Exposure to Ozone Modulates Human Airway Protease/ Antiprotease Balance Contributing to Increased Influenza A Infection Matthew J. Kesic1*, Megan Meyer1,4, Rebecca Bauer1,2, Ilona Jaspers1,2,3,4 1 Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina Chapel Hill, North Carolina, United States of America, 2 Curriculum in Toxicology, University of North Carolina Chapel Hill, North Carolina, United States of America, 3 Department of Pediatrics University of North Carolina Chapel Hill, North Carolina, United States of America, 4 Department of Microbiology and Immunology University of North Carolina Chapel Hill, North Carolina, United States of America Abstract Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA) is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI). Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs) to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility. Citation: Kesic MJ, Meyer M, Bauer R, Jaspers I (2012) Exposure to Ozone Modulates Human Airway Protease/Antiprotease Balance Contributing to Increased Influenza A Infection. PLoS ONE 7(4): e35108. doi:10.1371/journal.pone.0035108 Editor: Andrew Pekosz, Johns Hopkins University - Bloomberg School of Public Health, United States of America Received September 28, 2011; Accepted March 12, 2012; Published April 9, 2012 Copyright: 2012 Kesic et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The project described was in part supported by grant number HL095163 from the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, a grant from the Flight Attendant Medical Research Institute (FAMRI), as well as a grant from the Environmental Protection Agency (CR829522) (all to IJ). This research was also supported by grant number T32 ES007126-26 National Institute of Environmental Health Sciences Curriculum of Toxicology Training Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kesic@email.unc.edu Introduction Influenza A virus is responsible for the seasonal epidemics and reoccurring pandemics, which represents a worldwide threat to global public health [1]. It is a major cause of morbidity and mortality worldwide, as during the recent H1N1 pandemic. In the U.S. alone, over 100,000 individuals are hospitalized and over 20,000 people die every year due to influenza virus infection and related diseases [2,3]. Despite large-scale efforts in vaccination and antiviral therapies, the morbidity and mortality rates associated with influenza infections have not significantly changed in recent years [4,5]. In the context of potentially pandemic respiratory viral infections, it is important to identify molecular targets/pathways for therapeutic intervention to protect susceptible sub-populations. Epidemiologic studies show that exposure to inhaled oxidants such as cigarette smoke, diesel exhaust, and ozone can modulate immune function and increase susceptibility to respiratory viral infections [6,7,8,9,10,11]. Despite the extensive study into different factors influencing susceptibility to infection, the mechanism(s) by which inhaled oxidants modify viral pathogenesis are very complex and have yet to be fully elucidated. The ability of oxidants to cause lung injury/dysfunction is dependent, in part, on the delicate equilibrium that exists between oxidants and antioxidants. Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to readily detoxify reactive intermediates. Ozone is one of the most abundant components of air pollution in urban areas, and has been shown to be a potent inducer of oxidative stress causing airway inflammation and increased respiratory morbidities [8,9,12]. The Mexico City Metropolitan Area (MCMA), one of the most densely populated areas in the world, experiences high levels of air pollutants such as environmental ozone and particulate matter (PM) [13,14,15]. The MCMA is located 2240 m above sea level and is surrounded by mountains. Due to this geographical location, there is less available oxygen, making combustion less efficient, which produces more PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35108