Ozone Exposure on Influenza Infection Figure 2. Exposure to ozone does not affect cellular antiviral responses. Cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, cells were infected with influenza A/Bangkok/1/79. 24 h post-infection A) Total RNA was isolated and subjected to RT-PCR to quantify RIG-I transcripts and normalized to the expression of b-actin. B) TLR-3 transcripts and normalized to the expression of b-actin. C) IFN-a transcripts and normalized to the expression of b-actin. D) IFN-b transcripts and normalized to the expression of b- actin. NECs were obtained from six healthy volunteers (n = 6), and each experiment was performed in triplicate. Asterisk indicates statistical significance between test sample and control, * p,0.05. doi:10.1371/journal.pone.0035108.g002 possess the ability to cleave influenza virus in vitro [30,32,33,69]. Viral HA proteolytic cleavage is required for viral fusion and entry into the host cell. Proteases are regulated by antiproteases, such as SLPI and to a lesser extent A1AT. Studies have shown that a disruption of the protease/antiprotease balance is a hallmark of numerous lung diseases and pathologies including COPD, emphysema, asthma, and cancer [40,70,71,72]. Hennet et al, demonstrated a link between oxidative stress and protease expression which led to increased influenza infection in mice [39]. Linking this work with our current hypothesis, we investigated the protease/antiprotease balance on the epithelial surface in the context of an acute ozone exposure. For these experiments we exposed our NECs to either air or ozone for 4 h. 24 h post-exposure, both apical surface liquid and cell lysates were collected to characterize secreted/soluble and membrane-bound intracellular levels of HAT, TMPRSS2, and SLPI. Western blots revealed that intracellular levels of SLPI decreased slightly with ozone exposure, while HAT and TMPRSS2 expression remained relatively unchanged (Figure 5A). Although it is well known that SLPI is secreted and is present in normal human airway surface liquid, only recently was the transmembrane protease, HAT, found to be secreted in an in vitro overexpression system [33]. We found the levels of the secreted forms of HAT, TMPRSS2, and SLPI to be significantly changed by ozone exposure (Figure 5B). Under normal conditions, SLPI is in greater abundance than the proteases, yet 24 h post ozone exposure, we found a decrease in SLPI but a significant increase in both HAT and TMPRSS2. It is worth noting that the time at which these samples were taken corresponds to the exact time post ozone exposure when the cells were infected with influenza in the data shown in Figures 1, 2 and 3, thus representing the levels of protease and antiprotease present at the time of infection. To determine if disruption in the protease/ antiprotease equilibrium is due to oxidative stress imposed by the ozone exposure, we treated our NECs with the potent Nrf2 inducer EGCG prior to ozone challenge. Figure 5C shows that EGCG did increase levels of SLPI which corresponds with a decrease in protease expression. Finally, Figure 5D demonstrate that the addition of 1 mM EGCG significantly increases the trans- activation of the SLPI promoter which correlates with the increase in SLPI protein expression displayed in Figure 5C. Taken together, these results indicate that ozone-induced oxidative stress disrupts the protease/antiprotease balance, in favor of protease activity. Furthermore, we demonstrate that the equilibrium can be restored with the induction of Nrf2 via antioxidant supplementa- tion resulting in an increase in the antiprotease SLPI. Secreted proteases are functional for hemagglutinin cleavage We characterized the cleavage products of the viral HA protein to determine if the proteases produced by acute ozone exposure can cleave an intact influenza virion. We first investigated where HA cleavage takes place. NECs were exposed to air or ozone and 24 h post-exposure, cells were infected with Influenza A/Malaya/ 302/1954 H1N1. The cell lysates were analyzed by Western blotting for detection of the HA cleavage products. Figure 6A shows that the majority of the viral HA protein within the cell is cleaved and there is not a significant change with ozone exposure. In addition, Western blot analysis of apical washings incubated with virus showed that HA0 was cleaved and that there was an PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35108