polycyclic aromatic hydrocarbon (PAH) pollutants. For these reasons, Mexico City receives higher levels of environmental ozone and various other types of photochemical smog [13]. It was here that the first influenza pandemic of the 21st century emerged in March 2009. It was responsible for an estimated ,258,698 laboratory confirmed cases and roughly ,1,370 deaths by December 2009 [1,16,17]. The normal ‘‘Flu Season’’ occurs during the colder half of the year in each hemisphere [18]. Interestingly, this outbreak emerged during the dry and warmer months when ozone levels were significantly higher [19,20]. Possible mechanisms by which oxidative stress alters the airway environment leading to broadened cellular tropism and/or susceptibility to viral infections have been proposed. [21,22,23] The relationship between oxidative stress and the cellular protease/antiprotease balance has been considered to be a major contributor in the development of numerous airway pathologies [24]. Early studies demonstrated that the cleavage of influenza HA is essential for viral penetration into host cells and that this mechanism was mediated by cellular trypsin-like serine proteases. These proteases, in turn, are regulated by mucus antiproteases, such as SLPI and a1-antitrypsin (A1AT) [25,26,27,28,29]. Recent studies have identified specific cellular proteases and antiproteases that may be involved in influenza infection, which include transmembrane protease serine 2 (TMPRSS2), human airway trypsin-like protease (HAT), and secretory leukocyte proteinase inhibitor (SLPI) [30,31,32,33,34]. The expression of these proteases in the lung are necessary for cleavage of the viral HA surface protein, thus allowing viral fusion and entry into the host cell. Studies have shown a correlation between inflammation and oxidative stress which alters expression of these proteases and antiproteases [29,35]. Specifically, HAT has been shown to be released into the airway fluids under inflammatory conditions, particularly in asthmatics [31,36]. In addition, a murine study showed SLPI expression was suppressed and protease expression was increased in Nrf2-deficient mice which led to increased inflammation, further demonstrating a relationship between oxidative stress and protease expression [37]. Oxidative stress derived from cigarette smoke or addition of reactive oxygen intermediates has been shown to decrease antiprotease activity [35,38] and a murine study demonstrated that decreased antiprotease expression increased influenza infectivity [39]. The imbalance of protease/antiprotease expression caused by oxidative stress has been attributed to both an increase in inflammatory cells, which release proteolytic enzymes capable of destroying lung tissue, and a functional deficit of antiproteases due to oxidation of their active site [35,40]. In addition to influenza, studies have shown that regulated proteolysis is required for the spread/ propagation of many human viruses, including Human immuno- deficiency virus (HIV), Nipah, Ebola, severe acute respiratory syndrome coronavirus (SARS-CoV), and metapneumoviruses [41,42,43,44,45]. Although it has been shown that oxidative stress increases severity of viral infections, the exact mechanism as to how and why this happens and the role of ozone exposure in these responses are not fully understood. While the deleterious health effects of both ozone and influenza have been well documented, few studies have looked at the effects of ozone in the context of an influenza infection. Herein, we used our established cell culture model of differentiated human nasal epithelial cells (NECs) [46], exposed them to 0.4ppm of ozone, and determined the effects on susceptibility to an influenza A infection. We show that there is a delicate balance of proteases and antiproteases present in the human airway surface liquid. Additionally, we found exposure to ozone disrupts this equilibrium by enhancing protease secretion Ozone Exposure on Influenza Infection while inhibiting antiprotease expression leading to increased susceptibility to viral infection. We directly show that ozone increases soluble protease expression and that they are functional for cleaving the viral HA protein and activation of the intact influenza virion. Materials and Methods Ethics Statement Primary human nasal epithelial cells (NECs) were obtained from healthy adult volunteers. This protocol was approved by the University of North Carolina School of Medicine Institutional Review Board for Biomedical Research. In addition, written informed consent was provided by each study participants and/or their legal guardians. Nasal epithelial cell cultures, cell lines, and growth conditions Primary human nasal epithelial cells (NECs) were obtained as previously described [6]. Briefly, NECs were obtained from healthy adult volunteers by gently stroking the inferior surface of the turbinate several times with a Rhino-Probe curette (Arlington Scientific, Arlington, TX), which was inserted through a nasoscope. This protocol was approved by the University of North Carolina School of Medicine Institutional Review Board for Biomedical Research. NEC were expanded to passage 2 in bronchial epithelial growth medium (BEGM, Cambrex Bioscience Walkersville, Inc., Walkersville, MD) and then plated on collagen- coated filter supports with a 0.4 mM pore size (Trans-CLR; Costar, Cambridge, MA) and cultured in a 1:1 mixture of bronchial epithelial cell basic medium (BEBM) and DMEM-H with SingleQuot supplements (Cambrex), bovine pituitary extracts (13 mg/ml), bovine serum albumin (BSA, 1.5 mg/ml), and nystatin (20 units). Upon confluency, all-trans retinoic acid was added to the basolateral medium to establish air liquid interface (ALI) culture conditions (removal of the apical medium) to promote differentiation. Mucociliary differentiation was achieved after 18– 21 days post-ALI. We obtained the Madin Darby canine kidney (MDCK) cell line from the American Type Culture Collection (ATCC, Manassas, VA). The BEAS-2B cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct [47]. We obtained our BEAS-2B cells from the American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B cells were grown in keratinocyte basal medium (KBM) supplemented with 30 mg/ml bovine pituitary extract, 5 ng/ml human epidermal growth factor, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin. The primary human NECs were exposed under ALI conditions to filtered air or 0.4 ppm ozone for 4 h in the exposure chambers operated by the U.S. Environmental Protection Agency, Environ- mental Public Health Division. In noted experiments, 10 mM of a cell-permeable form of reduced glutathione, glutathione-ethylester (GSH-ET) (Sigma-Aldrich St. Louis. Mo) or 1 mM of EGCG (Sigma-Aldrich St. Louis. Mo) was added to the basolateral medium 30 min prior to ozone exposure, similar to our previous studies [6,46]. Virus-like particle (VLP) entry assay Construction of VLP expression plasmids. To generate the b-lactamase-M1 fusion expression plasmid (pCAGGS-b- lacM1 PR8) the influenza A/Puerto Rico/8/34/Mount Sinai (H1N1) M1 sequence was PCR amplified from the M1 expression vector pDZ-M (which was kindly provided by Dr. Adolfo Garcia- PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35108