Partially reduced metabolites of molecular oxygen, superoxide (O ÅÀ2 ) and hydrogen peroxide (H 2 O 2 ), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H 2 O 2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-c. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H 2 O 2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.

The dual oxidase isozymes Duox1 and Duox2 exhibit functional NADPH:O 2 oxidoreductase activity in thyroid and respiratory tract cells and are thought to be essential for H 2 O 2 generation in these tissues. However, it is not universally accepted that the heme peroxidase domains of the Duox isozymes are functional. To address this question, we modulated Duox2 expression in human tracheobronchial epithelial (TBE) cell culture systems and quantified peroxidase activity. We discovered that interferon-gamma (IFN-c) induced robust peroxidase activity in TBE cells that paralleled Duox2 expression. IFN-c-induced peroxidase activity was abolished in the presence of sodium azide, which implicated the activation of a heme peroxidase. IFN-c-induced peroxidase activity was abolished in TBE cell lines expressing anti-Duox2 short hairpin RNA transcripts. Together, these data unequivocally demonstrated that Duox2 contains a functional heme peroxidase in intact respiratory tract epithelium.

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RW. All-trans retinoic acid mediates DUOX2 expression and function in respiratory tract epithelium. Am J Physiol Lung Cell Mol Physiol 299: L215-L221, 2010. First published May 28, 2010 doi:10.1152/ajplung.00015.2010.-DUOX1 and DUOX2 are members of the NADPH oxidase family that are specifically regulated to produce hydrogen peroxide in epithelia of the thyroid, gastrointestinal tract, and respiratory tract. The determinants of DUOX1 or DUOX2 expression in various tissues have not been established. Using respiratory tract epithelial cells as a model, we investigated changes in DUOX mRNA and protein expression during the first 10 days of differentiation. By comparing a respiratory tract cell line, HBE1, with primary tracheobronchial epithelial (TBE) cells, we determined that DUOX2 was significantly expressed only in cell conditions that included all-trans retinoic acid (ATRA). In HBE1 cells, DUOX2 mRNA increased 6-fold after ATRA treatment. Similarly, ATRA induced a 19-fold increase in DUOX2 mRNA expression in primary TBE cells with parallel increases in DUOX protein and DUOXmediated H2O2 production as well. In addition, DUOX2 induction by rhinovirus required the presence of ATRA. ATRA had no effect on DUOX1 expression for all the conditions studied. Our data indicate that for respiratory epithelial cells, ATRA is important in the regulation of DUOX2 expression, function, and rhinovirus-mediated DUOX2 inducibility. DUOX1; gene regulation DUOX ISOZYMES DUOX1 AND DUOX2 are dual NADPH oxidase (NOX)-putative heme peroxidases expressed predominantly in epithelial tissues including epithelia of the thyroid, gastrointestinal tract, and respiratory tract (19). They are members of the NOX family that specifically produce hydrogen peroxide (H 2 O 2 ) in a regulated fashion. The functional significance of DUOX-mediated H 2 O 2 production and the relative contribution of each isoform for functional activity appears to be tissue-specific. For example, DUOX2 is the essential isoform for thyroid hormone synthesis (21), whereas DUOX1 appears to be the major isoform required for wound healing and innate host defense in the respiratory tract (6,7,16,22,28). In addition, the 5-to 10-fold higher levels of DUOX1 mRNA compared with DUOX2 mRNA in respiratory tract epithelium (17, 27) suggest DUOX1 is the predominant isoform for DUOXmediated H 2 O 2 production in the airway. However, DUOX2 likely has a higher capacity for H 2 O 2 generation compared with DUOX1 (2, 25) and may have a larger contribution to basal H 2 O 2 production than previously appreciated (15). How the relative expression levels and functional roles of each DUOX isoform are established in tissues remains undetermined.To begin to address this issue, we investigated some potential factors necessary for DUOX isoform expression in differentiating respiratory tract epithelia. Recent data suggest that DUOX expression in respiratory tract epithelia increases with increasing differentiation (11, 24). Furthermore, it has been proposed that DUOX expression is specifically expr...

The biological roles of the dual oxidases, DUOX1 and DUOX2, are dependent upon the tissue in which they are expressed. However, the mechanisms that control DUOX expression in these tissues are largely unexplored. Given the known role of DUOX for host defense in the gut and respiratory tract, we characterized potential mechanisms that control DUOX2 expression in response to interferon gamma (IFNγ) in respiratory tract epithelium. We discovered that IFNγ-mediated DUOX2 expression was regulated by a STAT-independent, JAK-independent pathway. These data provide insights into a novel IFNγ signaling pathway with potential importance for regulation of host defense responses.