Ozone Exposure on Influenza Infection Figure 1. Exposure to ozone enhances influenza infection. Cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. A) Basolateral supernatants collected 24 h post-exposure were analyzed for LDH levels. B) Basolateral supernatants collected 24 h post-exposure were analyzed for IL-6 levels. C) 24 h post-exposure, cells were infected with influenza A/Bangkok/1/79. Total RNA was isolated and subjected to RT-PCR to quantify Influenza HA transcripts and normalized to the expression of b-actin. D) 24 h post-exposure, cells were infected with influenza A/Bangkok/1/79. Apical supernatants collected 24 h post-infection were analyzed for determination of the viral titer indicated in log TCID50. 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.g001 HA expression. Interestingly, the addition of either EGCG or GSH-ET significantly inhibited viral HA transcription as com- pared to the vehicle control. We again collected apical washes to perform viral titer assays. Our results, shown on a log scale, indicate that both EGCG and GSH-ET significantly reduced viral replication (Figure 3B). Taken together, these results demonstrate that antioxidants, either through Nrf2 activation (EGCG) or by direct addition of an antioxidant (GSH-ET), counteract the oxidative stress induced by ozone exposure and can suppress influenza virus replication in NECs. Ozone exposure increases viral entry Ozone not only triggers intracellular oxidative stress, but has also been shown to directly affect and modify the cellular lipid membrane [66,67] and transmembrane molecules [68]. Since the influenza virion must first bind and enter the target cell prior to replication, we hypothesize that the increased susceptibility to influenza after ozone exposure most likely happens early in the virus lifecycle, upstream of viral replication. To determine which step(s) in the virus life cycle are affected by ozone exposure, we employed an enzymatic virus-like particle (VLP) assay. This assay quantitatively measures the amount of virus that enters the cells. Similar to our previous work [6], cells were infected with a VLP that only express the hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins along with a functional b-lactamase reporter fusion (PR8 b-lacM1 VLP) prior to loading with the fluorogenic substrate CCF2-AM . In cells in which the PR8 b- lacM1 VLP has entered, the CCF2-AM substrate will be cleaved, disrupting FRET of the substrate, and resulting in increased CCF2 emission at 447 nm. To determine the effects of ozone exposure on virus entry, NECs and the cell line MDCK as a control were exposed to air or ozone and 24 h post-exposure, cells were infected with PR8 b-lacM1 VLP. In this experiment, MDCK cells are used as a control cell line which does not produce proteases required to activate the viral HA protein. Figure 4 shows that exposure to ozone results in a significant increase in viral entry in the NECs alone. In summary, these data demonstrate that acute exposure to ozone enhances viral entry, consistent with the increased viral replication shown in Figure 1C. Ozone exposure disrupts host protease/antiproteases balance As stated earlier, recent reports have identified two endogenous human trypsin-like serine proteases (TMPRSS2 and HAT) that PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35108