Airway protease/antiprotease imbalance in atopic asthmatics contributes to increased Influenza A virus cleavage and replication
© Kesic et al.; licensee BioMed Central Ltd. 2012
Received: 26 June 2012
Accepted: 3 September 2012
Published: 19 September 2012
Asthmatics are more susceptible to influenza infections, yet mechanisms mediating this enhanced susceptibility are unknown. Influenza virus hemagglutinin (HA) protein binds to sialic acid residues on the host cells. HA requires cleavage to allow fusion of the viral HA with host cell membrane, which is mediated by host trypsin-like serine protease. We show data here demonstrating that the protease:antiprotease ratio is increased in the nasal mucosa of asthmatics and that these changes were associated with increased proteolytic activation of influenza. These data suggest that disruption of the protease balance in asthmatics enhances activation and infection of influenza virus.
KeywordsAsthma Influenza A Protease Antiprotease SLPI TMPRSS2 Hemagglutinin Susceptibility
The viral hemagglutinin (HA) protein expressed on the influenza virion surface is responsible for binding to sialic acid residues on the host cells. Fusion of the virus membrane to the host cell occurs only after HA is proteolytically cleaved, whereas virions with uncleaved HA (HA0) are non-infectious [1, 2]. After cleavage by a host trypsin-like serine protease, two protein fragments, HA1 and HA2, are produced. These proteases, in turn, are regulated by mucus antiproteases, such as secretory leukocyte protease inhibitor (SLPI) and α1-antitrypsin (A1AT) [2, 3]. Our group recently demonstrated that human nasal epithelial cells (NEC) secreted the cellular protease, transmembrane protease serine 2 (TMPRSS2) and the antiprotease, SLPI, and that oxidant-induced disruption of the protease/antiprotease balance, as characterized by increased expression and secretion of TMPRSS2, resulted in increased HA cleavage and viral replication .
Healthy volunteer (n=7)
Atopic asthmatic (n=6)*
Age (mean years, SD)
Skin prick test positivity (# of subjects, N)
3 African American
1 African American
1 Native Pacific Islander
BMI (Mean, SD)
To determine if the proteases present in the apical surface liquid from nasal lavage are functional and can cleave an intact influenza virion, NLF (50μg of total protein), was incubated with Influenza A/Malaya/302/1954 H1N1 and subsequently analyzed for the various forms of HA using immunoblotting. Figure 1C show NLF from both HVs and AAs was able to cleave HA0 to its products HA1 and HA2, but that NLF from AAs had increased proteolytic activity. Similar to our previous studies , we examined whether NLF from AAs and HVs differ in their ability to produce infectious virions using a modified infectivity viral titer assay. These experiments determine whether secreted proteases present in the NLF from HVs and AAs are able to facilitate multiple rounds of viral replication in Madin-Darby canine kidney cells (MDCKs), which require exogenously added protease to become infected and replicate influenza virus . NLF from HVs and AAs was incubated with influenza A/Bangkok/1/79 or with a mock control then added to the MDCK cells for analysis of influenza viral titers. Figure 1D shows that secreted proteases in NLF from healthy volunteers are able to activate influenza virions leading to viral entry and replication in MDCK cells and that these effects are increased in NLF from AAs. Taken together, these results demonstrate that NLF from AAs has a disruption in the secreted protease/antiprotease balance. Using the exact same samples, we also demonstrate that virus incubated with NLF from AA display a significant increase in influenza virus cleavage and replication in MDCK cells.
In conclusion, this is the first study to demonstrate that secreted proteases in NLF from humans can proteolytically activate influenza virions and that these activities are enhanced in AAs. We speculate that disruption of the epithelial protease/antiprotease balance in AAs is a plausible risk-factor for increased susceptibility of these individuals to influenza infection and potentially other viruses such as SARS-CoV and metapneumovirus, which also require proteolytic activation.
The project described was in part supported by National Institutes of Health grant KL2RR025746 (MH), National Institute of Environmental Health Sciences ES013611 (IJ), and Nation Heart, Lung, and Blood Institute HL095163 (IJ).
Secretory leukocyte protease inhibitor
Transmembrane protease serine 2
Nasal lavage fluid
Nasal epithelial cells
Madin-Darby canine kidney cells.
We thank Ms. Luisa E. Brighton and Martha Almond for their skillful assistance and Philip Bromberg for his critical review of the manuscript.
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