Aldrich St. Louis Mo) was used to inhibit trypsin protease activity in control samples. Protease inhibition assay Recombinant human SLPI (rhSLPI) (R&D Systems Minneap- olis MN) was added to serum-free DMEM containing 20 mg/ml trypsin and incubated for 60 min at room temperature prior to addition of 80 ml of virus. MDCK cells grown in round-bottom 96- well plates were inoculated with the above samples diluted in serum-free DMEM using 10-fold dilutions. After 3 days incuba- tion, a suspension of human erythrocytes (0.5%) was added to each well. Wells exhibiting hemagglutination were considered positive and virus titers were expressed as log TCID50. All experiments were performed in triplicate in three independent experiments. RT-PCR Total RNA was extracted using TRizol (Invitrogen) according to the supplier’s instruction. First-strand cDNA synthesis and real- time RT-PCR was performed as described previously [46,53] using commercially available primers and probes for HA (inventoried Taqman Gene Expression Assays) purchased from Applied Biosystems (Foster City, CA). Measurement of Lactate Dehydrogenase (LDH) and Interleukin-6 (IL-6) levels NECs were exposed to filtered air or 0.4 ppm ozone for 4 h. 24 h post exposure, basolateral supernatants were collected for the measurement of LDH concentration according to the manufac- turer’s recommendations (Takara Bio Inc. Madison, WI). Released LDH concentration was expressed as optical density. Measure- ments of IL-6 concentration were determined according to the manufacturer’s recommendations (BD Biosciences San Diego, CA). Released IL-6 concentration was expressed as pg/ml. SLPI trans-activation reporter assay To measure trans-activation of the SLPI promoter, 1.26105 BEAS-2B were cotransfected with either 0.1 mg of SLPI-luciferase reporter plasmid (which was kindly provided by Dr. Rosalia Simmen, Arkansas Children’s Hospital Research Institute) along with 0.02 mg of Thymidine kinase-Renilla luciferase by using Fugene 6 (Roche) according to the manufacturer’s recommenda- tions. After 24 h, cells were treated with 1 mM EGCG or DMSO. 8 h post-treatment cells were pelleted and lysed in passive lysis buffer (Promega, Madison, WI). The trans-activation activity was measured as luciferase light units as described previously [55]. All transfection experiments were performed in triplicate and normalized for transfection efficiency by using Renilla Luciferase. Statistical Analysis Unpaired Student’s t-test and One-way ANOVA were used for determination of statistically significant differences. The use of the term significant in text refers to a comparison of values for which p,0.05. Results Ozone exposure enhances viral replication Our group has recently demonstrated that oxidative stress increases susceptibility to influenza virus and that these responses may be mediated via increased viral entry [6,46]. Therefore, we wanted to evaluate how exposure to ozone affects viral entry and replication. We chose to examine influenza infection in human NECs exposed to 0.4ppm ozone for 4 h, based on our group’s Ozone Exposure on Influenza Infection recent in vivo [12,56] and in vitro [9] studies. To assess the effects of ozone on NECs, we determined LDH and IL-6 levels in basolateral supernatants as markers of cytotoxicity and inflamma- tion, respectively (Figure 1A and 1B). As expected, exposure to 0.4ppm of ozone for 4 h causes a significant increase in both inflammation and cytotoxicity [57]. Subsequently, 24 h post- exposure, cells were infected with influenza A/Bangkok/1/79 or a mock control. 24 h post-infection total RNA was subjected to real time RT-PCR to quantitate the influenza viral hemagglutinin transcript (HA) (Figure 1C). As depicted, we saw a significant increase in the amount of viral HA mRNA produced in the cells previously exposed to ozone as compared to the filtered air control. Similarly, by analyzing the apical washes for influenza viral titers 24 h post-infection, we saw a significant increase in viral titers in ozone exposed cells as compared to the control (Figure 1D). These results demonstrate that pre-exposure to ozone significantly increased viral replication in NECs. Ozone exposure does not affect cellular antiviral responses To determine the potential mechanism(s) mediating the enhancement of viral replication following acute ozone exposure, we assessed whether innate antiviral immune response mediators were modulated. Specifically, we analyzed the expression of interferon-b (IFN-b), interferon-a (IFN-a), retinoic acid inducible gene I (RIG-I), and Toll-Like recptor-3 (TLR-3). Human NECs were exposed to ozone and 24 h post-exposure were infected with influenza A. 24 h post-infection total RNA was isolated and RT- PCR was performed to quantitate the amount of each cellular transcript. Ozone alone had no significant effect on mRNA expression in any of the four antiviral genes as compared to the air control (Figure 2A–D). As expected, we found substantial inductions of these four antiviral mediators 24 h post-infection with influenza A, but without significant differences in ozone exposed compared to control cells. These data demonstrate that exposure to ozone does not alter baseline expression of host antiviral immune response genes, and does not interfere with their induction in response to influenza infection. Antioxidant supplementation suppresses viral replication Since we have shown that ozone exposure did not abrogate antiviral responses, we wanted to determine if the increase in influenza virus infection is due to the oxidative stress pathway/ mechanism induced by ozone exposure. To test this hypothesis, we treated our NECs with either a potent phase-II antioxidant inducer or a direct antioxidant. We have previously shown that oxidative stress increases susceptibility to influenza infection [6]. Previous reports have shown that Nrf2 gene expression and protein expression can be induced via antioxidant supplementation [58,59,60], more specifically by the addition of the polyphenolic catechin, epigallocatechin-3-gallate (EGCG). This compound has the ability to induce Nrf2 activation which in turn up-regulates the expression of multiple phase-II antioxidants [6,61,62]. Based on our previous published data [6,46], we determined that 1 mM of EGCG or 10 mM of GSH-ET is sufficient to detect an increase in Nrf2 expression or counteract oxidative stress induced by diesel exhaust exposure in NECs. These concentrations also correlate with levels of EGCG that are achievable in vivo [63,64,65]. NECs were treated with either 1 mM EGCG, 10 mM GSH-ET, or DMSO as a vehicle control for 30 min prior to a 4 h 0.4ppm ozone exposure. 24 h post-ozone exposure cells were infected with influenza A virus and total RNA was isolated 24 h post-infection to determine the level of influenza HA transcripts by RT-PCR. As shown in Figure 3A, exposure to ozone significantly increases viral PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35108