Immunomodulatory and anti-viral effects of statins in influenza H5N1 virus infection of human alveolar epithelial cells and peripheral blood–derived macrophages

Abstract

Background: Highly pathogenic avian influenza (HPAI) H5N1 virus panzootic in poultry continues to spread. It causes zoonotic human disease with a high (> 60%) fatality rate and continues to pose a pandemic threat. Based on clinical, animal, and in vitro cell studies, we and others have suggested that differences in viral replication competence, tissue tropism, and cytokine dysregulation between H5N1 and low pathogenic viruses may contribute to disease pathogenesis. Statins as HMG-CoA inhibitors act to reduce cholesterol and have been demonstrated to have anti-inflammatory and immune-modulatory activities. However, there is controversy about the benefits of statin use on influenza infection in mice and humans. In this study, we aimed to evaluate the effects of statin treatment in influenza infection using physiologically relevant in vitro models—human alveolar epithelial cells (AECs) and peripheral blood–derived macrophages (PBDMs). Materials and Methods: Primary human AECs and PBDMs were infected with HPAI H5N1 (A/HK/483/97) and seasonal H1N1 (A/HK/54/98) viruses in the presence or absence of statin (simvastatin and sevastatin) treatment. Virus replication was monitored by measuring infectious viral particles in cell culture supernatants using TCID50. Immuno-modulatory effects of statins were examined by measuring the mRNA and protein expression of cytokines and chemokines using qPCR and ELISA. In order to understand the intervention of statins and influenza infection, the gene expression profile of selected members of the sterol-biosynthesis pathway in influenza virus–infected AECs and PBDMs were also monitored. The responses of a variety of cytokine treatments on the genes of the sterol-biosynthesis pathway were investigated in AECs. Furthermore, the intracellular free cholesterol level was also examined by enzymatic assay in AECs infected with influenza virus. Results: We demonstrated that both simvastatin and mevastatin exhibited a dose-dependent inhibition of influenza virus replication for both HPAI H5N1 and seasonal H1N1 viruses in human AECs and PBDMs. The observed inhibitory effect of simvastatin and mevastatin occurred below the non-specific toxic effects to cells, which were measured by MTT assay. Treatment of simvastatin and mevastatin significantly suppressed H5N1 virus–induced pro-inflammatory cytokines such as TNF-α in PBDMs and chemokines, including IP-10 and MCP-1 secretion in both AECs and PBDMs at 24 hours post-infection. We further showed that human AECs and PBDMs infected with both HPAI H5N1 and seasonal H1N1 viruses had significant down-regulation of sterol pathway gene expression at 24 hours post-infection. AECs and PBDMs treated with IFN-γ or IFN-β but not IL-1β, TNF, or IL-6, showed down-regulation of sterol pathway gene expression. In addition, we found that the free cholesterol level was significantly reduced at 24 and 48 h post-H5N1 virus infection in AECs and in IFN-β–treated AECs. These results further support a specific modulation of the sterol metabolic pathway upon influenza virus infection. Conclusions: Taken together, the controversy about the beneficial effects of statin use in influenza infection and our data suggest that statins possess both the antiviral and immune-regulatory effects in H5N1-infected in vitro cell models. We also demonstrated a highly specific response of AECs and PBDMs through a coordinated negative regulation of multiple sterol pathway members upon influenza virus infection or treatment of interferon. Identification of a reduction in sterol pathway gene expression and cholesterol levels with IFN treatment in human AECs offers new insights on the host-mediated antiviral responses through the sterol metabolism pathway and opens new therapeutic options for human influenza disease.