Mouse beta-defensin 4 limits A(H1N1)09 influenza virus replication in respiratory tract of senescent mice

Abstract

Background: Mouse beta-Defesins (mBDs) are small molecules of cationic peptides with broad spectrum of anti-microbial properties and immune modulatory functions, which plays important roles against respiratory viral infections. The expression and function of mBDs in response to influenza infection has not been fully elaborated. Methods: The expression of mBD4 in mouse lung epithelial cell line LA4 and C57 BL/6 mouse respiratory tissues following A(H1N1)09 pandemic influenza virus infection were studied by quantitative RT-PCR, and also by immunofluorescence or immunohistochemistry staining. Virus replication and virus induced cytokines (tumour necrosis factor–alpha [TNF-α] and interleukin-6 [IL-6]) responses were studied and correlated with the induction of mBDs. Results: Firstly, mBD2, mBD3, and mBD4 were quickly increased in LA4 cells at 6 hours after inoculation with A(H1N1)09 virus and remained upregulated until 24 hours post infection. The induction of mBD4 was positively correlated with initial virus inoculation doses, with significantly higher expression induced in the cells inoculated with multiplicity of infection (MOI) of 10, comparing to the cells infected by MOI of 1. The time course for the induction of mBD4 correlated with the upregulation of inflammatory cytokine IL-6 and TNF-α. The expression of mBD4 in mouse respiratory tissue was studied and compared between young (6-8 weeks) and aged (72 weeks) mice. The results showed mBD4 were not detectable by immunohistochemical staining of formalin fixed young mice trachea and lung tissues, but showed there was stronger expression of mBD4 in epithelial cells lining trachea and bronchioles in aged mice, which indicated a higher basal expression of mBD4 in aged mice respiratory tissues. Upon infection with A(H1N1)09 influenza virus, a quick induction of mBD4 in young mice trachea tissue were observed at 12 hours p.i. and maintained at this level until day 4 p.i.. However, despite the higher basal level, there was no further induction of mBD4 in aged mice trachea tissues. For the lung tissue, delayed induction of mBD4 was observed in aged mice following A(H1N1)09 infection, but no increase was observed in the young mice lung tissues. Accordingly we also observed a lower viral load and cytokine levels in young mice. After giving the fusion mBD4 protein (0.126mg/kg) after infection of A(H1N1)09 in aged mice, we saw a reduced viral load in respiratory tissues. Conclusion: A(H1N1)09 influenza virus infection induces the expression of mBD4 in vitro and in vivo. Aged and young mice showed different pattern of change in mBD4 induction after infected by A(H1N1)09 influenza virus. The treatment of H1N1 infection in aged mice with fusion mBD4 could improve the outcome, which indicated mBD4 may play important roles in influenza infection.