Predicting Influenza and Rhinovirus Infections in Airway Cells Utilizing Volatile Emissions.

BACKGROUND: Respiratory viral infections are common and potentially devastating to patients with underlying lung disease. Diagnosing viral infections often requires invasive sampling, and interpretation often requires specialized laboratory equipment. Here, we test the hypothesis that a breath test could diagnose influenza and rhinovirus infections using an in vitro model of the human airway. METHODS: Cultured primary human tracheobronchial epithelial cells were infected with either influenza A H1N1 or rhinovirus 1B and compared with healthy control cells. Headspace volatile metabolite measurements of cell cultures were made at 12-hour time points postinfection using a thermal desorption-gas chromatography-mass spectrometry method. RESULTS: Based on 54 compounds, statistical models distinguished volatile organic compound profiles of influenza- and rhinovirus-infected cells from healthy counterparts. Area under the curve values were 0.94 for influenza, 0.90 for rhinovirus, and 0.75 for controls. Regression analysis predicted how many hours prior cells became infected with a root mean square error of 6.35 hours for influenza- and 3.32 hours for rhinovirus-infected cells. CONCLUSIONS: Volatile biomarkers released by bronchial epithelial cells could not only be used to diagnose whether cells were infected, but also the timing of infection. Our model supports the hypothesis that a breath test could serve to diagnose viral infections.

The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma.

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Early Life Wildfire Smoke Exposure Is Associated with Immune Dysregulation and Lung Function Decrements in Adolescence.

The long-term health effects of wildfire smoke exposure in pediatric populations are not known. The objectives of this study were to determine if early life exposure to wildfire smoke can affect parameters of immunity and airway physiology that are detectable with maturity. We studied a mixed-sex cohort of rhesus macaque monkeys that were exposed as infants to ambient wood smoke from a series of Northern California wildfires in the summer of 2008. Peripheral blood mononuclear cells (PBMCs) and pulmonary function measures were obtained when animals were approximately 3 years of age. PBMCs were cultured with either LPS or flagellin, followed by measurement of secreted IL-8 and IL-6 protein. PBMCs from a subset of female animals were also evaluated by Toll-like receptor (TLR) pathway mRNA analysis. Induction of IL-8 protein synthesis with either LPS or flagellin was significantly reduced in PBMC cultures from wildfire smoke-exposed female monkeys. In contrast, LPS- or flagellin-induced IL-6 protein synthesis was significantly reduced in PBMC cultures from wildfire smoke-exposed male monkeys. Baseline and TLR ligand-induced expression of the transcription factor, RelB, was globally modulated in PBMCs from wildfire smoke-exposed monkeys, with additional TLR pathway genes affected in a ligand-dependent manner. Wildfire smoke-exposed monkeys displayed significantly reduced inspiratory capacity, residual volume, vital capacity, functional residual capacity, and total lung capacity per unit of body weight relative to control animals. Our findings suggest that ambient wildfire smoke exposure during infancy results in sex-dependent attenuation of systemic TLR responses and reduced lung volume in adolescence.

Using the ROX Index to Predict Treatment Outcome for High-Flow Nasal Cannula and/or Noninvasive Ventilation in Patients With COPD Exacerbations.

BACKGROUND: The ratio of oxygen saturation index (ROX index; or SpO2 /FIO2 /breathing frequency) has been shown to predict risk of intubation after high-flow nasal cannula (HFNC) support among adults with acute hypoxemic respiratory failure primarily due to pneumonia. However, its predictive value for other subtypes of respiratory failure is unknown. This study investigated whether the ROX index predicts liberation from HFNC or noninvasive ventilation (NIV), intubation with mechanical ventilation, or death in adults admitted for respiratory failure due to an exacerbation of COPD. METHODS: We performed a retrospective study of 260 adults hospitalized with a COPD exacerbation and treated with HFNC and/or NIV (continuous or bi-level). ROX index scores were collected at treatment initiation and predefined time intervals throughout HFNC and/or NIV treatment or until the subject was intubated or died. A ROX index score of ≥ 4.88 was applied to the cohort to determine if the same score would perform similarly in this different cohort. Accuracy of the ROX index was determined by calculating the area under the receiver operator curve. RESULTS: A total of 47 subjects (18%) required invasive mechanical ventilation or died while on HFNC/NIV. The ROX index at treatment initiation, 1 h, and 6 h demonstrated the best prediction accuracy for avoidance of invasive mechanical ventilation or death (area under the receiver operator curve 0.73 [95% CI 0.66-0.80], 0.72 [95% CI 0.65-0.79], and 0.72 [95% CI 0.63-0.82], respectively). The optimal cutoff value for sensitivity (Sn) and specificity (Sp) was a ROX index score > 6.88 (sensitivity 62%, specificity 57%). CONCLUSIONS: The ROX index applied to adults with COPD exacerbations treated with HFNC and/or NIV required higher scores to achieve similar prediction of low risk of treatment failure when compared to subjects with hypoxemic respiratory failure/pneumonia. ROX scores < 4.88 did not accurately predict intubation or death.

Oxylipin concentration shift in exhaled breath condensate (EBC) of SARS-CoV-2 infected patients.

Infection of airway epithelial cells with severe acute respiratory coronavirus 2 (SARS-CoV-2) can lead to severe respiratory tract damage and lung injury with hypoxia. It is challenging to sample the lower airways non-invasively and the capability to identify a highly representative specimen that can be collected in a non-invasive way would provide opportunities to investigate metabolomic consequences of COVID-19 disease. In the present study, we performed a targeted metabolomic approach using liquid chromatography coupled with high resolution chromatography (LC-MS) on exhaled breath condensate (EBC) collected from hospitalized COVID-19 patients (COVID+) and negative controls, both non-hospitalized and hospitalized for other reasons (COVID-). We were able to noninvasively identify and quantify inflammatory oxylipin shifts and dysregulation that may ultimately be used to monitor COVID-19 disease progression or severity and response to therapy. We also expected EBC-based biochemical oxylipin changes associated with COVID-19 host response to infection. The results indicated ten targeted oxylipins showing significative differences between SAR-CoV-2 infected EBC samples and negative control subjects. These compounds were prostaglandins A2 and D2, LXA4, 5-HETE, 12-HETE, 15-HETE, 5-HEPE, 9-HODE, 13-oxoODE and 19(20)-EpDPA, which are associated with specific pathways (i.e. P450, COX, 15-LOX) related to inflammatory and oxidative stress processes. Moreover, all these compounds were up-regulated by COVID+, meaning their concentrations were higher in subjects with SAR-CoV-2 infection. Given that many COVID-19 symptoms are inflammatory in nature, this is interesting insight into the pathophysiology of the disease. Breath monitoring of these and other EBC metabolites presents an interesting opportunity to monitor key indicators of disease progression and severity.

Dual oxidase 2 bidirectional promoter polymorphisms confer differential immune responses in airway epithelia.

The dual oxidase enzymes, DUOX, localized to the respiratory tract epithelium, are important components of innate host defense against bacteria and virus. However, little is known regarding the regulation of DUOX transcription. To better understand DUOX2-mediated mechanisms of antiviral host defense in the airway epithelium, we designed a bidirectional promoter luciferase reporter system to identify important cis-regulatory regions in the human DUOX2/DUOXA2 promoter. In this report, we demonstrate that the genomic region between the translation start sites of DUOX2 and DUOXA2 functions as a bidirectional promoter in human airway tissue. We also identified key regulatory regions on the DUOX2/DUOXA2 promoter that were necessary for both bidirectional and unidirectional transcriptional activity. Importantly, we discovered two functionally important single-nucleotide polymorphisms (SNPs) within the promoter that differentially regulated DUOX2/DUOXA2 transcription in response to exogenous double-stranded DNA. One of these SNPs, rs269855 (enriched in people of African descent), conferred the highest level of DUOX2 promoter activity. The clinical sequelae for individuals who carry this polymorphism remain to be determined.

A 70-Year-Old Woman With Refractory Hypoxemia.

CASE PRESENTATION: A 70-year-old woman was transferred to our ED from an outside ED for hypoxemia. Three weeks earlier, an inpatient evaluation for syncope revealed a right intraventricular filling defect, multiple pulmonary nodules, pulmonary emboli, and a left breast mass. She underwent breast biopsy, was started on rivaroxaban, and was discharged with outpatient follow-up. She experienced progressively worsening dyspnea, prompting a return to the outside ED, where she was found to be severely hypoxemic and was intubated. Her medical history included diabetes, hypertension, hyperlipidemia, COPD, hypothyroidism, diastolic heart failure, and a 40+ pack-year smoking history.

Inactivation of SARS-CoV-2 in clinical exhaled breath condensate samples for metabolomic analysis.

Exhaled breath condensate (EBC) is routinely collected and analyzed in breath research. Because it contains aerosol droplets, EBC samples from SARS-CoV-2 infected individuals harbor the virus and pose the threat of infectious exposure. We report for the first time a safe and consistent method to fully inactivate SARS-CoV-2 in EBC samples and make EBC samples safe for processing and analysis. EBC samples containing infectious SARS-CoV-2 were treated with several concentrations of acetonitrile. The most commonly used 10% acetonitrile treatment for EBC processing failed to completely inactivate the virus in samples and viable virus was detected by the assay of SARS-CoV-2 infection of Vero E6 cells in a biosafety level 3 laboratory. Treatment with either 50% or 90% acetonitrile was effective to completely inactivate the virus, resulting in safe, non-infectious EBC samples that can be used for metabolomic analysis. Our study provides SARS-CoV-2 inactivation protocol for the collection and processing of EBC samples in the clinical setting and for advancing to metabolic assessments in health and disease.

All-trans retinoic acid mediates DUOX2 expression and function in respiratory tract epithelium.

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 DUOX-mediated H(2)O(2) 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.

IFNgamma mediates DUOX2 expression via a STAT-independent signaling pathway.

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 (IFNgamma) in respiratory tract epithelium. We discovered that IFNgamma-mediated DUOX2 expression was regulated by a STAT-independent, JAK-independent pathway. These data provide insights into a novel IFNgamma signaling pathway with potential importance for regulation of host defense responses.

Evidence-based review of data on the combination inhaler umeclidinium/vilanterol in patients with COPD.

The use of inhaled, fixed-dose, long-acting muscarinic antagonists (LAMA) combined with long-acting, beta2-adrenergic receptor agonists (LABA) has become a mainstay in the maintenance treatment of chronic obstructive pulmonary disease (COPD). One of the fixed-dose LAMA/LABA combinations is the dry powder inhaler (DPI) of umeclidinium bromide (UMEC) and vilanterol trifenatate (VI) (62.5 µg/25 µg) approved for once-a-day maintenance treatment of COPD. This paper reviews the use of fixed-dose combination LAMA/LABA agents focusing on the UMEC/VI DPI inhaler in the maintenance treatment of COPD. The fixed-dose combination LAMA/LABA inhaler offers a step beyond a single inhaled maintenance agent but is still a single device for the COPD patient having frequent COPD exacerbations and persistent symptoms not well controlled on one agent. Currently available clinical trials suggest that the once-a-day DPI of UMEC/VI is well-tolerated, safe and non-inferior or better than other currently available inhaled fixed-dose LAMA/LABA combinations for COPD.

Airway transcriptomic profiling after bronchial thermoplasty.

BACKGROUND: Bronchial thermoplasty is a nonpharmacological, device-based treatment option for a specific population of severe asthmatic subjects, but the underlying mechanisms are largely unknown. The purpose of this study is to identify potential altered pathways by bronchial thermoplasty using a transcriptomic approach. METHODS: Patients undergoing bronchial thermoplasty were recruited to the study, and a bronchial brushing sample was obtained before each bronchial thermoplasty session and sent for RNA sequencing. A variance component score test was performed to identify those genes whose expression varied after bronchial thermoplasty sessions. Differential gene expression meta-analysis of severe asthmatic subjects versus controls was performed using public repositories. Overlapping genes were included for downstream pathway and network analyses. RESULTS: 12 patients were enrolled in our study. A total of 133 severe asthma cases and 107 healthy controls from the public repositories were included in the meta-analysis. Comparison of differentially expressed genes from our study patients with the public repositories identified eight overlapping genes: AMIGO2, CBX7, NR3C2, SETBP1, SHANK2, SNTB1, STXBP1 and ZNF853. Network analysis of these overlapping genes identified pathways associated with neurophysiological processes. CONCLUSION: We have shown that bronchial thermoplasty treatment alters several gene networks that are important in asthma pathogenesis. These results potentially elucidate the disease-modifying mechanisms of bronchial thermoplasty and provide several targets for further investigation.

Volatile organic compound (VOC) emissions of CHO and T cells correlate to their expansion in bioreactors.

Volatile organic compound (VOC) emissions were measured from Chinese Hamster Ovary (CHO) cell and T cell bioreactor gas exhaust lines with the goal of non-invasively metabolically profiling the expansion process. Measurements of cellular 'breath' were made directly from the gas exhaust lines using polydimethylsiloxane (PDMS)-coated magnetic stir bars, which underwent subsequent thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) analysis. Baseline VOC profiles were observed from bioreactors filled with only liquid media. After inoculation, unique VOC profiles correlated to cell expansion over the course of 8 d. Partial least squares (PLS) regression models were built to predict cell culture density based on VOC profiles of CHO and T cells (R 2 = 0.671 and R 2 = 0.769, respectively, based on a validation data set). T cell runs resulted in 47 compounds relevant to expansion while CHO cell runs resulted in 45 compounds; the 20 most relevant compounds of each cell type were putatively identified. On the final experimental days, sorbent-covered stir bars were placed directly into cell-inoculated media and into media controls. Liquid-based measurements from spent media containing cells could be distinguished from media-only controls, indicating soluble VOCs excreted by the cells during expansion. A PLS-discriminate analysis (PLS-DA) was performed, and 96 compounds differed between T cell-inoculated media and media controls with 72 compounds for CHO cells; the 20 most relevant compounds of each cell line were putatively identified. This work demonstrates that the volatilome of cell cultures can be exploited by chemical detectors in bioreactor gas and liquid waste lines to non-invasively monitor cellular health and could possibly be used to optimize cell expansion conditions 'on-the-fly' with appropriate control loop systems. Although the basis for statistical models included compounds without certain identification, this work provides a foundation for future research of bioreactor emissions. Future studies must move towards identifying relevant compounds for understanding of underlying biochemistry.

Bench-to-bedside review: rare and common viral infections in the intensive care unit--linking pathophysiology to clinical presentation.

Viral infections are common causes of respiratory tract disease in the outpatient setting but much less common in the intensive care unit. However, a finite number of viral agents cause respiratory tract disease in the intensive care unit. Some viruses, such as influenza, respiratory syncytial virus (RSV), cytomegalovirus (CMV), and varicella-zoster virus (VZV), are relatively common. Others, such as adenovirus, severe acute respiratory syndrome (SARS)-coronavirus, Hantavirus, and the viral hemorrhagic fevers (VHFs), are rare but have an immense public health impact. Recognizing these viral etiologies becomes paramount in treatment, infection control, and public health measures. Therefore, a basic understanding of the pathogenesis of viral entry, replication, and host response is important for clinical diagnosis and initiating therapeutic options. This review discusses the basic pathophysiology leading to clinical presentations in a few common and rare, but important, viruses found in the intensive care unit: influenza, RSV, SARS, VZV, adenovirus, CMV, VHF, and Hantavirus.

Biological responses to asbestos inhalation and pathogenesis of asbestos-related benign and malignant disease.

Asbestos comprises a group of fibrous minerals that are naturally occurring in the environment. Because of its natural properties, asbestos gained popularity for commercial applications in the late 19th century and was used throughout the majority of the 20th century, with predominant use in the construction, automotive, and shipbuilding industries. Asbestos has been linked to a spectrum of pulmonary diseases, such as pleural fibrosis and plaques, asbestosis, benign asbestos pleural effusion, small cell lung carcinoma, non-small cell lung carcinoma, and malignant mesothelioma. There are several mechanisms through which asbestos can lead to both benign and malignant disease, and they include alterations at the chromosomal level, activation of oncogenes, loss of tumor suppressor genes, alterations in cellular signal transduction pathways, generation of reactive oxygen and nitrogen species, and direct mechanical damage to cells from asbestos fibers. While known risk factors exist for the development of asbestos-related malignancies, there are currently no effective means to determine which asbestos-exposed patients will develop malignancy and which will not. There are also no established screening strategies to detect asbestos-related malignancies in patients who have a history of asbestos exposure. In this article, we present a case that highlights the different biological responses in human hosts to asbestos exposure.

Effects of positive end-expiratory pressure and recruitment maneuvers in a ventilator-induced injury mouse model.

BACKGROUND: Positive-pressure mechanical ventilation is an essential therapeutic intervention, yet it causes the clinical syndrome known as ventilator-induced lung injury. Various lung protective mechanical ventilation strategies have attempted to reduce or prevent ventilator-induced lung injury but few modalities have proven effective. A model that isolates the contribution of mechanical ventilation on the development of acute lung injury is needed to better understand biologic mechanisms that lead to ventilator-induced lung injury. OBJECTIVES: To evaluate the effects of positive end-expiratory pressure and recruitment maneuvers in reducing lung injury in a ventilator-induced lung injury murine model in short- and longer-term ventilation. METHODS: 5-12 week-old female BALB/c mice (n = 85) were anesthetized, placed on mechanical ventilation for either 2 hrs or 4 hrs with either low tidal volume (8 ml/kg) or high tidal volume (15 ml/kg) with or without positive end-expiratory pressure and recruitment maneuvers. RESULTS: Alteration of the alveolar-capillary barrier was noted at 2 hrs of high tidal volume ventilation. Standardized histology scores, influx of bronchoalveolar lavage albumin, proinflammatory cytokines, and absolute neutrophils were significantly higher in the high-tidal volume ventilation group at 4 hours of ventilation. Application of positive end-expiratory pressure resulted in significantly decreased standardized histology scores and bronchoalveolar absolute neutrophil counts at low- and high-tidal volume ventilation, respectively. Recruitment maneuvers were essential to maintain pulmonary compliance at both 2 and 4 hrs of ventilation. CONCLUSIONS: Signs of ventilator-induced lung injury are evident soon after high tidal volume ventilation (as early as 2 hours) and lung injury worsens with longer-term ventilation (4 hrs). Application of positive end-expiratory pressure and recruitment maneuvers are protective against worsening VILI across all time points. Dynamic compliance can be used guide the frequency of recruitment maneuvers to help ameloriate ventilator-induced lung injury.

Posttranslational nitrotyrosination of alpha-tubulin induces cell cycle arrest and inhibits proliferation of vascular smooth muscle cells.

Hyperproliferation of vascular smooth muscle cells is a hallmark of atherosclerosis and related vascular complications. Microtubules are important for many aspects of mammalian cell responses including growth, migration and signaling. alpha-Tubulin, a component of the microtubule cytoskeleton, is unique amongst cellular proteins in that it undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyr-tubulin). Whereas the reversible detyrosination/tyrosination cycle of alpha-tubulin has been implicated in regulating various aspects of cell biology, the precise function of this posttranslational modification has remained poorly characterized. Herein, we provide evidence suggesting that alpha-tubulin detyrosination is a required event in the proliferation of vascular smooth muscle cells. Proliferation of rat aortic smooth muscle cells in response to serum was temporally associated with the detyrosination of alpha-tubulin, but not acetylation of alpha-tubulin; Glu-tubulin reached maximal levels between 12 and 18h following cell cycle initiation. Inclusion of 3-nitro-l-tyrosine (NO(2)Tyr) in the culture medium resulted in the selective nitrotyrosination of alpha-tubulin, that was paralleled by decreased elaboration of Glu-tubulin, decreased expression of cyclins A and E, decreased association of the microtubule plus-end binding protein EB1, and inhibited cell proliferation. Nitrotyrosination of alpha-tubulin did not induce necrotic or apoptotic death of rat aortic smooth muscle cells, but instead led to cell cycle arrest at the G(1)/S boundary coincident with decreased DNA synthesis. Collectively, these results suggest that the C-terminus of alpha-tubulin and its detyrosination are functionally important as a molecular switch that regulates cell cycle progression in vascular smooth muscle cells.

Duox2 exhibits potent heme peroxidase activity in human respiratory tract epithelium.

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-gamma) induced robust peroxidase activity in TBE cells that paralleled Duox2 expression. IFN-gamma-induced peroxidase activity was abolished in the presence of sodium azide, which implicated the activation of a heme peroxidase. IFN-gamma-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.

Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium.

Partially reduced metabolites of molecular oxygen, superoxide (O2-) and hydrogen peroxide (H2O2), 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 H2O2 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-gamma. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 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.