IntroductionHuman Rhinoviruses (HRVs) belong to the genus Enterovirus of the Picornaviridae family. There are over 160 serotypes of HRVs that are responsible for the common cold, which is a mild illness of the upper respiratory tract . HRVs may be associated with more severe diseases such as acute otitis media in children and sinusitis in adults . HRV infection has been also associated with different respiratory tract complications such as bronchiolitis, pneumonia, sinusitis and acute otitis media, and has been implicated in exacerbations of chronic respiratory disorders, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD) . Recently, experimental HRV infection in COPD patients established a causative relationship between virus infection and exacerbations, moreover these exacerbations were predicted by the World Health Organization to become one of the major causes of worldwide death in the next few decades .Due to the large number of serotypes, vaccination against HRVs is not feasible. Despite intensive efforts in antiviral research and development, no effective antiviral therapies have been approved for either the prevention or treatment of diseases caused by HRV . In this regard, many trials have been conducted to find antiviral components. For example, Pirodavir lowered HRV replication, whilst oral administration of Pleconaril led to a reduction in duration and severity of the illness . Treatment of HRV-induced exacerbations of asthma is still under clinical evaluation . Therefore, to date, the search for antiviral compounds against HRV is ongoing.Dexamethasone (DEX) has become one of the most commonly, and intensively used therapeutic agents in pediatric oncology . However, to date, no detailed study has been carried out on DEX’s antiviral activity against human rhinovirus 1B (HRV1B) in Hela cells.In this study the anti–HRV1B activity of DEX in HeLa cells, and in the lungs of HRV1B-infected mice using PCR after DEX treatment was studied. Furthermore, anti-HRV1B activity of DEX correlation with induction of autophagy activation via glucocorticoid receptor (GCR) was investigated.
1. Cell culture, viruses, and reagentsHuman rhinovirus 1B (HRV1B) was obtained from ATCC (Manassas, VA, USA) and propagated by infection at 33°C in HeLa cells. HeLa cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA, USA). DEX, rupintrivir (Rup), chloroquine diphosphate salt (CQ) and bafilomycin A1 (BAF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxycycline (DOX) was purchased from Clontech (Mountain View, CA, USA). For Tet-on system, the GCR shRNA-transfected HeLa cells were treated with doxycycline (2 mg/mL) in culture media every 2 days for 7 days.
2. Mice and virus infectionFour-week-old, female BALB/c mice were purchased from SPL animal company (Orient Bio Inc, Sungnam, Korea). Mice were maintained in accordance with the guidelines, and stabilized for 7 days at Kangwon National University. Mice were intranasally infected with HRV1B (1.8 × 107 pfu/mouse) 3 times with 10 μL/PBS at intervals of 10 minutes. DEX was intranasally administered at 0.1 mg/kg after HRV1B infection.
3. SRB assayThe SRB assay was used to measure antiviral activity as previously reported . The day before the experiment, HeLa cells (3 × 104 cells/well) were seeded in a 96-well culture plate. HeLa cells were used for HRV1B infection. After 24 hours, the HRV1B infection medium was replaced with 30 mM MgCl2, 1% FBS in MEM media. The HRV1B-infected HeLa cells were incubated at 33°C and 5% CO2 resulting in CPE. After 48 hours, the 96-well culture plate was washed with PBS, and fixed in 70% acetone (100 μL/well) for 30 minutes, followed by staining with 0.4% SRB (Sigma-Aldrich, St, Louis, MO, USA) in 1% acetic acid. The precipitated SRB crystals were solubilized with 10 mM unbuffered tris-based solution 100 μL/well. The absorbance was read on a SpectraMax i3 microplate reader (Molecular Devices, Palo Alto, CA, USA) at 562nm.
4. Real-time PCRTotal RNA was extracted from HeLa cells and lysate of mice lungs with a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed with RNase inhibitor, oligo (dT) 15 primers, dNTP mixture, and Moloney murine leukemia virus reverse transcriptase with 5 × buffer, according to an established protocol (Promega, Madison, WI, USA). Quantitative real- time PCR (qPCR) analysis was performed to amplify complementary deoxyribonucleic acid (cDNA), using the THUNDERBIRD® SYBR® qPCR mix (Toyobo, Osaka, Japan), and CFX96 optics module real-time PCR system (Bio-Rad, Hercules, CA, USA). The following primers: HRV 5′-NCR sense, 5′-TCC TCC GGC CCC TGA ATG-3′ and HRV 5′-NCR-antisense, 5′-GAA ACA CGG ACA CCC AAA G-3′; and human β-actin-sense, 5′-CCA TCA TGA AGT GTG ACG TGG-3′ and human β-actin-antisense, 5′-GTC CGC CTA GAA GCA TTT GCG-3′ were used. The PCR conditions were as follows: 95°C for 3 minutes for 1 cycle, and 95°C, 30 seconds; 60°C, 30 seconds; 72°C, 30 seconds; for 35 cycles.
5. Histological analysisThe HRV1B-infected lungs of mice were washed in PBS, and fixed with 4% (w/v) formaldehyde overnight. The HRV1B-infected lungs were dehydrated in serial gradients of ethanol and xylene, and embedded in paraffin. The tissues were sliced into 5-mm-thick sections and stained with hematoxylin and eosin. After HRV1B infection, the lungs with severe inflammation (score: 0–4) were graded for inflammation, edema, and cellular infiltration, according to the guidelines. A pathologist evaluated the degree of inflammation using a light microscope (200 X) and was blinded whilst scoring each slide. The means of the lung inflammatory score were graded for severity (absent, minimal, mild, moderate, marked).
6. Plasmid cloning and lentivirus productionPlasmid cloning and lentivirus production were carried out as described previously . To establish the Tet-inducible lentiviral shRNA expression system, the shRNA oligo was annealed and inserted into the Tet-pLKO-puro vector, which was gifted by Dmitri Wiederschain (plasmid #21915, Addgene, Cambridge, MA, USA). The Tet-pLKOblast vector was constructed with the blasticidin resistance gene. For shRNA sequences, TRCN0000245004 for GCRsh-1 was used. For lentivirus production in 293T cells, each lentiviral vector was co-transfected with pMD2.G and psPAX2 (gifted from Didler Trono: Addgene plasmid #12259, #12260), using Lipofectamine 2000.
7. Statistical analysisMultiple groups were compared using a 1-way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test using GraphPad Prism version 5 (GraphPad Software, La Jolla, CA, USA). Values of p < 0.05 were considered significant at a 95% confidence interval.
1. Dexamethasone showed antiviral activity against HRV1B in HeLa cellsTreatment of HRV1B-infected HeLa cells with DEX was performed at concentrations of 16, 80, 400 and 2000 nM. and showed cell viability of > 70% in a dose-dependent manner (Figure 1A). Treatment with Rup gave higher cell viability in HRV1B-infected HeLa cells (Figure 1A).The antiviral activity of DEX in HRV1B-infected HeLa cells assessed by RT-PCR, significantly reduced. HRV1B viral replication (Figure 1B). Treatment with Rup strongly reduced HRV1B viral replication (Figure 1B).
2. Dexamethasone ameliorates pulmonary inflammation and inhibits HRV1B replication in miceHistological evaluation of HRV1B-infected murine lung tissue showed characteristic HRV1B-infected lesions including necrotizing bronchiolitis, and interstitial pneumonia and scored a high histological score compared with lungs from control BALB/c mice which exhibited typical normal pulmonary tissue with a low histological score (Figures 2A and 2B). HRV1B-infected mice treated with DEX had moderate inflammation including increased necrosis, increased number of inflammatory cells and pulmonary edema and scored a moderate histological score (Figures 2A and 2B).To determine whether the decreased pulmonary inflammation was associated with decreased viral replication, RT-PCR was performed on lung tissue homogenate to assess HRV1B NCR gene expression. HRV1B-infected mice had increased viral replication, as detected by HRV1B NCR gene expression (Figure 2C). Treatment with DEX in HRV1B-infected mice decreased HRV1B NCR gene expression, suggesting a reduction in viral replication (Figure 2C).
3. Dexamethasone showed anti-HRV1B activity via GCR receptorTo assess whether the anti-HRV1B activity of DEX was mediated via the GCR in HeLa cells, the cells were conditionally depleted using the Tet-inducible knock out system. Treatment of GCR shRNA-transfected HeLa cells with DOX abolished the expression of GCR (Figure 3). The antiviral activity of DEX against HRV1B was significantly reduced in GCR knock-down HeLa cells (Figure 3), suggesting that the antiviral activity of DEX was mediated by GCR.
4. Dexamethasone inhibits HRV1B infection via activation of autophagyTo investigate whether autophagy activation by DEX correlated with the antiviral activity of DEX against HRV1B infection, autophagy was blocked with inhibitors including CQ or BAF. The results demonstrated that only treatment of DEX decreased cell viability in HRV1B-infected cells in a dose-dependent manner (Figure 4). Both CQ and BAF significantly attenuated the protective activity of DEX against HRV1B-infected cell viability (Figure 4).
DiscussionHRVs are involved in a range of clinical manifestations , but up to now viable options for the prevention or treatment of HRV infections remain limited. DEX has become one of the most commonly and intensively used therapeutic agents in pediatric oncology . Currently, no effective antiviral therapeutic effect of DEX has been reported for either the prevention or treatment of diseases caused by HRV infection. In the present study, the anti-HRV1B activity was evaluated in vitro. DEX significantly improved cell viability to > 70% in a dose-dependent manner in HRV1B-infected HeLa cells (Figure 1A). Furthermore, the antiviral activity of DEX was assessed by RT-PCR in HRV1B-infected HeLa cells. HRV1B viral replication was significantly reduced by DEX treatment (Figure 1B). Our results indicate that DEX exhibits anti–HRV1B virus activity in vitro.HRV predominantly infects cells of the upper respiratory tract, although HRV RNA has also been found in lower airways in experimental models of HRV infection . HRV is the etiological agent of approximately 50% to 70% of common colds . HRV infection is associated with bronchiolitis in human, pneumonia in the immunosuppressed and exacerbations of pre-existing pulmonary conditions such as asthma or chronic obstructive pulmonary disease [14,15]. In this study, only HRV1B-infected mice exhibited characteristic HRV1B-infected lesions including necrotizing bronchiolitis and interstitial pneumonia (Figure 2A), showing increment viral replication reflected by HRV1B NCR gene expression (Figure 2C). Following treatment with DEX, there was moderate inflammation, with increased necrosis and numbers of inflammatory cells and pulmonary edema in the lungs of HRV1B-infected mice with decreasing viral replication reflected by reduced HRV1B NCR gene expression (Figure 2C). Therefore, DEX greatly decreases HRV1B-associated inflammation and viral replication reflected by reduced HRV1B NCR gene expression in HRV1B-infected mice.Chronic inflammatory respiratory diseases are characterized by recurrent attacks of breathlessness and wheezing, which vary in severity and frequency from person to person . Glucocorticoids (GCs) are among the mainstays of treatment in asthmatics and exert their effects mainly via binding to their intracellular receptors (GCRs) . In this study, to assess whether the anti-HRV1B activity of DEX was mediated via the GCR, GCR in HeLa cells were conditionally depleted using the Tet-inducible knockout system. Treatment of DOX in GCR shRNA-transfected HeLa cells abolished the expression of GCR (Figure 3). DEX showed anti-HRV1B activity via mediation by the GCR (Figure 3).In our recent study, we reported that the glucocorticoid budesonide, showed anti-rhinoviral activity by inducing GCR-dependent autophagy activation . Another study reported that dexamethasone produced reactive oxygen species to activate autophagy . Autophagy is 1 of the 2 main cellular catabolic pathways, which is finely regulated to maintain cellular homeostasis . Moreover, autophagy strongly contributes to the innate and adaptive immune response . Autophagy plays an essential role in antiviral immune responses. Epstein-Barr virus (EBV), like other viruses that persist in the infected host, must interfere with this process to avoid its own elimination . The majority of herpesviruses have developed strategies to inhibit autophagy, especially in immune cells . In this current study, DEX treatment induced antiviral activity against HRV1B via autophagy activation (Figure 4).Collectively, DEX has demonstrated antiviral activity against HRV1B via GCR-dependent autophagy activation. Therefore, DEX is an attractive anti-HRV1B candidate with the potential to successfully prevent or treat HRV1B-associated malignancies.