PcEiger links the Imd/Relish pathway to ROS production in the intestine of the red swamp crayfish.

In the arthropod gut, commensal microbiota maintain the immune deficiency (Imd)/Relish pathway for expression of antimicrobial peptides, whereas pathogenic bacteria induce dual oxidase 2 (Duox2) for production of extracellular microbicidal reactive oxygen species (ROS). The Imd/Relish pathway and the Duox2/ROS system are regarded as independent systems. Here, we report that these two systems are bridged by the tumor necrosis factor (TNF) ortholog PcEiger in the red swamp crayfish Procambarus clarkii. PcEiger expression is induced by commensal bacteria or the Imd/Relish pathway. PcEiger knockdown alters bacterial abundance and community composition due to variations in the oxidative status of the intestine. PcEiger induces Duox2 expression and ROS production by regulating the activity of the transcription factor Atf2. Moreover, PcEiger mediates regulation of the Duox2/ROS system by commensal bacteria and the Imd/Relish pathway. Our findings suggest that the Imd/Relish pathway regulates the Duox2/ROS system via PcEiger in P. clarkii, and they provide insights into the crosstalk between these two important mechanisms for arthropod intestinal immunity.

Dual oxidase 1 promotes antiviral innate immunity.

Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.

Patients with Thyroid Dyshormonogenesis and DUOX2 Variants: Molecular and Clinical Description and Genotype-Phenotype Correlation.

Thyroid dyshormonogenesis (THD) is a heterogeneous group of genetic diseases caused by the total or partial defect in the synthesis or secretion of thyroid hormones. Genetic variants in DUOX2 can cause partial to total iodination organification defects and clinical heterogeneity, from transient to permanent congenital hypothyroidism. The aim of this study was to undertake a molecular characterization and genotype-phenotype correlation in patients with THD and candidate variants in DUOX2. A total of 31 (19.38%) patients from the Catalan Neonatal Screening Program presented with variants in DUOX2 that could explain their phenotype. Fifteen (48.39%) patients were compound heterozygous, 10 (32.26%) heterozygous, and 4 (12.90%) homozygous. In addition, 8 (26.67%) of these patients presented variants in other genes. A total of 35 variants were described, 10 (28.57%) of these variants have not been previously reported in literature. The most frequent variant in our cohort was c.2895_2898del/p.(Phe966SerfsTer29), classified as pathogenic according to reported functional studies. The final diagnosis of this cohort was permanent THD in 21 patients and transient THD in 10, according to reevaluation and/or need for treatment with levothyroxine. A clear genotype-phenotype correlation could not be identified; therefore, functional studies are necessary to confirm the pathogenicity of the variants.

Serotonin modulates insect gut bacterial community homeostasis.

BACKGROUND: Metazoan guts are in permanent contact with microbial communities. However, the host mechanisms that have developed to manage the dynamic changes of these microorganisms and maintain homeostasis remain largely unknown. RESULTS: Serotonin (5-hydroxytryptamine [5-HT]) was found to modulate gut microbiome homeostasis via regulation of a dual oxidase (Duox) gene expression in both Bactrocera dorsalis and Aedes aegypti. The knockdown of the peripheral 5-HT biosynthetic gene phenylalanine hydroxylase (TPH) increased the expression of Duox and the activity of reactive oxygen species, leading to a decrease in the gut microbiome load. Moreover, the TPH knockdown reduced the relative abundance of the bacterial genera Serratia and Providencia, including the opportunistic pathogens, S. marcescens and P. alcalifaciens in B. dorsalis. Treatment with 5-hydroxytryptophan, a precursor of 5-HT synthesis, fully rescued the TPH knockdown-induced phenotype. CONCLUSIONS: The findings reveal the important contribution of 5-HT in regulating gut homeostasis, providing new insights into gut-microbe interactions in metazoans.

Novel biallelic mutations in the DUOX2 gene underlying very early-onset inflammatory bowel disease: A case report.

Genetic variants affecting the function of dual oxidase 2 (DUOX2), the catalytic subunit of membrane-bound enzymes that produce hydrogen peroxide, are associated with very early-onset inflammatory bowel disease (VEO-IBD). We report the case of a 1-year-old boy diagnosed with VEO-IBD after presenting with bloody diarrhea. He had pancolitis and an extensive small intestinal ulcerative lesion at age 4 years. Infliximab treatment was successful but was discontinued due to delayed reaction. At age 7 years, treatment with ustekinumab was started, and remission has been maintained for more than 2 years. Whole-exome sequencing identified compound heterozygous missense DUOX2 variants of unknown significance (p.[R1212H];[F1490Y]). Protein expression in the whole-cell lysate and plasma membrane was lower in F1490Y-DUOX2 than in wild-type (WT)-DUOX2. Hydrogen peroxide generation upon ionomycin stimulation was lower in cells expressing R1212H-DUOX2 and F1490Y-DUOX2 than in those expressing WT-DUOX2. The novel, inherited, biallelic DUOX2 mutations may be molecular risk factors of VEO-IBD.

Lifespan extension by peroxidase and dual oxidase-mediated ROS signaling through pyrroloquinoline quinone in C. elegans.

Reactive oxygen species (ROS), originally characterized based on their harmful effects on cells or organisms, are now recognized as important signal molecules regulating various biological processes. In particular, low levels of ROS released from mitochondria extend lifespan. Here, we identified a novel mechanism of generating appropriate levels of ROS at the plasma membrane through a peroxidase and dual oxidase (DUOX) system, which could extend lifespan in Caenorhabditis elegans A redox co-factor, pyrroloquinoline quinone (PQQ), activates the C. elegans DUOX protein BLI-3 to produce the ROS H2O2 at the plasma membrane, which is subsequently degraded by peroxidase (MLT-7), eventually ensuring adequate levels of ROS. These ROS signals are transduced mainly by the oxidative stress transcriptional factors SKN-1 (Nrf2 or NFE2L2 in mammals) and JUN-1, and partially by DAF-16 (a FOXO protein homolog). Cell biology experiments demonstrated a similarity between the mechanisms of PQQ-induced activation of human DUOX1 and DUOX2 and that of C. elegans BLI-3, suggesting that DUOXs are potential targets of intervention for lifespan extension. We propose that low levels of ROS, fine-tuned by the peroxidase and dual oxidase system at the plasma membrane, act as second messengers to extend lifespan by the effect of hormesis.

Hydrogen peroxide-producing NADPH oxidases and the promotion of migratory phenotypes in cancer.

The cellular microenvironment plays a critical role in cancer initiation and progression. Exposure to oxidative stress, specifically hydrogen peroxide (H2O2), has been linked to aberrant cellular signaling through which the development of cancer may be promoted. Three members of the NADPH oxidase family (NOX4, DUOX1 and DUOX2) explicitly generate this non-radical oxidant in a wide range of tissues, often in support of the inflammatory response. This review summarizes the contributions of each H2O2-producing NOX to the invasive behaviors of tumors and/or the epithelial-mesenchymal transition (EMT) in cancer that plays an essential role in metastasis. Tissue localization in tumorigenesis is also highlighted, with patient-derived TCGA microarray data profiled across 31 cancer cohorts to provide a comprehensive guide to the relevance of NOX4/DUOX1/DUOX2 in cancer studies.

Molecular basis of the counteraction by calcium channel blockers of cyclosporine nephrotoxicity.

Nephrotoxicity is a serious side effect for the immunosuppressant drug cyclosporine A(CSA). In this study, we tested the hypothesis that administration of calcium channel blockers such as verapamil or nifedipine ameliorates renal CSA-induced renal dysfunction. Furthermore, our study investigates the roles of inflammatory, oxidative, and fibrotic pathways in CSA-induced renal dysfunction. Six groups of male rats ( n = 6/group) were used and received one of the following treatments for seven consecutive days: vehicle (Cremophor EL ip), CSA (25 mg·kg-1·day-1 ip), verapamil (2 mg·kg-1·day-1 ip), nifedipine (3 mg·kg-1·day-1 ip), CSA in the presence or absence of either verapamil, or nifedipine. Biochemical and histomorphometric analyses showed that rats treated with CSA exhibited clear signs of nephrotoxicity that included 1) proteinuria and elevations in serum creatinine and blood urea nitrogen, 2) mesangial expansion, 3) increases in glomerular and tubular type IV collagen expression, and 4) increases in the glomerulosclerosis and tubulointerstitial fibrosis indices. Although the single administration of nifedipine or verapamil had no significant effect on renal pathology, or its biochemical and physiological function, the concurrent use of either calcium channel blockers significantly and equipotently ameliorated the biochemical, morphological, and functional derangements caused by CSA. More importantly, we report that the oxidative (reactive oxygen species production, NADPH-oxidase activity, and dual oxidase 1/2 levels), fibrotic (transforming growth factor-ß1 expression), and inflammatory (NF-κB expression) manifestations of renal toxicity induced by CSA were significantly reversed upon administration of nifedipine or verapamil. Together, these results highlight the efficacy of calcium channel-blocking agents in attenuating CSA-induced nephrotoxicity and predisposing biochemical and molecular machineries.

Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production.

Human airway epithelial cells express pannexin 1 (Panx1) channels to release ATP, which regulates mucociliary clearance. Airway inflammation causes mucociliary dysfunction. Exposure of primary human airway epithelial cell cultures to IFN-γ for 48 h did not alter Panx1 protein expression but significantly decreased ATP release in response to hypotonic stress. The IFN-γ-induced functional down-regulation of Panx1 was due to the up-regulation of dual oxidase 2 (Duox2). Duox2 suppression by siRNA led to an increase in ATP release in control cells and restoration of ATP release in cells treated with IFN-γ. Both effects were reduced by the pannexin inhibitor probenecid. Duox2 up-regulation stoichiometrically increases H2O2 and proton production. H2O2 inhibited Panx1 function temporarily by formation of disulfide bonds at the thiol group of its terminal cysteine. Long-term exposure to H2O2, however, had no inhibitory effect. To assess the role of cellular acidification upon IFN-γ treatment, fully differentiated airway epithelial cells were exposed to ammonium chloride to alkalinize the cytosol. This led to a 2-fold increase in ATP release in cells treated with IFN-γ that was also inhibited by probenecid. Duox2 knockdown also partially corrected IFN-γ-mediated acidification. The direct correlation between intracellular pH and Panx1 open probability was shown in oocytes. Therefore, airway epithelial cells release less ATP in response to hypotonic stress in an inflammatory environment (IFN-γ exposure). Decreased Panx1 function is a response to cell acidification mediated by IFN-γ-induced up-regulation of Duox2, representing a novel mechanism for mucociliary dysfunction in inflammatory airway diseases.

Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR.

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

OBJECTIVE AND DESIGN: Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. MATERIAL OR SUBJECTS: Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. TREATMENT: A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. METHODS: Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. RESULTS: Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. CONCLUSIONS: Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

The Gastric Mucosa from Patients Infected with CagA+ or VacA+ Helicobacter pylori Has a Lower Level of Dual Oxidase-2 Expression than Uninfected or Infected with CagA-/VacA- H. pylori.

BACKGROUND: Helicobacter pylori (H. pylori) is a well-recognized gastroduodenal pathogen and class I carcinogen. Dual oxidase-2 (DUOX2), a member of NADPH oxidase family, has several critical physiological functions, including thyroid hormone biosynthesis and host mucosal defense. AIM: To investigate the effect of H. pylori infection on DUOX2 gene expression in human stomach. MATERIALS AND METHODS: The biopsies were obtained from patients who underwent endoscopic diagnosis. The patient serum was assayed for two virulence factors of H. pylori, CagA IgG and VacA. The inflammation in gastric mucosa was analyzed with histology. Real-time quantitative PCR was used to detect the expression of three members of NADPH oxidase, NOX1, NOX2, and DUOX2, as well as lactoperoxidase (LPO) in the gastric mucosa. NOX2, DUOX2, and myeloperoxidase (MPO) protein levels were quantified by Western blots or immunohistochemistry. RESULTS: The H. pylori-infected gastric mucosa had more severe inflammation than uninfected samples. However, the expression of DUOX2 mRNA and protein was lower in gastric mucosa of patients with H. pylori infection compared to the uninfected. Among the H. pylori-infected patients, those having CagA IgG or VacA in the serum had lower DUOX2 expression levels than those infected with H. pylori without either virulence factor. The NOX2 and MPO levels were higher in those patients infected with H. pylori irrespective of the virulence factors than those uninfected patients. NOX1 and LPO mRNA were undetectable in the gastric mucosa. CONCLUSION: CagA+ or VacA+ H. pylori in the stomach of patients may suppress DUOX2 expression to promote its own survival. Increased NOX2 could not eliminate H. pylori infection.

Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases.

BACKGROUND: The recent recognition that isoforms of the cellular NADPH-dependent oxidases, collectively known as the NOX protein family, participate in a wide range of physiologic and pathophysiologic processes in both the animal and plant kingdoms has stimulated interest in the identification, localization, and quantitation of their products in biological settings. Although several tools for measuring oxidants released extracellularly are available, the specificity and selectivity of the methods for reliable analysis of intracellular oxidants have not matched the enthusiasm for studying NOX proteins. SCOPE OF REVIEW: Focusing exclusively on superoxide anion and hydrogen peroxide produced by NOX proteins, this review describes the ideal probe for analysis of O2(-) and H2O2 generated extracellularly and intracellularly by NOX proteins. An overview of the components, organization, and topology of NOX proteins provides a rationale for applying specific probes for use and a context in which to interpret results and thereby construct plausible models linking NOX-derived oxidants to biological responses. The merits and shortcomings of methods currently in use to assess NOX activity are highlighted, and those assays that provide quantitation of superoxide or H2O2 are contrasted with those intended to examine spatial and temporal aspects of NOX activity. MAJOR CONCLUSIONS: Although interest in measuring the extracellular and intracellular products of the NOX protein family is great, robust analytical probes are limited. GENERAL SIGNIFICANCE: The widespread involvement of NOX proteins in many biological processes requires rigorous approaches to the detection, localization, and quantitation of the oxidants produced. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Proteinase activated receptor-2-mediated dual oxidase-2 up-regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma.

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase-activated receptor 2 (PAR-2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX-2, have been shown to be involved in airway inflammation in response to Toll-like receptor activation. However, the association between PAR-2 and DUOX-2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX-2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR-2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX-2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl-2, caspase-3). Challenge with CE led to up-regulation of DUOX-2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD-1068, a small molecule antagonist of PAR-2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX-2/ROS pathway; however, these effects were less pronounced than PAR-2 antagonism. The current study suggests that PAR-2 activation leads to up-regulation of the DUOX-2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.

Dual oxidase 2 is essential for house dust mite-induced pro-inflammatory cytokine production in human keratinocytes.

House dust mites (HDMs) are known to trigger chronic inflammation through Toll-like receptors (TLRs) and their signalling cascades. In this study, we found that TLR2 ligation by HDMs induced the activation of dual oxidase 2 (Duox2) and nuclear factor-κB (NF-κB), leading to the production of pro-inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDMs resulted in increases in interleukin-8 (IL-8) and chemokine (C-C motif) ligand 20 (CCL20) levels. However, pro-inflammatory cytokine production was abolished in keratinocytes transfected with TLR2 siRNA, indicating that HDM-induced cytokine production was mediated via TLR2 signalling. We also examined the function of Duox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM-mediated pro-inflammatory cytokine production. Human keratinocytes transfected with control siRNA or Duox1 siRNA showed no inhibition of IL-8 or CCL20 production in response to HDMs, whereas the silencing of Duox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of RelA/p65, a component of NF-κB, were induced by HDMs in human keratinocytes. Transfection of human keratinocytes with TLR2 siRNA or Duox2 siRNA resulted in the complete abolishment of RelA/p65 nuclear localization in response to HDMs. Taken together, these results indicate that the HDM-dependent TLR2-Duox2 signalling axis indeed promotes NF-κB activation, which induces IL-8 and CCL20 production and mediates epidermal keratinocyte inflammation.

Mutation screening of DUOX2 in Chinese patients with congenital hypothyroidism.

BACKGROUND: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder in infancy. Dual oxidase 2 gene (DUOX2) mutations have been reported to be one of the leading genetic causes of CH. AIM: The aim of this study was to screen for DUOX2 gene mutations among CH patients in the Guangxi Zhuang Autonomous Region of China and to define the relationships between DUOX2 genotypes and clinical phenotypes. MATERIALS AND METHODS: Blood samples were collected from 45 CH patients in Guangxi Zhuang Autonomous Region, China, and genomic DNA was extracted from peripheral blood leukocytes. All exons of the DUOX2 gene together with their exon-intron boundaries were screened by Sanger sequencing. RESULTS: Sequencing analysis of DUOX2 in 45 CH patients revealed ten different variants in thirteen individuals. The variants included five known mutations, namely c.3329G>A (p.R1110Q), c.1588A>T (p.K530X), c.2635G>A (p.E879K), c.2524C>T (p.R842X) and c.4027G>T (p.L1343F), and one novel frame shift variant c.3340delC (p.L1114SfsX56), as well as four novel missense variants c.903G>T (p.W301C), c.2048G>T (p.R683L), c.1736T>C (p.L579P) and c.3413C>A (p.A1138D). The variant p.K530X is highly recurrent in our patient cohort but the clinical phenotypes vary greatly among those carrying this variant. Most patients with monoallelic or biallelic DUOX2 pathogenic variants turned out to be cases of transient congenital hypothyroidism (TCH), while three patients with triallelic DUOX2 pathogenic variants were associated with permanent congenital hypothyroidism (PCH). CONCLUSIONS: The prevalence of DUOX2 pathogenic variants was high (29 %) among patients with CH in Guangxi, China. Monoallelic and biallelic DUOX2 pathogenic variants were mainly associated with TCH, while triallelic DUOX2 pathogenic variants were associated with PCH. Our study expanded the DUOX2 mutation spectrum, and functional studies of the novel mutations need to be conducted in the future.

Novel genetic variants in the TPO gene cause congenital hypothyroidism.

BACKGROUND: Mutations in the dual oxidase maturation factor 2 (DUOXA2) and thyroid peroxidase (TPO) genes have been reported to cause goitrous congenital hypothyroidism (GCH). The aim of this study was to determine the genetic basis of GCH in affected children. METHODS: Thirty children with GCH were enrolled for molecular analysis of the DUOXA2 and TPO genes. All subjects underwent clinical examination and laboratory testing. Genomic DNA was extracted from peripheral blood leukocytes, and Sanger sequencing was used to screen for DUOXA2 and TPO gene mutations in the exon fragments amplified from the extracted DNA. Family members of those patients with mutations were also enrolled and evaluated. RESULTS: Analysis of the TPO gene revealed six genetic variants, including two novel heterozygous mutations, c.1970T> C (p.I657T) and c.2665G> T (p.G889X), and four mutations that have been reported previously (c.670_672del, c.2268dup, c.2266T> C and c.2647C> T). Three patients harbored the same mutation c.2268dup. The germline mutations from four unrelated families were consistent with an autosomal recessive inheritance pattern. Conversely, no mutations in the DUOXA2 gene were detected. CONCLUSION: Two novel inactivating mutations (c.1970T> C and c.2665G> T) in the TPO gene were identified. The c.2268dup mutation occurred frequently. No mutations in the DUOXA2 gene were detected in this study.

IFN-γ-mediated reduction of large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction.

Effective mucociliary clearance (MCC) depends in part on adequate airway surface liquid (ASL) volume to maintain an appropriate periciliary fluid height that allows normal ciliary activity. Apically expressed large-conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels provide an electrochemical gradient for Cl(-) secretion and thus play an important role for adequate airway hydration. Here we show that IFN-γ decreases ATP-mediated apical BK activation in normal human airway epithelial cells cultured at the air-liquid interface. IFN-γ decreased mRNA levels of KCNMA1 but did not affect total protein levels. Because IFN-γ upregulates dual oxidase (DUOX)2 and therefore H2O2 production, we hypothesized that BK inactivation could be mediated by BK oxidation. However, DUOX2 knockdown did not affect the IFN-γ effect on BK activity. IFN-γ changed mRNA levels of the BK ß-modulatory proteins KCNMB2 (increased) and KCNMB4 (decreased) as well as leucine-rich repeat-containing protein (LRRC)26 (decreased). Mallotoxin, a BK opener only in the absence of LRRC26, showed that BK channels lost their association with LRRC26 after IFN-γ treatment. Finally, IFN-γ caused a decrease in ciliary beating frequency that was immediately rescued by apical fluid addition, suggesting that it was due to ASL volume depletion. These data were confirmed with direct ASL measurements using meniscus scanning. Overexpression of KCNMA1, the pore-forming subunit of BK, overcame the reduction of ASL volume induced by IFN-γ. Key experiments were repeated in cystic fibrosis cells and showed the same results. Therefore, IFN-γ induces mucociliary dysfunction through BK inactivation.

Dual oxidases control release of hydrogen peroxide by the gastric epithelium to prevent Helicobacter felis infection and inflammation in mice.

BACKGROUND & AIMS: Dual oxidases (DUOX) are conserved reduced nicotinamide adenine dinucleotide phosphate oxidases that produce H2O2 at the epithelial cell surface. The DUOX enzyme comprises the DUOX and DUOX maturation factor (DUOXA) subunits. Mammalian genomes encode 2 DUOX isoenzymes (DUOX1/DUOXA1 and DUOX2/DUOXA2). Expression of these genes is up-regulated during bacterial infections and chronic inflammatory diseases of the luminal gastrointestinal tract. The roles of DUOX in cellular interactions with microbes have not been determined in higher vertebrates. METHODS: Mice with disruptions of Duoxa1 and Duoxa2 genes (Duoxa(-/-) mice) and control mice were infected with Helicobacter felis to create a model of Helicobacter pylori infection--the most common human chronic infection. RESULTS: Infection with H. felis induced expression of Duox2 and Duoxa2 in the stomachs of wild-type mice, and DUOX protein specifically localized to the apical surface of epithelial cells. H. felis colonized the mucus layer in the stomachs of Duoxa(-/-) mice to a greater extent than in control mice. The increased colonization persisted into the chronic phase of infection and correlated with an increased, yet ineffective, inflammatory response. H. felis colonization also was increased in Duoxa(+/-) mice, compared with controls. We observed reduced expression of the H2O2-inducible katA gene in H. felis that colonized Duoxa(-/-) mice, compared with that found in controls (P = .0002), indicating that Duox causes oxidative stress in these bacteria. In vitro, induction of oxidative defense by H. felis failed to prevent a direct bacteriostatic effect at sustained levels of H2O2 as low as 30 µmol/L. CONCLUSIONS: Based on studies of Duoxa(-/-) mice, the DUOX enzyme complex prevents gastric colonization by H. felis and the inflammatory response. These findings indicate the nonredundant function of epithelial production of H2O2 in restricting microbial colonization.

N-Linked glycosylation of the superoxide-producing NADPH oxidase Nox1.

Nox1 is a membrane-integrated protein that belongs to the Nox family of superoxide-producing NADPH oxidases. Here we show that human Nox1 undergoes glycosylation at Asn-162 and Asn-236 in the second and third extracellular loops, respectively. Simultaneous threonine substitution for these residues completely abrogates the glycosylation, but does not prevent Nox1 from forming a heterodimer with p22(phox), trafficking to the cell surface, or producing superoxide. In the absence of p22(phox), Nox1 is transported to the plasma membrane mainly as a form with high mannose N-glycans, although their conversion into complex N-glycans is induced by expression of p22(phox). These findings indicate that glycosylation and subsequent N-glycan maturation of Nox1 are both dispensable for its cell surface recruitment. Superoxide production by unglycosylated Nox1 is largely dependent on p22(phox), which is abrogated by glutamine substitution for Pro-156 in p22(phox), a mutation leading to a defective interaction with the Nox1-activating protein Noxo1. Thus p22(phox) directly contributes to Nox1 activation in a glycosylation-independent manner, besides its significant role in Nox1 glycan maturation.