Variants of human CLDN9 cause mild to profound hearing loss.

Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.

Inhibition of a transcriptional repressor rescues hearing in a splicing factor-deficient mouse.

In mechanosensory hair cells (HCs) of the ear, the transcriptional repressor REST is continuously inactivated by alternative splicing of its pre-mRNA. This mechanism of REST inactivation is crucial for hearing in humans and mice. Rest is one of many pre-mRNAs whose alternative splicing is regulated by the splicing factor SRRM4; Srrm4 loss-of-function mutation in mice (Srrm4 bv/bv ) causes deafness, balance defects, and degeneration of all HC types other than the outer HCs (OHCs). The specific splicing alterations that drive HC degeneration in Srrm4 bv/bv mice are unknown, and the mechanism underlying SRRM4-independent survival of OHCs is undefined. Here, we show that transgenic expression of a dominant-negative REST fragment in Srrm4 bv/bv mice is sufficient for long-term rescue of hearing, balancing, HCs, alternative splicing of Rest, and expression of REST target genes including the Srrm4 paralog Srrm3 We also show that in HCs, SRRM3 regulates many of the same exons as SRRM4; OHCs are unique among HCs in that they transiently down-regulate Rest transcription as they mature to express Srrm3 independently of SRRM4; and simultaneous SRRM4-SRRM3 deficiency causes complete HC loss by preventing inactivation of REST in all HCs. Thus, our data reveal that REST inactivation is the primary and essential role of SRRM4 in the ear, and that OHCs differ from other HCs in the SRRM4-independent expression of the functionally SRRM4-like splicing factor SRRM3.

Overlapping Activities of Two Neuronal Splicing Factors Switch the GABA Effect from Excitatory to Inhibitory by Regulating REST.

A truncating mutation in the mouse Srrm4 gene, which encodes a neuronal splicing factor, causes alternative splicing defects selectively in the ear. The mechanism by which splicing is preserved in the brain of these mice is not known. Here, we show that SRRM3 limits the Srrm4 mutation-associated defects to the ear and that, in cortical neurons, overlapping SRRM3-SRRM4 activity regulates the development of interneuronal inhibition. In vitro, SRRM3 and SRRM4 regulate the same splicing events, but a mutation in mouse Srrm3 causes tremors and mild defects in neuronal alternative splicing, demonstrating unique SRRM3 roles in vivo. Mice harboring mutations in both Srrm3 and Srrm4 die neonatally and exhibit severe splicing defects. In these mice, splicing alterations prevent inactivation of the gene repressor REST, which maintains immature excitatory GABAergic neurotransmission by repressing K-Cl cotransporter 2. Thus, our data reveal that SRRM3 and SRRM4 act redundantly to regulate GABAergic neurotransmission by inactivating REST.

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.

NADPH Oxidases Are Essential for Macrophage Differentiation.

NADPH oxidases (NOXs) are involved in inflammation, angiogenesis, tumor growth, and osteoclast differentiation. However, the role of NOX1 and NOX2 in macrophage differentiation and tumor progression is still elusive. Here we report that NOX1 and NOX2 are critical for the differentiation of monocytes to macrophages, the polarization of M2-type but not M1-type macrophages, and the occurrence of tumor-associated macrophages (TAMs). We found that deletion of both NOX1 and NOX2 led to a dramatic decrease in ROS production in macrophages and resulted in impaired efficiency in monocyte-to-macrophage differentiation and M2-type macrophage polarization. We further showed that NOX1 and NOX2 were critical for the activation of the MAPKs JNK and ERK during macrophage differentiation and that the deficiency of JNK and ERK activation was responsible for the failure of monocyte-to-macrophage differentiation, in turn affecting M2 macrophage polarization. Furthermore, we demonstrated that the decrease in M2 macrophages and TAMs, concomitant with the reduction of cytokine and chemokine secretion, contributed to the delay in wound healing and the inhibition of tumor growth and metastasis in NOX1/2 double knockout mice compared with WT mice. Collectively, these data provide direct evidence that NOX1 and NOX2 deficiency impairs macrophage differentiation and the occurrence of M2-type TAMs during tumor development.

A mutation in the mouse ttc26 gene leads to impaired hedgehog signaling.

The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.

Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity.

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN(-)), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN(-) yields the bactericidal hypothiocyanite (OSCN(-)) in airway surface liquid (ASL). Although SCN(-) is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I(-)) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN(-), inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I(-) concentration, and this I(-) concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses.

Hepatocytes produce TNF-α following hypoxia-reoxygenation and liver ischemia-reperfusion in a NADPH oxidase- and c-Src-dependent manner.

Cell line studies have previously demonstrated that hypoxia-reoxygenation (H/R) leads to the production of NADPH oxidase 1 and 2 (NOX1 and NOX2)-dependent reactive oxygen species (ROS) required for the activation of c-Src and NF-κB. We now extend these studies into mouse models to evaluate the contribution of hepatocytes to the NOX- and c-Src-dependent TNF-α production that follows H/R in primary hepatocytes and liver ischemia-reperfusion (I/R). In vitro, c-Src-deficient primary hepatocytes produced less ROS and TNF-α following H/R compared with controls. In vivo, c-Src-KO mice also had impaired TNF-α and NF-κB responses following partial lobar liver I/R. Studies in NOX1 and p47phox knockout primary hepatocytes demonstrated that both NOX1 and p47phox are partially required for H/R-mediated TNF-α production. To further investigate the involvement of NADPH oxidases in the production of TNF-α following liver I/R, we performed additional in vivo experiments in knockout mice deficient for NOX1, NOX2, p47phox, Rac1, and/or Rac2. Cumulatively, these results demonstrate that NOX2 and its activator subunits (p47phox and Rac) control the secretion of TNF-α by the liver following I/R. Interestingly, in the absence of Kupffer cells and NOX2, NOX1 played a dominant role in TNF-α production following hepatic I/R. However, NOX1 deletion alone had little effect on I/R-induced TNF-α. Thus Kupffer cell-derived factors and NOX2 act to suppress hepatic NOX1-dependent TNF-α production. We conclude that c-Src and NADPH oxidase components are necessary for redox-mediated production of TNF-α following liver I/R and that hepatocytes play an important role in this process.

Conserved cysteine residues provide a protein-protein interaction surface in dual oxidase (DUOX) proteins.

Intramolecular disulfide bond formation is promoted in oxidizing extracellular and endoplasmic reticulum compartments and often contributes to protein stability and function. DUOX1 and DUOX2 are distinguished from other members of the NOX protein family by the presence of a unique extracellular N-terminal region. These peroxidase-like domains lack the conserved cysteines that confer structural stability to mammalian peroxidases. Sequence-based structure predictions suggest that the thiol groups present are solvent-exposed on a single protein surface and are too distant to support intramolecular disulfide bond formation. To investigate the role of these thiol residues, we introduced four individual cysteine to glycine mutations in the peroxidase-like domains of both human DUOXs and purified the recombinant proteins. The mutations caused little change in the stabilities of the monomeric proteins, supporting the hypothesis that the thiol residues are solvent-exposed and not involved in disulfide bonds that are critical for structural integrity. However, the ability of the isolated hDUOX1 peroxidase-like domain to dimerize was altered, suggesting a role for these cysteines in protein-protein interactions that could facilitate homodimerization of the peroxidase-like domain or, in the full-length protein, heterodimeric interactions with a maturation protein. When full-length hDUOX1 was expressed in HEK293 cells, the mutations resulted in decreased H2O2 production that correlated with a decreased amount of the enzyme localized to the membrane surface rather than with a loss of activity or with a failure to synthesize the mutant proteins. These results support a role for the cysteine residues in intermolecular disulfide bond formation with the DUOX maturation factor DUOXA1.

A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse.

Sensory hair cells are essential for hearing and balance. Their development from epithelial precursors has been extensively characterized with respect to transcriptional regulation, but not in terms of posttranscriptional influences. Here we report on the identification and functional characterization of an alternative-splicing regulator whose inactivation is responsible for defective hair-cell development, deafness, and impaired balance in the spontaneous mutant Bronx waltzer (bv) mouse. We used positional cloning and transgenic rescue to locate the bv mutation to the splicing factor-encoding gene Ser/Arg repetitive matrix 4 (Srrm4). Transcriptome-wide analysis of pre-mRNA splicing in the sensory patches of embryonic inner ears revealed that specific alternative exons were skipped at abnormally high rates in the bv mice. Minigene experiments in a heterologous expression system confirmed that these skipped exons require Srrm4 for inclusion into the mature mRNA. Sequence analysis and mutagenesis experiments showed that the affected transcripts share a novel motif that is necessary for the Srrm4-dependent alternative splicing. Functional annotations and protein-protein interaction data indicated that the encoded proteins cluster in the secretion and neurotransmission pathways. In addition, the splicing of a few transcriptional regulators was found to be Srrm4 dependent, and several of the genes known to be targeted by these regulators were expressed at reduced levels in the bv mice. Although Srrm4 expression was detected in neural tissues as well as hair cells, analyses of the bv mouse cerebellum and neocortex failed to detect splicing defects. Our data suggest that Srrm4 function is critical in the hearing and balance organs, but not in all neural tissues. Srrm4 is the first alternative-splicing regulator to be associated with hearing, and the analysis of bv mice provides exon-level insights into hair-cell development.

Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung.

Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO(3)(-) transport. Without CFTR, airway epithelial HCO(3)(-) secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.

Role of Nox isoforms in angiotensin II-induced oxidative stress and endothelial dysfunction in brain.

Angiotensin II (Ang II) promotes vascular disease through several mechanisms including by producing oxidative stress and endothelial dysfunction. Although multiple potential sources of reactive oxygen species exist, the relative importance of each is unclear, particularly in individual vascular beds. In these experiments, we examined the role of NADPH oxidase (Nox1 and Nox2) in Ang II-induced endothelial dysfunction in the cerebral circulation. Treatment with Ang II (1.4 mg·kg(-1)·day(-1) for 7 days), but not vehicle, increased blood pressure in all groups. In wild-type (WT; C57Bl/6) mice, Ang II reduced dilation of the basilar artery to the endothelium-dependent agonist acetylcholine compared with vehicle but had no effect on responses in Nox2-deficient (Nox2(-/y)) mice. Ang II impaired responses to acetylcholine in Nox1 WT (Nox1(+/y)) and caused a small reduction in responses to acetylcholine in Nox1-deficient (Nox1(-/y)) mice. Ang II did not impair responses to the endothelium-independent agonists nitroprusside or papaverine in either group. In WT mice, Ang II increased basal and phorbol-dibutyrate-stimulated superoxide production in the cerebrovasculature, and these increases were abolished in Nox2(-/y) mice. Overall, these data suggest that Nox2 plays a relatively prominent role in mediating Ang II-induced oxidative stress and cerebral endothelial dysfunction, with a minor role for Nox1.

Nox1 transactivation of epidermal growth factor receptor promotes N-cadherin shedding and smooth muscle cell migration.

AIMS: In atherosclerosis and restenosis, vascular smooth muscle cells (SMCs) migrate into the subendothelial space and proliferate, contributing to neointimal formation. The goal of this study was to define the signalling pathway by which Nox1 NAPDH oxidase mediates SMC migration. METHODS AND RESULTS: SMCs were cultured from thoracic aorta from Nox1(-/y) (Nox1 knockout, KO) and wild-type (WT) mice. In response to thrombin, WT but not Nox1 KO SMCs generated increased levels of reactive oxygen species (ROS). Deficiency of Nox1 prevented thrombin-induced phosphorylation of Src and the subsequent transactivation of the epidermal growth factor receptor (EGFR) at multiple tyrosine residues. Next, activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and matrix metalloproteinase-9 (MMP-9) by thrombin was inhibited by the EGFR inhibitor AG1478 and in Nox1 KO SMCs. Thrombin-induced shedding of N-cadherin from the plasma membrane was dependent on the presence of Nox1 and was blocked by AG1478 and an inhibitor of metalloproteinases. Migration of SMCs to thrombin was impaired in the Nox1 KO SMCs and was restored by expression of Nox1. Finally, treatment of WT SMCs with AG1478 abrogated Nox1-dependent SMC migration. CONCLUSIONS: The Nox1 NADPH oxidase signals through EGFR to activate MMP-9 and promote the shedding of N-cadherin, thereby contributing to SMC migration.

Activation of NADPH oxidase 1 increases intracellular calcium and migration of smooth muscle cells.

Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca(2+)](i)) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca(2+)](i), as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca(2+)](i) in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca(2+)](i) showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca(2+) entry, cells were either bathed in Ca(2+)-free medium or exposed to dihydropyridines to block L-type Ca(2+) channel activity. Treatment with nifedipine or removal of extracellular Ca(2+) reduced the thrombin-mediated increase of [Ca(2+)](i) in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca(2+)](i) in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca(2+) mobilization in SMCs, in part through regulation of Ca(2+) influx, to thereby promote cell migration.

Role for Nox1 NADPH oxidase in atherosclerosis.

OBJECTIVE: Examine the contribution of Nox1 NADPH oxidase to atherogenesis. METHODS AND RESULTS: Male apolipoprotein E deficient mice (ApoE(-/-)) and male mice deficient in both apolipoprotein E and Nox1 (ApoE(-/-) Nox1(-/y)) received an atherogenic diet for 18 weeks. Mean blood pressures, body weights, and serum cholesterol levels were similar between the two groups of mice. Deficiency of Nox1 decreased superoxide levels and reduced lesion area in the aortic arch from 43% (ApoE(-/-)) to 28% (ApoE(-/-) Nox1(-/y)). The reduction in lesion size at the level of the aortic valve in ApoE(-/-)/Nox1(-/y) was accompanied by a decrease in macrophage infiltration as compared to ApoE(-/-) mice. Carotid artery ligation in ApoE(-/-) mice induced accelerated intimal hyperplasia with decreased cellular proliferation and increased collagen content in the neointima of vessels deficient in Nox1. CONCLUSIONS: Nox1-derived ROS modify lesion composition and contribute to lesion size in a murine model of atherosclerosis.

Enhancement of respiratory mucosal antiviral defenses by the oxidation of iodide.

Recent reports postulate that the dual oxidase (DUOX) proteins function as part of a multicomponent oxidative pathway used by the respiratory mucosa to kill bacteria. The other components include epithelial ion transporters, which mediate the secretion of the oxidizable anion thiocyanate (SCN(-)) into airway surface liquid, and lactoperoxidase (LPO), which catalyzes the H(2)O(2)-dependent oxidation of the pseudohalide SCN(-) to yield the antimicrobial molecule hypothiocyanite (OSCN(-)). We hypothesized that this oxidative host defense system is also active against respiratory viruses. We evaluated the activity of oxidized LPO substrates against encapsidated and enveloped viruses. When tested for antiviral properties, the LPO-dependent production of OSCN(-) did not inactivate adenovirus or respiratory syncytial virus (RSV). However, substituting SCN(-) with the alternative LPO substrate iodide (I(-)) resulted in a marked reduction of both adenovirus transduction and RSV titer. Importantly, well-differentiated primary airway epithelia generated sufficient H(2)O(2) to inactivate adenovirus or RSV when LPO and I(-) were supplied. The administration of a single dose of 130 mg of oral potassium iodide to human subjects increased serum I(-) concentrations, and resulted in the accumulation of I(-) in upper airway secretions. These results suggest that the LPO/I(-)/H(2)O(2) system can contribute to airway antiviral defenses. Furthermore, the delivery of I(-) to the airway mucosa may augment innate antiviral immunity.

A key role for NOX4 in epithelial cell death during development of lung fibrosis.

UNLABELLED: The pathogenesis of pulmonary fibrosis is linked to oxidative stress, possibly generated by the reactive oxygen species (ROS) generating NADPH oxidase NOX4. Epithelial cell death is a crucial early step in the development of the disease, followed only later by the fibrotic stage. We demonstrate that in lungs of patients with idiopathic lung fibrosis, there is strong expression of NOX4 in hyperplastic alveolar type II cells. AIM: To study a possible causative role of NOX4 in the death of alveolar cells, we have generated NOX4-deficient mice. RESULTS: Three weeks after administration of bleomycin, wild-type (WT) mice developed massive fibrosis, whereas NOX4-deficient mice displayed almost normal lung histology, and only little Smad2 phosphorylation and accumulation of myofibroblasts. However, the protective effects of NOX4 deficiency preceded the fibrotic stage. Indeed, at day 7 after bleomycin, lungs of WT mice showed massive increase in epithelial cell apoptosis and inflammation. In NOX4-deficient mice, no increase in apoptosis was observed, whereas inflammation was comparable to WT. In vitro, NOX4-deficient primary alveolar epithelial cells exposed to transforming growth factor-ß(1) did not generate ROS and were protected from apoptosis. Acute treatment with the NOX inhibitors also blunted transforming growth factor-ß(1)-induced apoptosis. CONCLUSION: ROS generation by NOX4 is a key player in epithelial cell death leading to pulmonary fibrosis.

Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions.

A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN(-)) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN(-)). Airway epithelial cultures have been shown to secrete SCN(-) in a CFTR-dependent manner. Thus, reduced SCN(-) availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN(-) concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN(-) concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN(-) concentration was somewhat reduced. Among human subjects, SCN(-) concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN(-) levels had better lung function than those with low SCN(-) levels. Thus, although CFTR can contribute to SCN(-) transport, it is not indispensable for the high SCN(-) concentration in ASL. The correlation between lung function and SCN(-) concentration in CF patients may reflect a beneficial role for SCN(-).