HA cleavage and activation of the influenza virion, which correlated with enhanced viral entry and replication. Numerous host cell-dependent factors can affect and control influenza virus attachment and uptake by: (i) proteolytic cleavage of viral HA by host cell-derived serine proteases [30,33], (ii) host cell derived innate immune defense molecules aimed at inhibiting the infectious virions [25,76], and (iii) antiviral mediators limiting viral replication and shedding of virus particles [6,52]. We first focused on potential effects of ozone on host antiviral defense responses. Our data showed that exposure to ozone elicited the typical pro-inflammatory and cellular damage responses as seen with increased release of IL-6 and LDH. We found that the same exposure regimen also resulted in significant increase in viral replication. We initially hypothesized that this could be due to a disruption in one of the cellular antiviral response mechanism/ pathways, and assessed the transcription levels of 4 classical antiviral mediators; RIG-I, IFN-a, IFN-b, and TLR-3. Similar to our previous studies using exposure to diesel exhaust [46], we found that ozone did not decrease expression of these mediators or their response when challenged with influenza. In our previous study [46], we found that exposure to an oxidant pollutant increased influenza virus attachment and/or entry, but did not distinguish between these two steps in the viral infection cycle. Therefore, we focused our attention on steps upstream of viral replication to identify mechanisms by which oxidants, such as ozone, affect influenza infectivity. To dissect specific points in the virus life cycle upstream of viral replication that could determine the role ozone exposure plays in viral susceptibility, which ultimately dictates viral pathogenesis and outcome, we utilized our previously described enzymatic virus-like particle (VLP) assay [6]. Our data demonstrated that ozone exposure significantly increased influenza virus entry. This is consistent with our previous work showing that exposure to other oxidant pollutants and suppression of Nrf2 increases influenza virus entry [6,46]. Similar to these previous studies, data shown here demonstrated that the mechanism(s) through which ozone exposure alters influenza virus entry is mediated by oxidative stress. Since ozone directly interacts with the apical surface of the respiratory epithelium we hypothesize that ozone either directly, or via the induction of biologically active ozone reaction products, modifies either the expression or function of surface proteins, as previously been demonstrated for human surfactant protein A (SP- A) [77]. Among the most prominent proteins on the apical surface potentially regulating infection are the proteases. To date, at least five different proteases have been identified in the airways of animals and humans [78]. Despite many years of effort, the exact protease(s) that activates influenza has yet to be identified. Numerous groups have reported that the family of type II transmembrane serine proteases and trypsin-like proteases are responsible for viral cleavage [26,30,33,79]. Previous studies have examined these proteases in the context of influenza infections, specifically TMPRSS2 and HAT. Although these studies demon- strated that these proteases are capable of cleavage, the experiments were performed in cell lines such as MDCKs [33] or the colorectal adenocarcinoma cell line Caco-2 [32], neither of which are natural target cells for influenza. Since the cellular protease determines the tropism as well as efficiency of viral replication, we utilized fully differentiated human primary nasal epithelial cells (NECs) in this study, a natural host cell for influenza virus. We show that HAT and TMPRSS2 are not only present, but secreted from NECs (Figure 5B), and that ozone exposure enhances the release of the proteases into the apical compartment. This is consistent with reports showing that oxidative stress increases cellular protease activity [38]. Since proteases in the Ozone Exposure on Influenza Infection upper airway are inhibited by SLPI [78], we focused on this antiprotease and its expression in relation to HAT and TMPRSS2. Similar to previous reports, we report that SLPI is detected in the secreted airway and oxidative stress reduces the expression and function of the protein (Figure 5B) [35,80]. These data are consistent with our hypothesis that influenza virions can become activated in the airway surface liquid prior to binding to the epithelial surface. Virion activation in the airway prior to binding would eliminate the need for a membrane-bound protease thus broadening viral tropism from airway epithelium, that contain the necessary proteases, to any mammalian cell, since 2,6-linked sialic acid is expressed by most mammalian cells ranging from lymphoid to glial cells [81,82].This could explain the increased morbidity and mortality associated with the Mexico City 2009 H1N1 pandemic which resulted in increased infection of multiple cell types including distal airway epithelium and immune cells including alveolar macrophages [83,84] as compared to previous seasonal outbreaks. EGCG has been shown to have potent antioxidant capabilities; in part by inducing the expression of a number of antioxidant enzymes [85]. In vitro and in vivo studies have shown that this supplement induces the expression of phase II antioxidant genes such as GSTM1, which was associated with Nrf2-ARE signaling [58,86]. Whether and how activation of antioxidants expression is involved in the potential antiviral effects of EGCG is not known. Previous studies have indicated that it directly binds influenza virus and therefore prevents attachment and entry into host cells [63,87]. However, these studies were conducted in MDCK cells, which are not a natural host cell for influenza and require addition of exogenous proteases to achieve viral entry [63]. Iizuka et al, demonstrated that Nrf2-knockout mice display protease/antipro- tease imbalance leading to increased susceptibility to cigarette smoke-induced emphysema [37]. In addition, the same group showed that Nrf2 expression exerts a protective effect through the transcriptional activation of antiproteases [88]. Our data demon- strate that supplementation of differentiated nasal epithelial cells with EGCG from the basolateral side (to eliminate direct interaction with the virus during infection) significantly increases SLPI production (Figure 5C). It is worthy to note that we show a direct inverse relationship between the levels of secreted SLPI and secreted protease expression, suggesting a potential novel antiviral mechanism by which EGCG could be protective against influenza infections. We next determined that the secreted proteases are functional. We demonstrate that proteases present in the apical surface liquid were able to activate the virus (Figure 6B) and produce multiple rounds of viral replication (Figure 7A). In addition, apical supernatants from ozone-exposed epithelial cells had significantly higher viral titers, which correlate with the increased protease expression displayed in Figure 5B. In the final experiment we utilized rhSLPI and show a dose dependent protective response (Figure 7B). This demonstrates that that the antiprotease SLPI has a protective affect against viral activation and replication. In conclusion, this is the first study to demonstrate that secreted proteases from primary human respiratory nasal epithelium proteolytically activate influenza virions and that exposure to a common ambient air oxidant pollutant increases these effects. We speculate that individuals with increased airway oxidative stress, whether due to an underlying medical condition or exposure to high levels of air pollutants, would develop disruption of the epithelial protease/antiprotease balance and become more susceptible to infections with viruses like influenza, SARS-CoV, and metapneumovirus. Disruption in the protease/antiprotease balance has been described in several respiratory diseases PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35108