Exposure to Ozone Modulates Human Airway Protease/Antiprotease Balance Contributing to Increased Influenza A Infection

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.

Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs

“…A recent report showing that the TMPRSS2 gene is a marker of susceptibility to severe 2009 pandemic A(H1N1) influenza virus indicates that TMPRSS2 contributes to influ- enza virus pathogenesis in humans (8). In contrast, soluble activating proteases have been found in the secretions of the upper respiratory tract (20,21), and several groups have attempted to identify them. Secreted forms of TMPRSS2 and HAT have been observed, but their catalytic activities are uncertain (19,20,22 This may reflect, at least in part, the distinct specificity of each KLK for substrate recognition motifs.…”

“…In contrast, soluble activating proteases have been found in the secretions of the upper respiratory tract (20,21), and several groups have attempted to identify them. Secreted forms of TMPRSS2 and HAT have been observed, but their catalytic activities are uncertain (19,20,22 This may reflect, at least in part, the distinct specificity of each KLK for substrate recognition motifs. However, the amino acid sequence flanking the cleavage site does not appear to be the sole determinant of HA cleavage preference.…”

Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

“…Cultures at ALI were exposed to filtered air or 0.4 ppm O 3 for 4 hours in exposure chambers operated by the U.S. EPA, as previously described (20,24). The dose was selected for maximal innate immune response to O 3 with minimal cytotoxicity, and has been used previously by our group (25).…”

“…In addition, the lung microenvironment has been shown to influence Mac phenotype and function (16). However, most in vitro studies investigating the cellular inflammatory response to O 3 have used monoculture systems, which do not address the interaction between multiple cell types in the airway, and have limited applicability to in vivo situations (11,12,(17)(18)(19)(20) (22,23). Equal volumes of media were added to the apical side of epithelial cell monocultures.…”

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

“…cystic fibrosis, asthma, smoking), underlying mechanisms of a disease, as well as controlled exposures to air pollutants (e.g. ozone, diesel exhaust, cigarette smoke) 18,[21][22][23] . In addition, differentiated NECs grown on tissue culture inserts lend themselves to co-culture with other cells types (e.g.…”