syncytial virus (RSV) causes bronchiolitis in children followed by inflammation and asthmalike symptoms. The development of preventive therapy for this virus continues to pose a challenge. Fungal immunomodulatory proteins (FIPs) exhibit anti-inflammatory function. FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes. To determine whether FIP-fve affects the infection or consequence of immunity of RSV, we investigated viral titers of RSV and inflammatory cytokine levels in vivo and in vitro. Oral FIP-fve decreased RSV-induced airway hyperresponsiveness (AHR), airway inflammation, and IL-6 expression in bronchoalveolar lavage fluid (BALF) of BALB/c mice. RSV replication and interleukin 6 (IL-6) levels in RSV-infected HEp-2 cells were compared before and after FIP-fve treatment. FIP-fve inhibited viral titers on plaque assay and Western blot, as well as inhibited RSV-stimulated expression of IL-6 on ELISA and RT-PCR. The results of this study suggested that FIP-fve decreases RSV replication, RSV-induced inflammation and respiratory pathogenesis. FIP-fve is a widely used, natural compound from F. velutipes that may be a safe agent for viral prevention and even therapy. Ó 2014 Published by Elsevier B.V.
1. Introduction Respiratory syncytial virus (RSV) is the most common cause of severe acute lower respiratory illness in children, in some cases leading to infant hospitalization and even death (Nair et al., 2010; Swanson et al., 2011). In addition, RSV has been associated with long term complications, such as recurrent wheezing and asthma. Recently, prevention of RSV disease has become possible. However, prophylaxis with potent neutralizing monoclonal antibody, Palizivumab, is only available to the highest risk infants (DeVincenzo et al., 2010). The development of an accessible preventive therapy for RSV infection has so far been unsuccessful due to problems associated with stability, purity, reproducibility, tolerability, and potency (Wright et al., 2007). RSV infection has been thought to result in the viral triggering of an exaggerated Th2 immune response and exacerbated pulmonary ⇑ Corresponding authors at: Room 704, No. 110, Sec. 1, Chung Shan Medical University, Institute of Medicine, Chien-Kuo N. Road, Taichung 40203, Taiwan. Tel.: +886 4 24730022 11694; fax: +886 4 24751101. E-mail addresses: [email protected] (K.-H. Lue), [email protected] (J.-L. Ko). http://dx.doi.org/10.1016/j.antiviral.2014.08.006 0166-3542/Ó 2014 Published by Elsevier B.V.
disease. The correlation between viral load and proinflammatory cytokines IL-6 and IL-8 suggests that if viral load is reduced by a robust antiviral, disease severity decreases even if the disease is mediated by these proinflammatory cytokines (DeVincenzo et al., 2010). In the presence of decreased NF-kB reporter activity in lung, liver, and spleen tissues challenged with lipopolysaccharides, there is attenuation of inflammatory gene expressions (cyclooxygenase2, inducible nitric-oxide synthase, and TLR4) and inflammatory cytokine secretion (Karpurapu et al., 2011). The role of NF-jB in mediating inflammation has been established using genetic approaches and chemical inhibitors (Ghosh and Hayden, 2008). Epithelial cells of infected tissue or tissue-resident haematopoietic cells, such as mast cells or dendritic cells (DCs), initiate the inflammatory response by triggering pro-inflammatory pathways through NF-jB in response to inflammatory stimuli (Hayden et al., 2006). RSV-triggered IKK activation, p65 nuclear accumulation, and DNA binding are characteristic of the classical pathway of NF-jB activation (Fink et al., 2008; Harris and Werling, 2003). Phosphorylation at Ser536 is essential for RSV-induced p65-mediated promoter transactivation in airway epithelial cells (Fink et al., 2008).
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
Fungal immunomodulatory protein (FIP-fve) has been isolated and purified from the edible golden needle mushroom (Flammulina velutipes) (Ko et al., 1995). It is comprised of 114 amino acid residues and characterized by a molecular mass of 13 kDa, a high degree of amino acid sequence homology with LZ-8 and 70 invariant amino acid residues (Ko et al., 1995). FIP-fve has been found to stimulate the production of IFN-c in PBMCs (Wang et al., 2004). Th1 cells produce IFN-c, which is essential for the eradication of intracellular pathogens (Teixeira et al., 2005). It has been demonstrated that glial cells are derived from neural progenitor cells with neurotropic JHM strain of mouse hepatitis virus infection and that treatment of infected cells with IFN-c inhibits viral replication in a dosedependent manner (Whitman et al., 2009). Strategies aimed at balancing neonatal Th1 and Th2 responses can facilitate the treatment of allergies (Dubois et al., 2010). Therefore, the aims of this study were to assess the ability of FIP-fve to disrupt RSV infection or the following inflammatory responses and to investigate the effects of FIP-fve on the viral load of RSV and RSV-induced inflammation. 2. Materials and methods 2.1. Intranasal immunization and RSV challenge Female BALB/c mice (6 weeks old) were purchased from the National Laboratory Animal Center, Taiwan, and animal experiments were approved by the Animal Use Committee of Chung Shan Medical University. The mice were divided into three groups of four mice each, as follows: (1) normal control group with oral and intranasal administration of PBS; (2) RSV group with oral administration of PBS and intranasal administration of 2 105 plaque-forming units (PFU) of purified RSV (in endotoxin-free PBS) for 6 days as previously described (Han et al., 2010); (3) FIP-fve/RSV group with oral administration of 10 ml/kg FIP-fve once a day from 2 days before RSV infection and intranasal administration of 2 105 PFU of purified RSV (in endotoxin-free PBS) for 6 days. The effects of FIP-fve on RSV-induced airway hyperresponsiveness (AHR) were measured using whole-body plethysmograph (Buxco, Troy, NY) with increasing doses of inhaled methacholine (Sigma– Aldrich, St. Louis, MO) as previously described (Stark et al., 2005). Immediately after measurement of AHR, lungs were lavaged through the trachea with 1 ml of normal saline to collect bronchoalveolar lavage fluid (BALF). BALF was aspirated and stored at – 70 °C until assay. The animals were sacrificed and the lungs were fixed in formalin and embedded in paraffin. Lung tissue sections were cut from the paraffin blocks and stained with hematoxylin and eosin or specific anti-RSV antibodies (Millipore Cat. No. 5006). 2.2. Measurement of cytokines The levels of IL-6 and IFN-r in BALF were assayed according to the manufacturer’s protocol (Mouse Interleukin-6, eBioscience catalog number 88-7064-88 and Mouse IFN-gamma DuoSet, R&D system catalog number DY485-05, respectively). The levels of IL-6 in medium of HEp-2 cells were assayed according to the manufacturer’s instructions (Human Interleukin-6, eBioscience Catalog Number 88-7066-88). 2.3. H Score The intensity of immunoreactivity was assessed by integrated optic density in a fixed field with a total pixel area of 150 150 on a scale of 0–3 using the Pro Plus 6.1 image analysis program (Media Cybernetic, Inc., Silver Spring, MD). The proportion of positive cells was calculated for each field. Three images of representative areas were acquired and at least 100 cells per tissue section were manually counted on a computer screen. The proportion of
125
positive cells was scored as follows: none, 0; less than one tenth, 0.1; less than one half, 0.5; and greater than one half, 1. Finally, a semi-quantitative H score of RSV immunoreactivity was determined by multiplying the four-tiered intensity score by the fourtiered proportion score of positive cells in a section. Minimum H score was 0 and maximum H score was 3. 2.4. Plaque-forming unit assay Plaque-forming unit assay for detecting abortive RSV replication was performed as previously described. Briefly, HEp-2 cells were seeded onto 12-well tissue culture plates at 3 105 cells/ well. The plates were incubated in a humidified incubator until the cell monolayer achieved confluence. The media were aspirated from each well and washed twice with PBS. Pretreatment with 300 ll FIP-fve per well at various doses (0, 7.5, 30 lM) was carried out for 2 h. Then, 300 ll diluted virus were added to each well, followed by mixing and incubation of the plates at 37 °C, 5% CO2 for 1 h. After the incubation period, the remaining media were aspirated from each well. This was followed by overlaying of 2.5 ml of 0.75% methyl cellulose in 2% FBS-medium in each well. The plates were further incubated at 37 °C in a humidified 5% CO2 incubator for 6 days. After 6 days, the plates were fixed and stained with crystal violet-methanol at room temperature for 20 min. RSV specific immunostaining of RSV plaques was performed on EnVision + Dual Link System-HRP (Dako North American, Inc.). 2.5. RT-PCR and real-time PCR The detailed protocols for RNA isolation and RT-PCR have been previously described (Wang et al., 2011). cDNA was reversetranscribed from 3 lg total cellular RNA using random hexamer primers and murine leukemia virus reverse transcriptase (Promega). The primer sequences for PCR amplification were: RSV sense 50 -AGCAAAGTCAAGTTGAATGATAC-30 and antisense 50 -GGCTGTAAGACCAGATCTATC-30 ; human IL-6 sense 50 -ATGAAGTTTCTCTCCGCAAGAGACTTCCAGCCAG-30 and antisense 50 CTAGGTTTGCCGAGTAGACCTCATAGTGACC-30 ; human b-actin sense 50 -CAGGGAGTGATGGTGGGCA-30 and antisense 50 -CAAACATCATCTGGT CATCTTCTC-30 . Two microliters of cDNA were amplified in a reaction volume of 50 lL containing 0.5 units of Taq polymerase (Promega), 400 lM dNTPS, 10 mM Tris–HCl (pH 8.0), 1.5 mM MgCl2, 50 mM KCl and 10 pmol of each primer. Using ABI PRISM 7000 real-time PCR system and Smart Quant Green Master Mix (Protech, PT-GL-SQGR), real-time PCR was performed in triplicate in a 10 lL reaction volume. The primer sequences for real-time PCR amplification were: RSV sense 50 -AGGATTGTTTATGAATGCCTATGGT-30 and antisense 50 -GCTTTTGGGTTGTTCAATATATGGTAG30 ; human IL-6 sense 50 -GCACTGGCAGAAAACAACCT-30 and antisense 50 -CAGGGGTGGTTATTGCATCT-30 ; human b-actin sense 50 -TCATCACCATTGGCAATGAG-30 and antisense 50 -CACTGTGTTGGCGTACAGGT-30 ; mouse b-actin sense 50 -CCACACCCGCCACCAGTTCG-30 and antisense 50 -CCCATTCCCACCATCACACC-30 . 2.6. Statistical analysis Statistical analysis was performed using ANOVA. All values are presented as means ± standard deviation. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Effects of FIP-fve treatment on RSV infection in BALB/c mice First, we purified FIP-fve from F. velutipes. Purity was confirmed on SDS–PAGE analysis with Coomassie blue staining (Supplemen-
126
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
tary Fig. 1A). To assess the cytotoxic effects of FIP-fve, HEp-2 cells were treated with various concentrations of FIP-fve for 24 h. The results showed no significant cellular toxicity (Supplementary Fig. 1B). To determine whether FIP-fve administered during RSV infection affects the consequences of infection in mice, daily FIPfve was administered orally from 2 days before RSV infection to 6 days after RSV infection, followed by inhalation of methacholine. A decrease in AHR was observed when compared with RSV group (Fig. 1A). Airway inflammation was assessed by cytokines in BALF and evaluated on ELISA. The levels of IL-6 (Fig. 1B) and IFN-c (Fig. 1C) in BALF increased significantly in the RSV group when compared with the untreated control group. The FIP-fve/RSV group demonstrated lower levels of IL-6 (NC, 470.0 ± 21.5 pg/ml; RSV, 1157.3 ± 187.6 pg/ml; FIP, 854.3 ± 19.3 pg/ml; P < 0.05) and higher levels of IFN-c (NC, non-detected; RSV, 69.7 ± 13.6 pg/ml; FIP, 251.2 ± 96.6 pg/ml; P < 0.05) in BALF. Levels of IL-4, IL-10 and TGF-b did not differ among the FIP-fve/RSV group and the remaining two study groups (below the level of detection; data not shown). We also detected RSV content in lungs of mice on real time quantitative PCR. The whole lungs of mice were homogenized followed by extraction of mRNA. The mRNA level of RSV in lung decreased to 0.6-fold in FIP-fve/RSV group when compared with RSV group (as 1.0). However, this difference did not reach statistical significance (Fig. 1D). Tissue sections were fixed in formalin and histopathological analyses of the lungs were performed. Infection of mice with RSV resulted in the development of cell infiltration of peribronchial and perivascular airway tissue. FIP-fve administered during infection reduced this goblet cell metaplasia (Fig. 2A–C). Immunohistochemistry of paraffin-embedded lungs was performed using RSV-specific antibody. Uninfected mice did
not demonstrate positive staining (Fig. 2D). RSV rate was reduced in lung tissues of FIP-fve/RSV group when compared with RSV group (Fig. 2E and F). The H scores of RSV immunostaining were significantly higher in the lung tissues of RSV group (H score = 1.2) when compared with the lung tissues of normal group (H score = 1.2) and significantly lower in the lung tissues of FIP-fve/ RSV group when compared with the lung tissues of normal group (H score = 0.2) (Fig. 2G). 3.2. Effects of FIP-fve on plaque formation We examined the efficacy of FIP-fve as an antiviral agent against RSV in vitro. Pre-treated HEp-2 cells were incubated with FIP-fve at 0, 7.5, or 30 lM for 2 h, then infected with 0.0002 MOI RSV for 1 h. Post-treated HEp-2 cells were infected with 0.0002 MOI RSV, followed by incubation with various concentrations of FIP-fve. Pre- and post-treated HEp-2 cells were overlaid with 0.75% methylcellulose-DMEM medium with 0, 7.5, or 30 lM FIPfve after aspiration of the content. The plates were incubated for 6 days then stained with crystal violet. In the pre-treated group, the plaque count was 65.7 ± 2.1 for the control group and decreased to 25.7 ± 1.2 and 10.3 ± 1.2 in 7.5 lM and 30 lM FIPfve treated cells, respectively (IC50 = 6.2 lM). In the post-treated group, the plaque count was 68.0 ± 4.0 for the control group and decreased to 51.3 ± 3.5 and 33.7 ± 2.5 in 7.5 lM and 30 lM FIPfve treated cells, respectively (IC50 = 29.7 lM) (Fig. 3A and B). We also detected RSV plaques with RSV specific immunostaining. Monolayers of HEp-2 cells were pretreated with FIP-fve for 2 h and infected with 0.0003 MOI RSV for 1 h, followed by removal of the contents. The untreated and treated cultures were overlaid
Fig. 1. Effects of FIP-fve on AHR and cytokines in mouse BALF. BALB/c mice (6–8 weeks old) were inoculated with 2 105 PFU of RSV. FIP-fve at 10 ml/kg was administered for 2 days before RSV infection and for 6 days after infection. (A) Airway responsiveness to inhaled methacholine at 0, 5, 10, 20, and 40 mg/ml was measured. IL-6 (B) and IFNc (C) levels in BALF were assessed on day 6 after infection. (D) RSV content in mouse lung on real time quantitative PCR. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄ p < 0.01, ⁄⁄⁄p < 0.001.
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
127
Fig. 2. Effects of FIP-fve treatment on airway responsiveness in RSV-infected mice. BALB/c mice (6–8 weeks old) were inoculated with 2 105 PFU of RSV. FIP-fve at 10 kg/ml was administered 2 days before RSV infection and for 6 days after infection. BALB/c mice lungs were harvested for histologic analyses and revealed histopathological changes such as infiltration induced by RSV. Compared with sham-inoculated control mice (A), RSV-infected mice (B) and RSV-infected mice with FIP-fve administration (C) developed characteristic peribronchial and perivascular airway tissue infiltration. On immunohistochemical staining (IHC) with RSV-specific antibody at 1/100 dilution alveolar epithelium was negative in control group (D). There was visible staining in RSV group (E) and FIP-fve/RSV group (F). (G) H scores of RSV immunostaining. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
with 0.75% methylcellulose-DMEM medium and incubated for 6 days after infection. The plates were fixed followed by staining with RSV specific antibody. The plaque count was 111.0 ± 7.0 for the control group and decreased to 17.3 ± 4.7 and 0 in 7.5 lM and 30 lM FIP-fve treated cells, respectively (Fig. 3C and D).
3.3. FIP-fve reduces the replication of RSV and RSV-induced IL-6 We examined the effects of FIP-fve on RSV and IL-6 in HEp-2 cells. Following pre-treatment or post-treatment with FIP-fve and infection with RSV at 0.1, 0.5 or 3 MOI for 24 h, RSV G protein
128
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
Fig. 3. Effects of FIP-fve treatment on RSV replication in HEp-2 cells. Pre-treated HEp-2 cell monolayers (3 105 cells/well) were incubated with FIP-fve at 0, 7.5, or 30 lM for 2 h before or after RSV for 1 h. After RSV treatment, pre- and post-treated HEp-2 cells were overlaid with 0.75% methylcellulose-DMEM medium with 0, 7.5, or 30 lM FIP-fve after aspiration of the content. The plates were incubated in 5% CO2 at 37 °C for 6 days. (A and B) Fixing and staining with 0.1% crystal violet. (C and D) Fixing and staining with RSV specific antibody. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.
expression in HEp-2 cells was detected on Western blot. The data showed that pre-treatment with FIP-fve inhibits RSV G protein expression. For the pre-treated group, the IC50 value was 7.5 lM at 3 MOI RSV infection (Fig. 4A). This value was not reduced in the post-treated group. For the post-treated group, the IC50 value was >30 lM at 3 MOI RSV infection (Fig. 4B). Pre-treatment with FIP-fve inhibited RSV replication after 24 h infection. We next treated HEp-2 cells with FIP-fve for 2 h, followed by infection with RSV at 0.1 and 0.5 MOI for 24 h, 48 h and 72 h, respectively. FIP-fve decreased the expression of RSV G protein at 48 h and 72 h but not at 24 h (Fig. 4C). We analyzed RSV infection via confocal microscopy. HEp-2 cells were pre-treated with FIP-fve for 2 h then infected with 3 MOI RSV for 1 h, followed by fixing and staining with RSV specific antibody. The data showed that FIP-fve does not affect RSV infection (Fig. 4D). The IL-6 level in the cell culture supernatant was induced to 58684.9 qg/ml following RSV infection. After 7.5 lM and 30 lM FIP-fve treatment, IL-6 levels decreased to 41342.8 pg/ml and 16276.0 pg/ml, respectively (Fig. 4E). On RT-PCR, the mRNA levels of RSV decreased to 0.9-fold at 7.5 lM and 0.4-fold at 30 lM FIP-fve treatment. The mRNA levels of IL-6 in HEp-2 decreased to 0.6-fold at 7.5 lM and 0.3-fold at 30 lM FIP-fve treatment when compared with RSV-infected HEp-2 cells, respectively (Fig. 4F). On real-time quantitative PCR, the mRNA levels of RSV were 1.2-fold at 7.5 lM and 0.6-fold at 30 lM FIP-fve treatment. The mRNA levels of IL-6 decreased to
0.4-fold at 7.5 lM and 0.1-fold at 30 lM FIP-fve treatment when compared with RSV-infected HEp-2 cells, respectively (Fig. 4G). 3.4. FIP-fve inhibits RSV-induced NF-jB translocation in A549 cells RSV induces the inflammation of respiratory system. The HEp-2 cells are located on upper respiratory tract. Our previous data showed that FIP-fve inhibited RSV-induced inflammation in HEp2 cells. It has been reported that RSV induced lung inflammation (Ishioka et al., 2011). We further investigated whether FIP-fve inhibited RSV-induced lung cell inflammation by pulmonary alveolar epithelial cells (A549 cell line). To investigate whether FIP-fve inhibits RSV-induced inflammation through NF-jB pathway, laser excited confocal microscope fluorescence method was used to achieve p65 translocation. A549 cells were seeded onto 24 wells with coverslip overnight followed by FIP-fve pre-treatment for 2 h and RSV infection for 8 h. Treatment of A549 cells with RSV induced a 5.6-fold increase in the mean fluorescence intensity of NF-jB/p65 in the nucleus when compared with control cells and significant decreases in FIP-fve content (2.4-fold at 7.5 lM and 1.8-fold at 30 lM) (Fig. 5A and B). We also investigated TNF-ainduced NF-jB translocation as controls. A549 cells were pre-treated with FIP-fve for 8 h followed by TNF-a (50 gg/ml) for 20 min. FIP-fve showed inhibitory ability in TNF-a-induced NF-jB translocation (Fig. 5C and D).
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
129
Fig. 4. FIP-fve inhibits expression of RSV and IL-6 in HEp-2 cells. HEp-2 cells (2 105 cells/well) were (A) pre-treated with FIP-fve (0, 7.5 30 lM) for 2 h then infected or not infected with RSV (MOI = 3) for 1 h. (B) Post-treatment with FIP-fve with RSV infection (MOI = 3) or without RSV infection for 1 h. Then, 0.5 ml 10% FBS-DMEM were added at 1 h after infection for 24 h. Western blot was performed to analyze RSV protein expression. (C) HEp-2 cells (2 105 cells/well) were infected or not infected with RSV (MOI = 0.1, 0.5) for one hour followed by post-treatment with FIP-fve (0, 7.5 30 lM) for 2 h. After infection for one hour, 0.5 ml 10% FBS-DMEM were added. The cells were incubated for 24, 48 and 72 h. Western blot was performed to analyze RSV protein expression. (D) HEp-2 cells (3 104 cells/well) were seeded onto 24 wells with coverslip. These HEp-2 cells were pretreated with FIP-fve (0, 7.5, 30 lM) for 2 h then inoculated with RSV at MOI of 3 for 1 h. This was followed by staining with RSV specific antibody and FITC-conjugated anti-mouse antibody (Green). (E) Conditioned media were subjected to ELISA to measure amounts of secreted IL-6 (mean value from three independent experiments). Total cellular RNA from HEp-2 cells was analyzed using (F) RT-PCR and (G) real-time PCR for RSV, IL-6 and b-actin expressions. The experiments were performed in triplicate. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
4. Discussion We have shown that FIP-fve suppresses AHR and IL-6 secretion in BALF of RSV-infected BALB/c mice. We have also demonstrated that FIP-fve inhibits plaque formation and IL-6 expression induced by RSV infection. Moreover, FIP-fve down regulates the nuclear translocation of NF-jB. We suggest that FIP-fve inhibits RSV replication and RSV-induced inflammation via reduction in NF-jB translocation.
Py-Im polyamide 1 binds to the jB sites within the IL-6 and IL-8 promoters. Incubation of A549 cells with Py-Im polyamide 1, followed by TNF-a stimulation, results in a significant reduction in both IL-6 and IL-8 expressions (Raskatov et al., 2012). In our study, FIP-fve exhibited similar inhibition of TNF-a-induced NF-jB translocation in HEp-2 cells (data not shown). TLR4 with small interfering RNA attenuates high glucose-induced IjB/NF-jB activation and inhibits downstream synthesis of IL-6 and CCL-2. RSV induces
130
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
Fig. 5. Effects of FIP-fve treatment on NF-jB translocation in RSV-infected cells. (A and B) A549 cells (1 104 cells/well) were seeded onto 24 wells with coverslip. A549 cells were pretreated with FIP-fve (0, 7.5, 30 lM) for 2 h then inoculated with RSV at MOI of 3. A549 cells were stained with Texas RedÒ-X phalloidin conjugate after 8 h of infection to detect NF-jB. (C and D) A549 cells (1 104 cells/well) were seeded onto 24 wells with coverslip. A549 cells were pretreated with FIP-fve (0, 7.5, 30 lM) for 8 h then inoculated with TNF-a (50 gg/ml) for 20 min. Cells with NF-jB translocation are indicated by the white arrows. Cells without NF-jB translocation are indicated by the yellow arrows. The experiments were performed in triplicate. The data are expressed as mean ± SD. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
p65Ser536 phosphorylation resulting from the recognition of replicative viral RNA by RIG-I and TRAF6 functions downstream of RIG-I to trigger NF-jB pathway (Yoboua et al., 2010). In our study, FIP-fve inhibited RSV-induced IL-6 secretion by blocking the NF-jB pathway. In a recent study, the expression and barrier function of tight junction molecules claudin-4 and occludin were induced in addition to IL-8 and TNF-a in human nasal epithelial cells after RSV infection. The increase in tight junction molecules caused budding and replication of RSV, which was regulated through a protein kinase C d/hypoxia-inducible factor-1a/NF-jB pathway (Masaki et al., 2011). The results of the present study suggested that FIP-fve reduces RSV replication by downregulating NF-jB translocation. FIP-fve directly interacts with the target substrate by binding to polysaccharides near the substrates, possibly the TLR4 receptor. LZ-8 is an immunomodulatory protein from Ganoderma lucidum with 63% protein sequence identity with FIP-fve (Liu et al., 2012). It has been reported that anti-TLR4 mAb treatment of human dendritic cells significantly blocks rLZ-8-induced IL-12 p40 and IL-10 production. However, these effects are abolished by anti-TLR1, TLR2, and -TLR3 mAb (Lin et al., 2009). It has also been shown that rLZ-8 primes macrophages via TLR4-independent modus. Moreover, rLZ-8 binding to the surface of a TLR4-deficient macrophage has been demonstrated using fluorescence (Yeh et al., 2010). We speculated that FIP-fve inhibits IL-6 production partly via TLR-4/ MD2 pathway. It has been reported that TLR-4 enhances IFN-c production (Kim and Chung, 2012), and increased IFN signaling attenuates viral replication (Collins and Graham, 2008). The NS1 and NS2 proteins of RSV mediate resistance to the antiviral action of IFN-a/b (Valarcher et al., 2003). These are possible reasons for the pronounced effects of FIP-fve when administered prior to
infection. Moreover, FIP-fve is a stable protein that can be taken orally. FIP-fve has been shown to be resistant to digestive enzymes in simulated gastric fluid and simulated intestinal fluid (Ou et al., 2009). In summary, our findings provide clear evidence that FIP-fve is a potent suppressor of not only RSV infection but also subsequent inflammation. We propose that FIP-fve, a natural compound, is a safe and stable antiviral agent.
Conflict of interest statement The authors have no financial conflicts of interest. Acknowledgments We would like to thank Dr. Yen-Hung Chow for providing A2 strain RSV and HEp-2 cell line. Confocal microscopy was performed at the Instrument Center of Chung Shan Medical University. This study was funded by grants from the National Science Council, Ministry of Education, and Chung Shan Medical University Hospital (CSH-2011-D-001, CSH-2012-D-002). This manuscript was copy edited by Cheryl Robbins, a professional native English speaking editor.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral. 2014.08.006.
Y.-C. Chang et al. / Antiviral Research 110 (2014) 124–131
References Collins, P.L., Graham, B.S., 2008. Viral and host factors in human respiratory syncytial virus pathogenesis. J. Virol. 82, 2040–2055. DeVincenzo, J.P., Wilkinson, T., Vaishnaw, A., Cehelsky, J., Meyers, R., Nochur, S., Harrison, L., Meeking, P., Mann, A., Moane, E., Oxford, J., Pareek, R., Moore, R., Walsh, E., Studholme, R., Dorsett, P., Alvarez, R., Lambkin-Williams, R., 2010. Viral load drives disease in humans experimentally infected with respiratory syncytial virus. Am. J. Respir. Crit. Care Med. 182, 1305–1314. Dubois, A., Deruytter, N., Adams, B., Kanda, A., Delbauve, S., Fleury, S., Torres, D., Francois, A., Petein, M., Goldman, M., Dombrowicz, D., Flamand, V., 2010. Regulation of Th2 responses and allergic inflammation through bystander activation of CD8+ T lymphocytes in early life. J. Immunol. 185, 884–891. Fink, K., Duval, A., Martel, A., Soucy-Faulkner, A., Grandvaux, N., 2008. Dual role of NOX2 in respiratory syncytial virus- and sendai virus-induced activation of NFkappaB in airway epithelial cells. J. Immunol. 180, 6911–6922. Ghosh, S., Hayden, M.S., 2008. New regulators of NF-kappaB in inflammation. Nat. Rev. Immunol. 8, 837–848. Han, J., Jia, Y., Takeda, K., Shiraishi, Y., Okamoto, M., Dakhama, A., Gelfand, E.W., 2010. Montelukast during primary infection prevents airway hyperresponsiveness and inflammation after reinfection with respiratory syncytial virus. Am. J. Respir. Crit. Care Med. 182, 455–463. Harris, J., Werling, D., 2003. Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response. Cell. Microbiol. 5, 671– 680. Hayden, M.S., West, A.P., Ghosh, S., 2006. NF-kappaB and the immune response. Oncogene 25, 6758–6780. Ishioka, T., Kimura, H., Kita, H., Obuchi, M., Hoshino, H., Noda, M., Nishina, A., Kozawa, K., Kato, M., 2011. Effects of respiratory syncytial virus infection and major basic protein derived from eosinophils in pulmonary alveolar epithelial cells (A549). Cell Biol. Int. 35, 467–474. Karpurapu, M., Wang, X., Deng, J., Park, H., Xiao, L., Sadikot, R.T., Frey, R.S., Maus, U.A., Park, G.Y., Scott, E.W., Christman, J.W., 2011. Functional PU.1 in macrophages has a pivotal role in NF-kappaB activation and neutrophilic lung inflammation during endotoxemia. Blood 118, 5255–5266. Kim, H.S., Chung, D.H., 2012. TLR4-mediated IL-12 production enhances IFN-gamma and IL-1beta production, which inhibits TGF-beta production and promotes antibody-induced joint inflammation. Arthritis Res. Ther. 14, R210. Ko, J.L., Hsu, C.I., Lin, R.H., Kao, C.L., Lin, J.Y., 1995. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur. J. Biochem. 228, 244–249. Lin, Y.L., Liang, Y.C., Tseng, Y.S., Huang, H.Y., Chou, S.Y., Hseu, R.S., Huang, C.T., Chiang, B.L., 2009. An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and MAPK pathways. J. Leukoc. Biol. 86, 877–889. Liu, Y.F., Chang, S.H., Sun, H.L., Chang, Y.C., Hsin, I.L., Lue, K.H., Ko, J.L., 2012. IFNgamma induction on carbohydrate binding module of fungal immunomodulatory protein in human peripheral mononuclear cells. J. Agric. Food Chem. 60, 4914–4922. Masaki, T., Kojima, T., Okabayashi, T., Ogasawara, N., Ohkuni, T., Obata, K., Takasawa, A., Murata, M., Tanaka, S., Hirakawa, S., Fuchimoto, J., Ninomiya, T., Fujii, N.,
131
Tsutsumi, Himi, T., Sawada, N., 2011. A nuclear factor-kappaB signaling pathway via protein kinase C delta regulates replication of respiratory syncytial virus in polarized normal human nasal epithelial cells. Mol. Biol. Cell 22, 2144–2156. Nair, H., Nokes, D.J., Gessner, B.D., Dherani, M., Madhi, S.A., Singleton, R.J., O’Brien, K.L., Roca, A., Wright, P.F., Bruce, N., Chandran, A., Theodoratou, E., Sutanto, A., Sedyaningsih, E.R., Ngama, M., Munywoki, P.K., Kartasasmita, C., Simoes, E.A., Rudan, I., Weber, M.W., Campbell, H., 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375, 1545–1555. Ou, C.-C., Hsiao, Y.-M., Wang, W.-H., Ko, J.-L., Lin, M.-Y., 2009. Stability of fungal immunomodulatory protein, FIP-gts and FIP-fve, in IFN-c production. Food Agric. Immunol. 20, 319–332. Raskatov, J.A., Meier, J.L., Puckett, J.W., Yang, F., Ramakrishnan, P., Dervan, P.B., 2012. Modulation of NF-kappaB-dependent gene transcription using programmable DNA minor groove binders. Proc. Natl. Acad. Sci. U.S.A. 109, 1023–1028. Stark, J.M., Khan, A.M., Chiappetta, C.L., Xue, H., Alcorn, J.L., Colasurdo, G.N., 2005. Immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection. J. Infect. Dis. 191, 387–395. Swanson, K.A., Settembre, E.C., Shaw, C.A., Dey, A.K., Rappuoli, R., Mandl, C.W., Dormitzer, P.R., Carfi, A., 2011. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl. Acad. Sci. U.S.A. 108, 9619–9624. Teixeira, L.K., Fonseca, B.P., Vieira-de-Abreu, A., Barboza, B.A., Robbs, B.K., Bozza, P.T., Viola, J.P., 2005. IFN-gamma production by CD8+ T cells depends on NFAT1 transcription factor and regulates Th differentiation. J. Immunol. 175, 5931– 5939. Valarcher, J.F., Furze, J., Wyld, S., Cook, R., Conzelmann, K.K., Taylor, G., 2003. Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins. J. Virol. 77, 8426–8439. Wang, P.H., Hsu, C.I., Tang, S.C., Huang, Y.L., Lin, J.Y., Ko, J.L., 2004. Fungal immunomodulatory protein from Flammulina velutipes induces interferongamma production through p38 mitogen-activated protein kinase signaling pathway. J. Agric. Food Chem. 52, 2721–2725. Wang, P.H., Ko, J.L., Yang, S.F., Lin, L.Y., 2011. Implication of human nonmetastatic clone 23 Type 1 and its downstream gene lipocalin 2 in metastasis and patient’s survival of cancer of uterine cervix. Int. J. Cancer 129, 2380–2389. Whitman, L., Zhou, H., Perlman, S., Lane, T.E., 2009. IFN-gamma-mediated suppression of coronavirus replication in glial-committed progenitor cells. Virology 384, 209–215. Wright, P.F., Karron, R.A., Belshe, R.B., Shi, J.R., Randolph, V.B., Collins, P.L., O’Shea, A.F., Gruber, W.C., Murphy, B.R., 2007. The absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines. Vaccine 25, 7372–7378. Yeh, C.H., Chen, H.C., Yang, J.J., Chuang, W.I., Sheu, F., 2010. Polysaccharides PS-G and protein LZ-8 from Reishi (Ganoderma lucidum) exhibit diverse functions in regulating murine macrophages and T lymphocytes. J. Agric. Food Chem. 58, 8535–8544. Yoboua, F., Martel, A., Duval, A., Mukawera, E., Grandvaux, N., 2010. Respiratory syncytial virus-mediated NF-kappa B p65 phosphorylation at serine 536 is dependent on RIG-I, TRAF6, and IKK beta. J. Virol. 84, 7267–7277.
