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. 2013 Jul 23;8(7):e69670.
doi: 10.1371/journal.pone.0069670. Print 2013.

Roflumilast inhibits respiratory syncytial virus infection in human differentiated bronchial epithelial cells

Manuel Mata  1 , Isidoro Martinez, Jose A Melero, Herman Tenor, Julio Cortijo
Affiliations

Roflumilast inhibits respiratory syncytial virus infection in human differentiated bronchial epithelial cells

Manuel Mata et al. PLoS One. .

Abstract

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H2O2 and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H2O2 formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.

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Conflict of interest statement

Competing Interests: Dr. Hermann Tennor has declared competing interest. In concrete he is employed by Takeda Corporation, this does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Roflumilast N-oxide (RNO) alleviated viral burden following RSV infection, reduced RSV-induced ICAM-1 expression and restored cilia motility in well-differentiated human bronchial epithelial cells (WD-HBE).
WD-HBE were infected with RSV (2 x 106 PFU/insert) or mock-infected (control) in the presence of Roflumilast N-oxide (RNO; 0.1-100nM) or vehicle and maintained in air-liquid culture over another 10 days for viral quantification or 15 days for ICAM-1 and cilia motility evaluation. RSV abundance in WD-HBE was determined by immunocytochemical detection of RSV F and G proteins (panel A) or real-time RT-PCR based quantification of RSV nucleoprotein RNA (panel B). In panel B viral nucleoprotein RNA was related to host cell GAPDH and normalized to RSV infection with vehicle (=1). In mock-infected WD-HBE, RSV nucleoprotein RNA was below detection limit. ICAM1 expression was determined by Real Time RT-PCR using mock-infected cells as controls (panel C). Cilia motility was evaluated by HSDV using three independent videos of each experimental replicate. Results represent mean ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p< 0.05 compared to mock-infected cells. # p<0.05 compared to infected cells.
Figure 2
Figure 2. RSV compromised the number of β-tubulin IV labeled ciliated cells and the expression of Foxj1 and Dnai2: Reversal by roflumilast N-oxide.
WD-HBE were infected with RSV at 2 x 106 PFU/insert or mock infected and cultured in the presence of roflumilast N-oxide (RNO; 0.1-100nM) or vehicle (0.1% DMSO) over 10 days. To evaluate the number of ciliated cells sections from WD-HBE cells were immunostained for β tubulin IV as described in Methods. In (A) representative microphotographs from mock infected, RSV infected with vehicle, RSV infected with 1nM roflumilast N-oxide, RSV infected with 100nM roflumilast N-oxide sections are shown. β-tubulin IV staining is red (rhodamine), nuclei are blue (DAPI). The microphotographs are representative from three separate infections. For quantitative analyses the numbers of β-tubulin IV staining (ciliated) cells were counted in ten different sections of three separate infections and expressed as percent of ciliated cells in mock-infected, untreated controls (=100%) (panel B). At day 10 after infection total RNA of WD-HBE was extracted and the expression of Foxj1 (panel C) and Dnai2 (panel D) was quantified by real time RT-PCR in mock-infected (white bars) or RSV infected cells in the presence of roflumilast N-oxide (RNO; 0.1-100 nM; grey bars) or vehicle (black bars). Results are depicted as the means ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p< 0.05 compared to mock-infected cells. #p<0.05 versus RSV infected cells.
Figure 3
Figure 3. Roflumilast N-oxide mitigates enhanced MUC5AC and hCLCA1 mRNA expression and the expression and release of IL-13 following infection with RSV.
WD-HBE were infected with RSV at 2 x 106 PFU/insert in the presence of roflumilast N-oxide (0.1-100nM) or vehicle (0.1% DMSO) or mock infected and cultured until day 10 when measurements were performed. Total RNA was extracted and analyzed by real time RT-PCR for MUC5AC (panel A), hCLCA1 (panel B) and IL-13 (panel C) in mock (white bars) or RSV infected cultures in the absence (black bars) or presence (stippled bars) of roflumilast N-oxide (RNO). IL-13 release was evaluated by luminex in culture supernatants sampled as described in the Methods section. Data represent the means ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p< 0.05 compared to mock-infected cells. # p<0.05 compared to RSV infected cells.
Figure 4
Figure 4. RSV infection stimulated the expression and release of IL-8, IL-6 and TNFα from WD-HBE: Prevention by Roflumilast N-oxide.
WD-HBE were mock-infected or infected with RSV at 2 x 106 PFU/insert in the presence of Roflumilast N-oxide (RNO; 0.1-100nM) or vehicle (0.1% DMSO). Measurements of IL-8 (panel A), IL-6 (panel B) and TNFα (panel C) mRNA transcripts (upper panels) and released protein (lower panels) were performed at day 10 after infection. For quantitative mRNA expression analyses total RNA was extracted, reverse transcribed and analyzed by real time RT-PCR. For measurements of released cytokines apical supernatants from an one hour incubation as described in Methods were collected and analyzed using Luminex. Results are depicted as the means ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p< 0.05 compared to mock-infected cells. # p<0.05 compared to RSV infected cells.
Figure 5
Figure 5. Roflumilast N-oxide supported the anti-oxidative apparatus compromised in RSV-infected WD-HBE and reduced ROS.
WD-HBE were mock-infected or infected with RSV at 2 x 106 PFU/insert in the presence of Roflumilast N-oxide (RNO) at 1 nM or 100 nM or vehicle (0.1% DMSO). In order to quantify Nrf2 mRNA transcripts total RNA was extracted from RSV or mock infected cultures at days 2, 4, 10 and 15 after infection, reverse transcribed and analyzed by Real Time RT-PCR (panel A). HO1 (panel B) and GPX (panel C) mRNA were analyzed with the identical procedure at day 10 after infection. Again at day 10 after RSV or mock infection Total Antioxidant Capacity (TAC) (panel D) and H2O2 release (panel E) were analyzed in cell lysates using ABTS® or Amplex Red® reagents, respectively as indicated in the Methods. Results are shown as the means ± SEM of three independent infections. Each condition was evaluated by triplicate in three independent wells. Cultures from three different patients were used. * p<0.05 compared to mock-infected cells. # p<0.05 compared to infected cells.
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This work was supported by grants SAF2008-03113/SAF2011-26443 (JC), PI10/02294 (MM), and CIBERES (CB06/06/0027) from the Ministry of Economy and Competitiveness and the Health Institute ‘Carlos III’ of the Spanish Government and research grants from Regional Government (GV2007/287 and AP073/10, from GeneralitatValenciana). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.