Impaired mouse beta-defensin 4 (mBD4) response to pH1N1 infection associated with high pulmonary viral replication in immunocompromised mice

Abstract

Beta-defensins are cationic peptides that can be induced by proper stimulations and show broad anti-microbial activities. Secreted by the epithelial cells lining the respiratory tract, beta-defensins are considered to be key elements that mount early innate immune responses to influenza infection. In retrospect, influenza pandemics emerged irregularly and caused millions of death globally. Despite scientists have made great efforts to develop strategies to combat influenza, influenza virus especially influenza A virus is still a leading threat to the public health. Especially those immunocompromised individuals tend to develop severe diseases upon influenza infections. Shortage of influenza vaccines supply and emergence of drug resistant virus strains caused great difficulties to combat the next influenza pandemic. Beta-defensins are potentially effective anti-influenza antivirals produced by the host nevertheless the expression of beta-defensins in airway epithelial cells and the roles they played during influenza virus infection are still not well illustrated. Firstly, in vitro expression and antiviral functions of mouse beta-defensins were studied in a lung epithelial cell line, LA 4 cells. Immunofluorescence assay (IFA) showed very low basal levels of mBD2, 3 and 4 in uninfected LA4 cells. Infection of LA 4 cells with A/HK/415742/2009 (H1N1) mouse-adapted strain (A(H1N1)pdm09-mut) virus induced a quick and strong induction of the three mBDs on mRNA levels in a dose dependent manner which was shown by qPCR .IFA showed only mBD4 was induced significantly on protein levels in LA4 cells after influenza infection. The levels of induction of mBDs were associated with the susceptibility of LA4 to different subtype of influenza virus because H7N9 influenza virus induced stronger mBD4 responses compared with pH1N1 virus. There were strong correlations between the expression of mBDs and pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) andinterleukin6 (IL-6). The anti-influenza activity of mBD4 was tested in MDCK cells with recombinant Trx-fusion mBD4. The results indicated the mechanism of action against pH1N1 virus was mainly block viral entry. The expression and distribution of mBD2, 3 and 4 were studied in the respiratory tissues in immune component normal adult mice and immunocompromised aged mice and obese mice before and after A(H1N1)pdm09-mutvirus infection. qPCR results suggested that senescent mice and obese mice showed delayed and attenuated induction of mBDs in response to pH1N1 virus infection though they possessed higher basal levels of mBDs. Higher viral load and more severe lung inflammation in senescent and obese mice were revealed by plaque assay and ELISA detection of TNF-α and IL-6. Intranasal administration of recombinant mBD4 could reduce viral load and lung infiltration after lethal dose viral infection at early time points in the immunocompromised mouse models which could be proved by plaque assay and histological examination of lung tissues. Treatment with recombination mBD4 showed markedly improved survival rate in obese mice, while no improvement of survival rate in influenza infected senescent mice. These series of studies demonstrated that mBD4 played important roles during influenza infection and explored the possibility to use mBD4 as an effective antiviral against influenza in immunocompromised individuals.