Induction and antiviral activity of human β-defensin 3 against adenovirus and influenza A virus in vitro infection of human airway epithelial cells

Abstract

BackgroundRespiratory pathogens such as adenoviruses (AdV) and influenza A viruses (IAV) can cause serious upper and lower respiratory infections in infants, the elderly, and immunocompromised individuals. However, options for antiviral drugs targeting respiratory viruses are limited. Human beta-defensins (hBDs) are disulfide-rich peptides that demonstrate broad antimicrobial activity against bacteria, fungi, and some viruses, while also playing immunomodulatory roles. Among defensins, human beta-defensins (hBDs) are the most prevalent, with six peptides identified: hBD-1 through hBD-6. Nonetheless, the production of hBD-3 during respiratory virus infections has not been extensively studied, nor has the effect of hBD-3 on these infections. ObjectiveThis study investigated whether infection with AdV-5 or IAV induces hBD-3 expression in human airway epithelial cells and whether a recombinant form of hBD-3 (rhBD-3) can inhibit in vitro infection by these viruses, one DNA virus and one RNA virus. MethodsIn vitro models of human airway epithelial cells (A549 and HEp-2) infected with AdV-5 and IAV were established. In vitro expression of hBD-3 mRNA was assessed by RT-PCR; hBD-3 protein was examined using immunofluorescence and Western blot. The inhibition of viral infection by hBD-3 was quantified using cytopathic effect and plaque reduction assays. Additionally, epithelial cells of animal origin, MDCK and MDBK, were similarly tested for rHBD-3 antiviral activity against IAV infection. ResultsIAV and AdV infections significantly increased hBD-3 mRNA levels in A549 and HEp-2 cells. Immunofluorescence and Western blotting confirmed the presence of hBD-3 protein on the surface of IAV-infected cells. Cells infected with AdV-5 produced less hBD-3 protein. Additionally, rhBD-3 inhibited both AdV-5 and IAV infections in both cell lines at concentrations of 7.5-50 g/mL, with the strongest effect at the highest concentration. Lastly, rhBD-3 also inhibited IAV infection in MDBK and MDCK cells within the same range. ConclusionsIn vitro infection of human airway epithelial cells with IAV and AdV induces hBD-3 mRNA and protein expression. In turn, rhBD-3 inhibits viral infection in a dose-dependent manner in HEp-2 and A549 cells against these viruses, an RNA virus and a DNA virus. hBD-3 is expressed and localized on the surface of infected cells. rHBD-3 also inhibits IAV infection in bovine and canine epithelial cells. Our findings suggest that hBD-3 plays a broad-spectrum role in defense against respiratory viruses in humans and animals and modulates the innate immune responses. PerspectiveThis work supports the use of hBD-3 as a natural, broad-spectrum antiviral agent, either alone or in combination, for treating viral infections, particularly emerging respiratory agents such as IAV and coronaviruses, for viral pandemic preparedness and response. Data SummaryThere are no supporting external data. Impact StatementThis study demonstrates in vitro induction of human beta defensin type 3 (hBD-3) in human airway epithelial cells (A549 and HEp-2 cells) during viral infection by the influenza A virus (IAV) and the human adenovirus type 5 (AdV-5), and inhibition of the viral replication on these cells by the hBD-3 in a recombinant form; this defensin also showed viral inhibition on bovine and canine epithelial cells; further, these are viruses from two very different families; one is an enveloped RNA virus, and the other is a naked DNA virus. Both results are novel observations in the biomedical literature. Our results suggest that this defensin plays a principal role in innate defense during natural respiratory viral infections in humans, and support the use of hBD-3 alone or in combination as a broad-spectrum antiviral agent for the treatment of these infections, especially for emerging agents such as IAV and coronaviruses, for viral pandemic preparedness and response. In fact, few antiviral drugs are currently approved for treating respiratory virus infections, with only specific inhibitors of influenza and respiratory syncytial viruses.