Ozone Exposure on Influenza Infection Figure 3. Antioxidant supplementation suppresses viral replication. Cultures of differentiated human epithelial cells were treated with EGCG, GSH-ET, or DMSO as a vehicle control for 30 min before exposure to either 0.4ppm of ozone or air for 4 h. A) 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. B) 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 6 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.g003 increase in the cleavage of HA from the ozone exposed cells as measured by densitometry. Quantification of the H2 cleavage products demonstrated that ozone exposure leads to enhanced Figure 4. Ozone exposure increases viral entry. Cultures of differentiated human epithelial cells and MDCK cells used as controls, were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post ozone exposure cells were infected with PR8 b-lacM1 VLP. At 4 h post- infection, cells were loaded with CCF2-AM substrate and assayed for viral entry. 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.g004 proteolytic activation of the virion. This data demonstrate that HA cleavage can take place in both the intra- and extracellular environment and that ozone exposure increases the proteolytic activation of the virus in the airway (Figure 6B). To characterize the function of TMPRSS2 and HAT in HA cleavage, we depleted the proteases via immunoprecipitation prior to addition of the virus. Figure 6C shows the apical washes from NECs that were immunoprecitipated with anti-HAT, anti-TMPRSS2, and IgG as a control. The cleared supernatants were incubated with Influenza A/Malaya/302/1954 H1N1. As shown in Figure 6D, there is little difference in the cleavage of the HA protein when the protease are depleted separately, but we demonstrate a significant decrease in the cleavage of HA when both proteases were removed. This data show that the protease(s) found in the apical surface liquid are able to cleave an intact influenza A virion. In addition we show that there are additional proteases secreted into the airway liquid space that are able to activate the virus. Secreted proteases are functional and can activate Influenza A virions to propagate a viral infection Although we show that SLPI, HAT, and TMPRSS2 are secreted by NECs, the question remains as to whether they are functional. To test functionality and the role they play in a viral infection, we employed a modified infectivity viral titer assay. These experiments are very similar to the viral titer assays mentioned earlier, but with a few modifications. Similar to the PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35108