DUOX Genes Research Articles

Histone lactylation-ROS loop contributes to light exposure-exacerbated neutrophil recruitment in zebrafish

Light serves as a crucial external zeitgeber for maintaining and restoring physiological homeostasis in most organisms. Disrupting of light rhythms often leads to abnormal immune function, characterized by excessive inflammatory responses. However, the underlying regulatory mechanisms behind this phenomenon remain unclear. To address this concern, we use in vivo imaging to establish inflammation models in zebrafish, allowing us to investigate the effects and underlying mechanisms of light disruption on neutrophil recruitment. Our findings reveal that under sustained light conditions (LL), neutrophil recruitment in response to caudal fin injury and otic vesicle inflammation is significantly increased. This is accompanied by elevated levels of histone (H3K18) lactylation and reactive oxygen species (ROS) content. Through ChIP-sequencing and ChIP‒qPCR analysis, we discover that H3K18 lactylation regulates the transcriptional activation of the duox gene, leading to ROS production. In turn, ROS further promote H3K18 lactylation, forming a positive feedback loop. This loop, driven by H3K18 lactylation-ROS, ultimately results in the over recruitment of neutrophils to inflammatory sites in LL conditions. Collectively, our study provides evidence of a mutual loop between histone lactylation and ROS, exacerbating neutrophil recruitment in light disorder conditions, emphasizing the significance of maintaining a proper light-dark cycle to optimize immune function.

Dual oxidase 2 (duox 2) participates in the intestinal antibacterial innate immune responses of Procambarus clarkii by regulating ROS levels

Dual oxidase (Duox) a member of the nicotinamide adenine dinucleotide phosphate oxidase (NOX) family can induce the production of reactive oxygen species (ROS). In vertebrates, the duox gene was indicated to be associated with the mucosal immunity. The roles of the duox gene in invertebrates were mainly studied in insects for the function of maintaining intestinal flora balance. In recent years, some studies have reported that Duox is involved in regulating the production of ROS and plays an important role in defending against the intestinal pathogen infection. However, the molecular mechanism has not been fully illuminated. In this study, a duox 2 involved in the production of H2O2 was identified for the first time in P. clarkii. Mature Pc-Duox 2 is a 7-transmembrane protein molecule that includes PHD, FAD, and NAD domains. Pc-duox 2 was mainly expressed in hemocytes and intestinal tissue. Its expression levels were obviously upregulated after intramuscular or oral infection with V. harveyi. In the RNAi assay, the upregulated trends of H2O2 and total ROS levels in crayfish intestine were significantly suppressed when Pc-duox 2 was knocked down. Compared with the slightly affected SOD activity, the upregulated CAT activity was suppressed more obviously in the crayfish intestine. Furthermore, Pc-duox 2 had an important effect on the maintenance of the structural stability of crayfish the intestine. Further research revealed that the knockdown of Pc-duox 2 could cause an obvious suppression in the upregulated levels of Toll signalling pathway-related genes, including Pc-toll 1, Pc-toll 3, Pc-dorsal, Pc-ALF 5, Pc-crustin 1, and Pc-lysozyme. Ultimately, these changes triggered the accelerated death of crayfish. Overall, we speculated that Pc-duox 2 played an important role in antibacterial innate immunity in the crayfish intestine by regulating the total ROS level.

Circadian clock1a coordinates neutrophil recruitment via nfe212a/duox-reactive oxygen species pathway in zebrafish

Neutrophil recruitment to inflammatory sites appears to be an evolutionarily conserved strategy to fight against exogenous insults. However, the rhythmic characteristics and underlying mechanisms of neutrophil migration on a 24-h timescale are largely unknown. Using the advantage of invivo imaging of zebrafish, this study explored how the circadian gene clock1a dynamically regulates the rhythmic recruitment of neutrophils to inflammatory challenges. We generated a clock1a mutant and found that neutrophil migration is significantly increased in caudal fin injury and lipopolysaccharide (LPS) injection. Transcriptome sequencing, chromatin immunoprecipitation (ChIP), and dual-luciferase reporting experiments suggest that the clock1a gene regulates neutrophil migration by coordinating the rhythmic expression of nfe212a and duox genes to control the reactive oxygen species (ROS) level. This study ultimately provides a visual model to expand the understanding of the rhythmic mechanisms of neutrophil recruitment on a circadian timescale in a diurnal organism from the perspective of ROS.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential “nitrite stress–Duox activation–ROS production” pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for “nitrite stress–Duox activation–ROS production–Dorsal activation–AMP synthesis” that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

Microbiota aggravates the pathogenesis of Drosophila acutely exposed to vehicle exhaust

Vehicle exhaust (VE) is the primary cause of urban air pollution, which adversely affects the respiratory system, exacerbates lung diseases, and results in high mortality rates. However, the underlying mechanism of the pathogenesis is largely unclear. Here, we developed a Drosophila model to systematically investigate the effects of VE on their health and physiology. We found that VE significantly impaired life span and locomotion in Drosophila. Interestingly, there was an increase in bacterial load in the guts upon VE exposure, suggesting VE is able to induce dysbiosis in the guts. Microbiota depletion can ameliorate the impairment of life span and locomotion. VE causes permeability of intestinal epithelial cells and increases proliferation of intestinal cells, suggesting VE disrupts intestinal homeostasis. We elucidate the underlying mechanism by which VE triggers Imd and DUOX gene expression. Taken together, this Drosophila model provides insight into the pathogenesis of Drosophila exposure to VE, enabling us to better understand the specific role of microbiota.

3.5-GHz radiofrequency electromagnetic radiation promotes the development of Drosophila melanogaster

With the rapidly increasing popularity of 5G mobile technology, the effect of radiofrequency radiation on human health has caused public concern. This study explores the effects of a simulated 3.5 GHz radiofrequency electromagnetic radiation (RF-EMF) environment on the development and microbiome of flies under intensities of 0.1 W/m2, 1 W/m2 and 10 W/m2. We found that the pupation percentages in the first 3 days and eclosion rate in the first 2 days were increased under exposure to RF-EMF, and the mean development time was shortened. In a study on third-instar larvae, the expression levels of the heat shock protein genes hsp22, hsp26 and hsp70 and humoral immune system genes AttC, TotC and TotA were all significantly increased. In the oxidative stress system, DuoX gene expression was decreased, sod2 and cat gene expression levels were increased, and SOD and CAT enzyme activity also showed a significant increase. According to the 16S rDNA results, the diversity and species abundance of the microbial community decreased significantly, and according to the functional prediction analysis, the genera Acetobacter and Lactobacillus were significantly increased. In conclusion, 3.5 GHz RF-EMF may enhance thermal stress, oxidative stress and humoral immunity, cause changes in the microbial community, and regulate the insulin/TOR and ecdysteroid signalling pathways to promote fly development.

Dual Oxidase Mutant Retards Mauthner-Cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish.

Dual oxidase (duox)-derived reactive oxygen species (ROS) have been correlated with neuronal polarity, cerebellar development, and neuroplasticity. However, there have not been many comprehensive studies of the effect of individual duox isoforms on central-axon regeneration in vivo. Here, we explored this question in zebrafish, an excellent model organism for central-axon regeneration studies. In our research, mutation of the duox gene with CRISPR/Cas9 significantly retarded the single-axon regeneration of the zebrafish Mauthner cell in vivo. Using deep transcriptome sequencing, we found that the expression levels of related functional enzymes in mitochondria were down-regulated in duox mutant fish. In vivo imaging showed that duox mutants had significantly disrupted mitochondrial transport and redox state in single Mauthner-cell axon. Our research data provide insights into how duox is involved in central-axon regeneration by changing mitochondrial transport.

Anopheles stephensi Dual Oxidase Silencing Activates the Thioester-Containing Protein 1 Pathway to Suppress Plasmodium Development

We characterized the dual oxidase (Duox) gene in the major Indian malaria vector Anopheles stephensi, which regulates the generation of reactive oxygen species. The AsDuox gene encodes for a 1,475-amino-acid transmembrane protein that contains an N-terminal noncytoplasmic heme peroxidase domain, a calcium-binding domain, seven transmembrane domains, and a C-terminal cytoplasmic NADPH domain. Phylogenetic analyses revealed that A. stephensi Duox protein is highly conserved and shares 97–100% amino acid identity with other anopheline Duoxes. AsDuox is expressed in all the developmental stages of A. stephensi and the pupal stages revealed relatively higher expressions. The Duox gene is induced in Plasmodium-infected mosquito midguts, and RNA interference-mediated silencing of this gene suppressed parasite development through activation of the thioester-containing protein 1 pathway. We propose that this highly conserved anopheline Duox, being a Plasmodium agonist, is an excellent target to control malaria parasite development inside the insect host.

Zebrafish duox mutations provide a model for human congenital hypothyroidism.

ABSTRACTThyroid dyshormonogenesis is a leading cause of congenital hypothyroidism, a highly prevalent but treatable condition. Thyroid hormone (TH) synthesis is dependent on the formation of reactive oxygen species (ROS). In humans, the primary sources for ROS production during thyroid hormone synthesis are the NADPH oxidases DUOX1 and DUOX2. Indeed, mutations in DUOX1 and DUOX2 have been linked with congenital hypothyroidism. Unlike humans, zebrafish has a single orthologue for DUOX1 and DUOX2. In this study, we investigated the phenotypes associated with two nonsense mutant alleles, sa9892 and sa13017, of the single duox gene in zebrafish. Both alleles gave rise to readily observable phenotypes reminiscent of congenital hypothyroidism, from the larval stages through to adulthood. By using various methods to examine external and internal phenotypes, we discovered a strong correlation between TH synthesis and duox function, beginning from an early larval stage, when T4 levels are already noticeably absent in the mutants. Loss of T4 production resulted in growth retardation, pigmentation defects, ragged fins, thyroid hyperplasia/external goiter and infertility. Remarkably, all of these defects associated with chronic congenital hypothyroidism could be rescued with T4 treatment, even when initiated when the fish had already reached adulthood. Our work suggests that these zebrafish duox mutants may provide a powerful model to understand the aetiology of untreated and treated congenital hypothyroidism even in advanced stages of development.This article has an associated First Person interview with the first author of the paper.

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

BackgroundNADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not been examined. In this study, we analyzed the evolution of this gene family in arthropods. A thorough search of genomes and transcriptomes was performed enabling us to browse most branches of arthropod phylogeny.ResultsWe have found that the subfamilies NOX5 and DUOX are present in all arthropod groups. We also show that a NOX gene, closely related to NOX4 and previously found only in mosquitoes (NOXm), can also be found in other taxonomic groups, leading us to rename it as NOX4-art. Although the accessory protein p22-phox, essential for NOX1-4 activation, was not found in any of the arthropods studied, NOX4-art of Aedes aegypti encodes an active protein that produces H2O2. Although NOX4-art has been lost in a number of arthropod lineages, it has all the domains and many signature residues and motifs necessary for ROS production and, when silenced, H2O2 production is considerably diminished in A. aegypti cells.ConclusionsCombining all bioinformatic analyses and laboratory work we have reached interesting conclusions regarding arthropod NOX gene family evolution. NOX5 and DUOX are present in all arthropod lineages but it seems that a NOX2-like gene was lost in the ancestral lineage leading to Ecdysozoa. The NOX4-art gene originated from a NOX4-like ancestor and is functional. Although no p22-phox was observed in arthropods, there was no evidence of neo-functionalization and this gene probably produces H2O2 as in other metazoan NOX4 genes. Although functional and present in the genomes of many species, NOX4-art was lost in a number of arthropod lineages.

Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin.

The dual oxidases (Duox) are involved in hydrogen peroxide generation, which is essential for thyroid hormone synthesis, and therefore they are markers of thyroid function. During inflammation, cytokines upregulate DUOX gene expression in the airway and the intestine, suggesting a role for these proteins in innate immunity. It was previously demonstrated that interleukin-4 (IL-4) upregulates DUOX gene expression in thyrocytes. Although the role of IL-4 in autoimmune thyroid diseases has been studied extensively, the effects of IL-4 on thyroid physiology remain largely unknown. Therefore, a new animal model was generated to study the impact of IL-4 on thyroid function. Transgenic (Thyr-IL-4) mice with thyroid-targeted expression of murine IL-4 were generated. Transgene expression was verified at the mRNA and protein level in thyroid tissues and primary cultures. The phenotype of the Thyr-IL-4 animals was characterized by measuring serum thyroxine (T4) and thyrotropin levels and performing thyroid morphometric analysis, immunohistochemistry, whole transcriptome sequencing, quantitative reverse transcription polymerase chain reaction, and ex vivo thyroid function assays. Thyrocytes from two Thyr-IL-4 mouse lines (#30 and #52) expressed IL-4, which was secreted into the extracellular space. Although 10-month-old transgenic animals had T4 and thyrotropin serum levels in the normal range, they had altered thyroid follicular structure with enlarged follicles composed of elongated thyrocytes containing numerous endocytic vesicles. These follicles were positive for T4 staining the colloid, indicating their capacity to produce thyroid hormones. RNA profiling of Thyr-IL-4 thyroid samples revealed modulation of multiple genes involved in inflammation, while no major leukocyte infiltration could be detected. Upregulated expression of Duox1, Duoxa1, and the pendrin anion exchanger gene (Slc26a4) was detected. In contrast, the iodide symporter gene Slc5a5 was markedly downregulated resulting in impaired iodide uptake and reduced thyroid hormone levels in transgenic thyroid tissue. Hydrogen peroxide production was increased in Thyr-IL-4 thyroid tissue compared with wild-type animals, but no significant oxidative stress could be detected. This is the first study to show that ectopic expression of IL-4 in thyroid tissue upregulates Duox1/Duoxa1 and Slc26a4 expression in the thyroid. The present data demonstrate that IL-4 could affect thyroid morphology and function, mainly by downregulating Slc5a5 expression, while maintaining a normal euthyroid phenotype.

DuOx2 Promoter Regulation by Hormones, Transcriptional Factors and the Coactivator TAZ

The production of H₂O₂, which is essential to thyroid hormone synthesis, involves two NADPH oxidases: dual oxidases 1 and 2 (DuOx1 and DuOx2). A functional study with human DuOx genes and their 5′-flanking regions showed that DuOx1 and -2 promoters are different from thyroid-specific gene promoters. Furthermore, their transcriptional activities are not restricted to thyroid cells. While regulation of Tg (thyroglobulin) and TPO (thyroperoxidase) expression have been extensively studied, DuOx2 promoter regulation by hormones and transcriptional factors need to be more explored. Herein we investigated the role of TSH, insulin and insulin-like growth factor 1 (IGF-1), as well as the cAMP effect on DuOx2 promoter (ptx41) activity in transfected rat thyroid cell lines (PCCL3). We also assessed DuOx2 promoter activity in the presence of transcriptional factors crucial to thyroid development such as TTF-1 (thyroid transcription factor 1), PAX8, CREB, DREAM, Nkx2.5 and the coactivator TAZ in HeLa and HEK 293T-transfected cells. Our results show that TSH and forskolin, which increase cAMP in thyroid cells, stimulated DuOx2 promoter activity. IGF-1 led to pronounced stimulation, while insulin induction was not statistically different from DuOx2 promoter basal activity. All transcriptional factors selected for this work and coactivator TAZ, except DREAM, stimulated DuOx2 promoter activity. Moreover, Nkx2.5 and TAZ synergistically increased DuOx2 promoter activity. In conclusion, we show that DuOx2 expression is regulated by hormones and transcription factors involved in thyroid organogenesis and carcinogenesis, reinforcing the importance of the control of H₂O₂ generation in the thyroid.

Roles of DUOX-Mediated Hydrogen Peroxide in Metabolism, Host Defense, and Signaling

Among the NADPH oxidases, the dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially called thyroid oxidases, based on their high level of expression in thyroid tissue. Genetic alterations causing inherited hypothyroidism clearly demonstrate their physiological implication in thyroid hormonogenesis. However, a growing list of biological functions triggered by DUOX-dependent reactive oxygen species (ROS) in highly differentiated mucosae have recently emerged. A role of DUOX enzymes as ROS providers for lactoperoxidase-mediated killing of invading pathogens has been well established and a role in bacteria chemorepulsion has been proposed. Control of DUOX expression and activity by inflammatory molecules and immune receptor activation consolidates their contributions to innate immune defense of mucosal surfaces. Recent studies conducted in ancestral organisms have identified effectors of DUOX redox signaling involved in wound healing including epithelium regeneration and leukocyte recruitment. Moreover, local generation of hydrogen peroxide (H2O2) by DUOX has also been suggested to constitute a positive feedback loop to promote receptor signaling activation. A correct balance between H2O2 generation and detoxification mechanisms must be properly maintained to avoid oxidative damages. Overexpression of DUOX genes has been associated with an increasing number of chronic inflammatory diseases. Furthermore, H2O2-mediated DNA damage supports a mutagenic function promoting tumor development. Despite the high sequence similarity shared between DUOX1 and DUOX2, the two isoforms present distinct regulations, tissue expression and catalytic functions. The phenotypic characterization of novel DUOX/DUOXA invalidated animal models will be very useful for defining their medical importance in pathological conditions.

Activation of Dual Oxidases Duox1 and Duox2

Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.

Silencing of DUOX NADPH Oxidases by Promoter Hypermethylation in Lung Cancer

The development of lung cancer is associated with aberrant promoter methylation and thus transcriptional silencing of many tumor suppressor genes or genes critical for cellular maintenance. Here we report that the NADPH oxidases DUOX1 and DUOX2, which are one of the main sources for reactive oxygen species production in the airway, are frequently silenced in human lung cancer. Screening of lung cancer cell lines revealed loss of DUOX1 and DUOX2 expression, which was restored after treatment with 5-aza 2'-deoxycytidine. Two genes, DUOXA1 and DUOXA2, which are transcriptionally and functionally linked to DUOX, also showed coordinated down-regulation in lung cancer cells and lung cancer specimen. Bisulfite sequencing and methylation-specific PCR revealed that CpG-rich promoter regions in both DUOX genes are hypermethylated. Epigenetic modification of at least one DUOX gene was detected in 50% of primary adenocarcinomas. Immunohistochemical analysis of airway sections derived from cancerous and matched healthy tissues confirmed down-regulation of Duox in the ciliated epithelial cells lining the respiratory tract. Reintroduction of functional Duox1 into lung cancer cell lines increased cell migration and wound repair without affecting cell growth. Our results suggest that an area on chromosome 15 that includes DUOX1, DUOX2, and their maturation factors is a frequent target for epigenetic silencing in lung cancer.

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