It is widely recognized that imbalances in the lung antioxidant system can lead to oxidative stress, which in its most severe form, potentiates influenza-induced morbidity and even mortality 
. Nevertheless, remarkably little is known about the nature of antioxidant enzymes that are most prevalent in the lung, or about how infection impacts the levels of antioxidant enzymes. Based upon published expression profiles, we have identified seven antioxidant proteins that are particularly abundant in the lung and we have traced their spatio-temporal profiles during the course of infection. Interestingly, we observed dramatic shifts in the levels of antioxidants during infection, and we found that some of the most significant changes are due to loss of influenza virus permissive cells, including bronchial Clara cells and alveolar AT2 cells.
To learn about the roles of Clara and AT2 cells in modulating the levels of antioxidant enzymes, we used immunohistochemistry to analyze the antioxidant expression in these cells. Among the 7 key lung-abundant antioxidant enzymes, Clara cells strongly express two intracellular antioxidants, Cat and Prdx6. Clara cells have several functions, one of which is to protect bronchioles by providing a lining of cells and by secreting a variety of proteins, including CCSP and surfactant A (SPA) 
. In addition, Clara cells also able to break down harmful substances inhaled into the lungs and to detoxify them in smooth endoplasmic reticulum via cytochrome P450 enzymes, which generate ROS 
. It is thus reasonable to conjecture that high levels of intracellular antioxidant enzymes protect Clara cells against ROS stress that results both from a high oxygen tension and from Clara cell-specific cytochrome P450 enzymes.
Although Prdx6 is robustly expressed in Clara cells and Clara cells become depleted, the overall levels of Prdx6 stay relatively constant. Analysis of the dynamics of Prdx6 during disease progression shows that the levels of Prdx6 rise significantly within the alveolar spaces. These data are consistent with antioxidant rebalancing, or compensatory expression. This response is not driven by viral replication, since Prdx6 induction was observed in both infected and uninfected cells. Importantly, Prdx6 levels rise in AT1 cells, which cover more than 95% of the surface of alveoli and account for gas exchange 
. Therefore, even a relatively small increase in the levels of Prdx6 when analyzed by immunohistochemistry, can lead to a significant overall increase in the levels of Prdx6 in the lung, thus compensating for the drop in Prdx6 due to the loss of Clara cells. Rat AT1 is known to act not only as a barrier for gas exchange but also as a barrier to oxidative injury via secretion of apolipoprotein E and Tf 
. Likewise, induction of Prdx6 in mouse AT1 cells may also provide a critical defense against infection-induced oxidative stress.
Prdx6 is a unique bifunctional enzyme with GSH peroxidase activity (which reduces hydrogen peroxide and phospholipid hydroperoxide to oxidized glutathione and water) and phospholipase A2 (PLA2
) activity, whose products signal a pro-inflammatory response 
. Its lipid peroxidation-reducing activity enables the resolution of oxidized lipids in the cell membrane, which would otherwise have the capacity to amplify damage through a chain reaction of oxidation and lipid breakdown products. Therefore, Prdx6 has direct protective effect against toxicity and apoptosis 
. Interestingly, Prdx6 demonstrates lung-specific functions via the interaction with SPA that translocates Prdx6 into lamellar body and extracellular space 
. In addition, SPA regulates PLA2
activity of Prdx6 and alters its ability to degrade the major phospholipid of surfactant 
. Finally, PLA2
activity of Prdx6 also plays a critical role in oxidative stress- and TNF-induced apoptosis 
. Further studies on the mechanism of Prdx6 induction in AT1 cells and its potential role in suppressing influenza-induced apoptotic cell death are in progress.
After infecting Clara cells, the virus moves deeper into the lung and replicates in AT2 cells, which are found in the alveoli. AT2 cells are known as secretory cells which release the components of surfactant and extracellular matrix 
and extracelluar antioxidants Gpx3 and Sod3 
. These observations are consistent with a role for AT2 cells in providing antioxidant protection to cells coated with AT2-produced surfactant (e.g., AT1 cells). Thus, AT2 cells represent another target cell type that, when virally infected, might lead to loss of extracellular antioxidant enzymes. Unexpectedly, however, we did not observe any effects of AT2 cell depletion on antioxidant protein expression levels in the lungs. In particular, GPX3 was increased at the time of drastic loss of AT2 cells. Although both Sod3 and Gpx3 are secreted by AT2 cells in the lungs, they are also secreted by kidney proximal tubule cells 
. Immunostained lung sections demonstrated that Gpx3, which is normally at the basement membrane of blood vessels, becomes diffuse and is subsequently increased throughout the alveolar spaces. Therefore, it is possible that plasma Gpx3 gains access to the lungs due to increased permeability during infection. Thus, the reduction in AT2-secreted Gpx3 is likely masked by the influx of antioxidants in the plasma, thus providing alternative mechanism for maintaining the balance of antioxidants during infection.
In summary, here we have identified novel lung-abundant antioxidant enzymes and we have shown that influenza virus infection impacts their spatio-temporal distribution. Both Clara and AT2 cells are the major influenza-permissive cell types in the lungs, and their loss lead to reduction of key lung antioxidants. However, we present two examples wherein loss of expression of antioxidants is balanced by expression by other cell types. Specifically, both the Clara-enriched Prdx6 and the AT2-enriched Gpx3 are maintained, possibly by compensatory expression system during infection. Taken together, this work reveals several novel lung-specific antioxidants and new mechanisms by which the lungs maintain their levels of antioxidants during infection. Influenza toxicity can be caused by oxidative stress, which can be exacerbated by imbalances in the levels of lung-abundant antioxidants. A deeper understanding of the biology and the responses of lung-abundant antioxidants to influenza is therefore fundamental to our understanding of influenza-induced morbidity and mortality.