Ozone Exposure on Influenza Infection Figure 5. Ozone exposure modulates cellular protease and anti-protease levels. Cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, samples were collected and subjected to Western blot analysis to detect SLPI, HAT, TMPRSS2, and b-actin. A) Total cellular lysates were analyzed for intracellular SLPI, TMPRSS2, and HAT protein expression by Western blot. Membrane was stripped and analyzed for b-actin as a loading control. B) 24 h post-exposure, apical supernatants were analyzed for secreted SLPI, TMPRSS2, and HAT protein levels. C) Cultures of differentiated human epithelial cells were treated with EGCG or DMSO as a vehicle control for 30 min before exposure to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, apical supernatants were analyzed for secreted SLPI, TMPRSS2, and HAT protein levels. D) Function activity of the SLPI promoter was determined using a dual-luciferase reporter assay. BEAS-2B cells were co-transfected with SLPI-Luc and TK-Renilla. Cells were treated with EGCG or DMSO as a vehicle control. Cells were harvested 8 h post-treatment and analyzed for luciferase expression. The values represent luciferase production from a representative experiment performed in triplicate. NECs were obtained from four healthy volunteers (n = 4). Densitometry was used to quantitate the amounts of protein, and the numbers below the gel indicate the ozone/air or EGCG/DMSO sample ratio. doi:10.1371/journal.pone.0035108.g005 Figure 6. Secreted proteases are functional for Hemagglutinin cleavage. Cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. A) 24 h post-exposure, cells were infected with influenza A/Malaya/302/1954. 24 h post-infection, total cellular lysates were analyzed for intracellular cleavage of the viral HA protein by Western blot. B) 24 h post-exposure, apical supernatants were collected and incubated with influenza A/Malaya/302/1954. Cleavage of the viral HA protein was analyzed by Western blot. C) Cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, apical supernatants were immunoprecipitated (IP) with anti- HAT, anti- TMPRSS2, or an isotype control (IgG). Protein levels were analyzed by Western blot. D) Apical supernatants of differentiated human epithelial cells, were immunoprecipitated (IP) with anti- HAT, anti- TMPRSS2, or an isotype control (IgG) followed by incubation with influenza A/ Malaya/302/1954. Cleavage of the viral HA protein was analyzed by Western blot. NECs were obtained from four healthy volunteers (n = 4). Densitometry was used to quantitate the amounts of cleaved H2 protein, and the numbers below the gel indicate the ozone/air ratio. doi:10.1371/journal.pone.0035108.g006 PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35108