Doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza H3N2 virus: Involvement of matrix metalloproteinases

Abstract

Acute respiratory distress syndrome, a severe form of acute lung injury (ALI), is a major cause of death during influenza pneumonia. We have provided evidence for the involvement of recruited neutrophils, their toxic enzymes such as myeloperoxidase and matrix metalloproteinases (MMPs), and neutrophil extracellular traps in aggravating alveolar-capillary damage. In this study, we investigated the effects of doxycycline (DOX), an inhibitor of MMPs, on influenza-induced ALI. BALB/c mice were infected with a sublethal dose of mouse-adapted virulent influenza A/Aichi/2/68 (H3N2) virus, and administered daily with 20 mg/kg or 60 mg/kg DOX orally. The effects of DOX on ALI were determined by measuring inflammation, capillary leakage, and MMP activities. Furthermore, levels of T1-α (a membrane protein of alveolar type I epithelium) and thrombomodulin (an endothelial protein) in the bronchoalveolar lavage fluid were evaluated by Western blot analysis. Our results demonstrate significantly decreased inflammation and protein leakage in the lungs after DOX treatment. Levels of MMP-2 and MMP-9 activity, T1-α and thrombomodulin were also diminished in the DOX-treated group. These findings were corroborated by histopathologic analyses, which demonstrated significant reduction in lung damage. Although DOX treatment reduced ALI, there were no effects on virus titers and body weights. Taken together, these results demonstrate that DOX may be useful in ameliorating ALI during influenza pneumonia. Further studies are warranted to determine whether DOX can be used in combination with anti-viral agents to alleviate severe influenza pneumonia.

Introduction

Influenza A viruses pose significant public health problems with frequent outbreaks worldwide (Ivan et al., 2012). The majority of deaths associated with influenza pneumonia are attributed to the sequelae of acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) (Yokoyama et al., 2010). We previously demonstrated that depletion of macrophages results in excessive influx of neutrophils during influenza pneumonia, and contributes to ALI/ARDS with severe hypoxemia. In contrast, depletion of neutrophils leads to only mild lung injury. We have also provided evidence for generation of neutrophil extracellular traps (NETs) that can aggravate alveolar-capillary damage (Narasaraju et al., 2011). Human cases and animal models of influenza pneumonia reveal prominent neutrophilic infiltration within the affected areas, thus implicating their role in lung injury (Wang et al., 2008). The recruitment and activation of neutrophils, together with their released toxic products such as matrix metalloproteinases (MMPs), myeloperoxidase (MPO), elastase, and reactive oxygen intermediates (ROI) can contribute to lung injury. Gelatinases (including MMP-2 and MMP-9) are zinc-dependent endopeptidases, degrade major components of the basement membrane such as gelatin and collagen IV, and exert deleterious effects on the epithelium and endothelium in the thin alveolar-capillary barrier (O'Connor and FitzGerald, 1994).

Gelatinases are implicated in many pathologic conditions including ARDS, cancer, and pulmonary fibrosis (Malemud, 2006, O'Connor and FitzGerald, 1994, Rundhaug, 2003). Infection of different cell lines with influenza virus can activate MMPs. Infection of Madin-Darby canine kidney (MDCK) cells with influenza virus increases MMP-2 activity and decreases MMP-9 activity, whereas MMP-9 activity in Vero cells increases with infection (Yeo et al., 1999). The induction of MMP-9 by tumor necrosis factor-α (TNFα) in the brain has been linked with encephalitis due to influenza infection. Cigarette smoke exposure prior to influenza virus infection further enhances the activities of MMP-2 and MMP-9 (Gualano et al., 2008). Although induction of MMPs during influenza is documented, their functional roles and mechanisms remain unknown. Several studies have shown protective effects of MMP inhibitors during ventilation-induced lung injury and ARDS. Chemically-modified tetracyclines (CMTs), doxycycline (DOX), and sivelestat sodium have been tested for their ameliorative effects on ALI. Treatment with a combination of sivelestat sodium and an antiviral agent (oseltamivir) indicated promising results in a patient infected with the novel 2009 swine-origin influenza (Quispe-Laime et al., 2010). Via its MMP inhibitory actions, DOX has been reported to be protective in various pulmonary conditions such as toluene diisocyanate-induced asthma, lipopolysaccharide-induced ALI and pulmonary fibrosis (Fujita et al., 2006, Fujita et al., 2007, Lee et al., 2004, Liu et al., 2006). The aim of this study was to investigate whether DOX, an inhibitor of MMPs, exerts ameliorative effects in a murine model of ALI induced by a virulent mouse-adapted strain (P10) of human influenza H3N2 virus (Narasaraju et al., 2009).