assays before, we utilized the well-characterized MDCK cell line which does not produce the proper proteases to activate the influenza virion, which can be overcome by the addition of exogenous trypsin. In contrast to the previous titer experiments above, we did not add exogenous trypsin to enable multicycle replication of influenza virus in these cells. These experiments allowed us to test if secreted proteases present in the apical surface liquid from NECs exposed to air or ozone were able to facilitate multiple rounds of viral replication in MDCKs. We show that the secreted proteases present in the apical supernatants from NECs are functional and can propagate viral entry and replication. We also demonstrate that the ozone-exposed NEC supernatants displayed significantly greater infection kinetics than the air control (Figure 7A). This correlates with the increase in protease expression post ozone exposure (Figure 5B), viral entry (Figure 4), and viral replication (Figure 1C and 1D). In addition we show that this is a protease-mediated event, since the addition of a known serine protease inhibitor, PMSF, significantly inhibits viral replication. To demonstrate that SLPI has the ability to protect airway epithelium from influenza infection by inhibiting proteolytic cleavage, we incubated various amounts of recombinant human SLPI (rhSLPI) to our traditional viral titer assay. Briefly, we incubated our viral titer media, serum-free DMEM containing 20 mg/ml of exogenous trypsin, with varying amounts of rhSLPI prior to addition of influenza. As shown in Figure 7B, addition of rhSLPI significantly inhibits multicycle viral replication. We conclude that the addition of rhSLPI directly inhibits trypsin- mediated cleavage of viral HA leading to decreased infection and replication. Taken together, these data demonstrate that exposure Ozone Exposure on Influenza Infection to ozone disrupts the protease/antiprotease balance resulting in greater secretion of endogenous proteases leading to increased viral cleavage/activation culminating in enhanced viral entry and replication. Discussion Numerous epidemiological studies link increased ambient ozone levels with respiratory viral infections [1,73,74]. However, the role of ozone exposure in viral replication and pathogenesis remains to be fully defined. Because ambient ozone is a major source of oxidative stress in the airway epithelium and this is the primary site for influenza infections, it is important to determine the role ozone exposure plays in viral infections. Although it has been shown that ozone exposure can modulate many aspects of the immune response [8], how these alterations influence a viral infection have yet to be elucidated. Recent studies have demonstrated the importance of the protease/antiprotease balance in the context of normal lung homeostasis, and have shown that oxidative stress can disrupt this delicate equilibrium [40,75]. It has been well documented that many lung pathologies are dependent on the regulatory interplay between oxidative stress and protease expression [40,70,71]. Although there is speculation that Mexico City’s increased influenza-related morbidity and mortality rates were due to exposures to higher levels of ozone, little research has been done looking at the effects of pre-exposure to ozone in the context of an influenza infection in humans. Herein, we demonstrate that acute ozone exposure disrupts the protease/ antiprotease balance resulting in increased secreted protease expression in primary human epithelial cells, resulting in increased Figure 7. Secreted proteases are functional and can activate virions to propagate a viral infection. A) Cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure apical supernatants were incubated with influenza A/ Bangkok/1/79 for 30 min, and then analyzed for determination of the viral titer indicated in log TCID50. B) Increasing amounts of rhSLPI (8 nM, 40 nM, 200 nM, 1000 nM) were incubated with media containing 20 mg/ml trypsin for 60 min, then analyzed for determination of the viral titer indicated in log TCID50. NECs were obtained from four healthy volunteers (n = 4), and each experiment was performed in triplicate. Data from a representative experiment performed in triplicate. Asterisk indicates statistical significance between test sample and control, * p,0.05. doi:10.1371/journal.pone.0035108.g007 PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35108