Elsevier

Antiviral Research

Volume 83, Issue 2, August 2009, Pages 191-200
Antiviral Research

Macrolide antibiotics inhibit respiratory syncytial virus infection in human airway epithelial cells

https://doi.org/10.1016/j.antiviral.2009.05.003Get rights and content

Abstract

To examine the effects of macrolide antibiotics on RS virus infection in airways, human tracheal epithelial cells were pre-treated with bafilomycin A1 and clarithromycin, and infected with RS virus. Viral titers in supernatant fluids and RNA of RS virus, and concentrations of cytokines in supernatant fluids, including interleukin-6 increased with time after infection. Bafilomycin A1 and clarithromycin reduced viral titers in supernatant fluids of RS virus, RNA of RS virus, the susceptibility to RS virus infection, and concentrations of cytokines induced by virus infection. N-acetyl-S-geranylgeranyl-l-cysteine, an inhibitor for a small GTP binding protein of RhoA, isoform A of the Ras-homologus (Rho) family, an active form of which is associated with RS virus infection via binding to its fusion protein (F protein), reduced viral titers in supernatant fluids and RNA of RS virus. Bafilomycin A1 and clarithromycin inhibited RhoA activation induced by lysophosphatidic acid in the cells. Fasudil, an inhibitor of Rho kinase, also reduced viral titers in supernatant fluids and RNA of RS virus. These findings suggest that macrolide antibiotics may inhibit RS virus infection, partly through the reduced expression of F protein receptor, activated RhoA, and the inhibition of subsequent Rho kinase activation in human airway epithelial cells.

Introduction

Respiratory syncytial (RS) virus is one of the important pathogens of common colds (Hayden and Gwaltney, 1988), and is the major cause of viral lower respiratory tract disease in infants and young children (Collins and Crowe, 2006). A relationship was reported between wheezing-associated respiratory illness and RS virus outbreaks in children (Henderson et al., 1979). RS virus infection was also suggested to be an important illness in the elderly and high-risk adults (Falsey et al., 2005, Zambon et al., 2001), and associated with the development of exacerbations of chronic obstructive pulmonary disease (COPD) (Guidry et al., 1991).

RS virus F glycoprotein, the part of the virus that binds to the receptor for RS virus (Collins and Crowe, 2006), can interact with the activated intracellular protein RhoA (Budge and Graham, 2004, Pastey et al., 1999), the isoform A of a small guanosine triphosphatase (GTPase) of the Ras superfamily (Rho, Ras-homologus) (Takai et al., 2001). The F protein promotes fusion of viral and cellular membranes with subsequent transfer of viral genome material into the cell, and promotes syncytial formation of the infected cells (Collins and Crowe, 2006). Pastey et al. (2000) demonstrated the inhibitory effects of a RhoA-derived peptide on syncytium formation induced by RS virus. RhoA signaling is also suggested to relate to cell-to-cell fusion and syncytium formation after RS virus infection (Gower et al., 2005). However, clinically available anti-RS virus agents have not been well studied.

RhoA has various functions including stimulus-evoked cell adhesion and motility, enhancement of contractile response and cytokinesis (Narumiya, 1996, Takai et al., 2001). RhoA functions are modulated by a variety of agents including bafilomycin A1, one of the macrolide antibiotics and a specific inhibitor of the vascular-ATPase (V-ATPase) (Palokangas et al., 1997), and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) (Lu et al., 2004). Macrolide antibiotics bafilomycin A1, erythromycin, and clarithromycin inhibit infection of rhinovirus (RV) (Jang et al., 2006, Suzuki et al., 2001a, Suzuki et al., 2002), the major cause of the common cold (Racaniello, 2006), in human airway epithelial cells by the reduction of intercellular adhesion molecule-1 (ICAM-1), the receptor for a major group of RV, and by affecting the acidification of endosomes, where RV RNA enters into the cytoplasm of infected cells. However, the inhibitory effects of macrolides on the infection of RS virus are still uncertain.

Neutrophilic and eosinophilc inflammation in the exacerbations of bronchial asthma and COPD are associated with a variety of mediators including interleukin (IL)-6 and IL-8, the production and secretion of which are stimulated by RS virus in airway epithelial cells as shown previously (Noah and Becker, 1993, Tripp et al., 2005). Bafilomycin A1, erythromycin and clarithromycin reduce pro-inflammatory cytokines including IL-6 after RV infection in airway epithelial cells (Jang et al., 2006, Suzuki et al., 2001a, Suzuki et al., 2002). Macrolide antibiotics have clinical benefits in improving the quality of life in refractory asthma patients (Simpson et al., 2008) and reducing COPD exacerbations (Seemungal et al., 2008). However, the inhibitory effects of macrolides on cytokine production after RS virus infection are still uncertain.

We therefore examined the inhibitory effects of bafilomycin A1 and clarithromycin, macrolide antibiotics, on RS virus infection. We also examined the effects of bafilomycin A1 and clarithromycin on the production of cytokines, and the RhoA activation to clarify the mechanisms responsible for the inhibition of RS virus infection.

Section snippets

Media components

Reagents for cell culture media were obtained as follows: Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, phosphate-buffered saline (PBS), and fetal calf serum (FCS) were from GIBCO-BRL Life Technologies, Palo Alto, CA; ultroser G (USG) was from BioSepra, Cergy-Saint-Christophe, France.

Human tracheal epithelial cell culture

Isolation and culture of the human tracheal surface epithelial cells were performed as described previously (Suzuki et al., 2001a, Suzuki et al., 2002, Terajima et al., 1997) with some modification (

Effects of macrolides on RS virus infection of human tracheal epithelial cells

Hep-2 cells did not show any syncytial formation when culture medium 2 h after removing inoculum and washing cells was added to the cells (data not shown), but supernatant fluids 1 day after infection produced syncytial formation on the cells (Fig. 1A). Exposing confluent human tracheal epithelial cell monolayers to RS virus (0.5 × 10−3 TCID50 units/cell) consistently led to infection. RS virus was detected in supernatants fluids 1 day after infection, and the viral content progressively increased

Discussion

In the present study, we have shown that viral titers in supernatant fluids and RNA of RS virus in the human tracheal epithelial cells increased with time, and bafilomycin A1, one of the macrolide antibiotics and a specific inhibitor of the vascular-ATPase (V-ATPase) (Palokangas et al., 1997) and a widely used macloride antibiotic clarithromycin reduced viral titers of RS virus in supernatant fluids concentration-dependently, RNA of RS virus replication, and the susceptibility to RS virus

Conflict of interest statement

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Acknowledgments

The authors thank Mr. Grant Crittenden for reading the manuscript. This study was partly supported by Abott-Japan Pharmaceutical Co., Ltd., Taisho-Toyama Pharmaceutical Co., Ltd. Japan, Health and Labour Sciences Research Grants for Research on Measures for Intractable Diseases from the Ministry of Health, Labour and Welfare (H20nannchiippann35) and the Respiratory Failure Research Group from the Ministry of Health, Labour and Welfare, Japan.

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