Duox2 Expression Research Articles

DUOX2 As a Potential Prognostic Marker which Promotes Cell Motility and Proliferation in Pancreatic Cancer.

DUOX2 has been reported to highly express in several types of cancers. However, the prognostic significance and the biological function of DUOX2 expression with pancreatic cancer (PC) still remain unclear. The present study is aimed at investigating whether DUOX2 could act as a novel biomarker of prognosis and evaluating its effect on PC cell progression. The mRNA and protein expression of DUOX2 in PC cells and tissues were assessed by quantitative real-time PCR (RT-qPCR) and immunohistochemistry. The effect of DUOX2 expression on PC cell motility and proliferation was evaluated in vitro. The correlation between DUOX2 mRNA expression and clinicopathological features and its prognostic significance were analyzed according to the Gene Expression Profiling Interactive Analysis (GEPIA) website based on The Cancer Genome Atlas (TCGA) and the GTEx databases combined with our clinical information. According to bioinformatics analysis, we forecasted the upstream transcription factors (TFs) and microRNA (miRNA) regulatory mechanism of DUOX2 in PC. The expression of DUOX2 at transcriptional and protein level was dramatically increased in PC specimens when compared to adjacent nontumor specimens. Functionally, DUOX2 knockdown inhibited cell motility and proliferation activities. Our clinical data revealed that the patients had better postoperative overall survival (OS) with lower expression of DUOX2, which is consistent with GEPIA data. Multivariate analysis revealed that high DUOX2 expression was considered as an independent prognostic indicator for OS (P = 0.031). Based on Cistrome database, the top 5 TFs of each positively and negatively association with DUOX2 were predicted. hsa-miR-5193 and hsa-miR-1343-3p targeting DUOX2 were forecasted from TargetScan, miRDB, and DIANA-TarBase databases, which were negatively correlated with OS (P = 0.043 and P = 0.0088, respectively) and DUOX2 expression (P = 0.0093 and P = 0.0032, respectively) in PC from TCGA data. These findings suggest that DUOX2 acts as a promising predictive biomarker and an oncogene in PC, which could be a therapeutic target for PC.

Protection Against Radiation-Induced Duox1 and Duox2 Upregulation in Rat's Lung Tissues by a Combination of Curcumin and L-Selenomethionine

Background: It has been proposed that increased levels of pro-inflammatory and pro-fibrotic cytokines play a key role in radiation-induced lung injury. Interleukin-4 (IL-4) and IL-13 are two pro-fibrotic cytokines that promote the production of free radicals through stimulation of Duox1 and Duox2. In this experimental study, we aimed to evaluate the expression of IL4Ra1, Duox1, IL13Ra2, and Duox2 genes following rat’s lung irradiation. Objectives: Also, we detected the modulatory effect of a combination of curcumin and L-selenomethionine on the expression of these genes. Methods: Twenty male rats were divided into four groups as G1: control (no treatment or radiation); G2: treatment with a combination of curcumin and L-selenomethionine; G3: radiation; G4: radiation plus a combination of curcumin and L-selenomethionine. sixty-seven days after irradiation, rats were killed for detecting the expression of IL4Ra1, IL13Ra2, Duox1, and Duox2. Results: The results showed no detectable expression for IL13Ra2, while the expression of IL4Ra1, Duox1, and Duox2 was increased. Treatment with a combination of curcumin and L-selenomethionine could attenuate the expression of these genes. Conclusions: This study proposes that upregulation of Duox1 and Duox2 may be involved in radiation-induced lung injury. Treatment with a combination of curcumin and L-selenomethionine may be useful for the mitigation of lung injury through modulation of these genes.

INFLAMMATORY CYTOKINES AND MICROBIAL LIGANDS AND METABOLITES INTERACT TO MODULATE DUOX2 EXPRESSION AND ACTIVITY

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) is characterized by a dysregulated crosstalk between the host and the microbiome that leads to the development of inflammation and dysbiosis. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, particularly of butyrate-producing species such as Faecalibacterium prausnitzii. The epithelial NADPH oxidase dual oxidase 2 (DUOX2), which prevents bacterial colonization of the mucosa through the production of hydrogen peroxide (H2O2), is the only gene consistently altered in IBD patients before the onset of disease. However, the involvement of DUOX2 in IBD is not well understood. We aimed to define how inflammation and the microbiota regulate DUOX2 activity. Methods C57BI/6J males raised in specific-pathogen free (SPF) and germ-free (GF) conditions underwent a model of dextran sulfate sodium (DSS)-induced colitis for 6 days. We obtained colon specimens for histopathology and isolation of colon epithelial cells (CEC). We stimulated colonoids from wild-type (WT), toll-like receptor 4-KO (Tlr4-KO), and Duox2-KO mice with IFNγ, heat-killed adherent invasive Escherichia coli (AIEC) strain LF82, and heat-killed F. prausnitzii strain A2-165 and determined expression of Duox2, Duoxa2, and production of H2O2. We also treated colonoids with butyrate, a microbial metabolite with anti-inflammatory properties. Extracellular H2O2 production was analyzed by means of the Amplex Red assay, whereas gene expression was determined by qPCR. Results SPF mice undergoing DSS-induced colitis developed overt inflammation that was accompanied by upregulation of Duox2 and Duoxa2, as well as increased production of H2O2 in freshly isolated CECs. DSS-treated GF mice developed a mild inflammation that also caused increased H2O2 production and Duoxa2 upregulation. WT colonoid stimulation with IFNγ and the Crohn’s disease-associated pathobiont AIEC induced Duox2 and Duoxa2 expression, whereas heat-killed F. prausnitzii did not. Similarly, both IFNγ and AIEC promoted epithelial production of H2O2 in WT colonoids but not Duox2-KO colonoids, indicating that epithelial release of H2O2 in response to these stimuli is mediated by DUOX2. Response to AIEC additionally required functional TLR4. Although heat-killed F. prausnitzii did not alter H2O2 production, its metabolite butyrate caused a significant blockade in the release of H2O2 in response to both IFNγ and AIEC. Conclusions Our results show that both inflammation and pathobionts induce the expression and activity of DUOX2, which begets more inflammation. We posit that specific depletion of pathobionts or restitution with butyrate-producing bacteria such as F. prausnitzii may be beneficial in IBD.

Non-thermal dielectric-barrier discharge plasma induces reactive oxygen species by epigenetically modifying the expression of NADPH oxidase family genes in keratinocytes

We have previously shown that non-thermal dielectric-barrier discharge (DBD) plasma induces the generation of reactive oxygen species (ROS) in cells; however, the underlying mechanism has not been elucidated. This study aimed to identify the mechanisms through which DBD plasma induces the expression of NADPH oxidase (NOX) family members by epigenetic modification in human keratinocytes (HaCaT). Cell exposure to DBD plasma in 10% oxygen and 90% argon resulted in the generation of ROS, triggering oxidative stress that manifested in various forms, including lipid membrane peroxidation, DNA base modification, and protein carbonylation. DBD plasma upregulated the expression of NOX1, NOX5, and DUOX2 at the mRNA and protein levels; and siRNAs targeting NOX1, NOX5, and DUOX2 attenuated the generation of DBD plasma-induced ROS. DBD plasma upregulated the transcriptional activators TET1, MLL1, and HAT1 and downregulated the transcriptional repressors DNMT1, EZH2, and HDAC1. Additionally, DBD plasma increased the binding of transcriptional activators and decreased the binding of transcriptional repressors to the DUOX2 promoter. Methyl-specific polymerase chain reaction and bisulfite sequencing indicated that DBD plasma decreased methylation at the DUOX2 promoter. These results suggest that DBD plasma induces ROS generation by enhancing the expression of the NOX system through epigenetic DNA and histone modifications.

Abstract 3348: Exogenous DNA upregulates DUOX2 expression in pancreatic cancer via activation of the cGAS-STING signaling pathway

Abstract Dual oxidase 2 (DUOX2) is a transmembrane protein that produces hydrogen peroxide in the extracellular environment, mediates innate immunity at mucosal surfaces, contributes to chronic inflammation-related tissue injury and angiogenesis, and can promote the initiation of epithelial-derived cancers. In pancreatic adenocarcinomas (PDAC), the composition of the tumor microbiome significantly affects patient outcomes. However, it remains unclear how microbes in the tumor microbiome affect PDAC on a cellular level. In recent years, the cGAS-STING signaling pathway has been identified as a crucial mediator of anti-viral defense by recognizing the presence of DNA in the cytosol and subsequently producing Type I interferons (IFNs) as well as a host of other anti-viral genes. Using Western blot and qPCR, we show that the introduction of exogenous DNA into the cytosol significantly upregulates DUOX2 expression in human PDAC cells after activating cGAS-STING signaling. Expression of DUOX2 occurs most strongly 2 days post-transfection of DNA, while cGAS-STING signaling is activated within hours. Treatment of BxPC-3 cells with exogenous cyclic GMP-AMP (cGAMP) for 24 hrs increases DUOX2 expression in a concentration-dependent manner, suggesting the necessity of cGAS activity for DUOX2 induction. Furthermore, Western analysis of phospho-NF-κB p65 in BxPC-3 cells demonstrates activation of NF-κB that mirrors the time-dependent induction of DUOX2. In CFPAC-1 cells, another PDAC line, Jak-Stat signaling is activated along with the components of cGAS-STING. These data suggest that DNA-induced DUOX2 upregulation in PDAC cells may be mediated in a cell context-specific fashion, beyond the canonical components of the cGAS-STING pathway. Upregulation of DUOX2 by exogenous DNA is not observed in colon cancer cell lines. In summary, these data suggest that extracellular DNA could contribute to a DUOX2-induced, H2O2-mediated pro-inflammatory milieu that specifically contributes to the pathogenesis of PDAC, in part through activation of the cGAS-STING pathway. Citation Format: Stephen L. Wang, Yongzhong Wu, Smitha Antony, Agnes Juhasz, Jennifer Meitzler, Goujian Jiang, Iris Dahan, Jiamo Lu, Krishnendu Roy, James Doroshow. Exogenous DNA upregulates DUOX2 expression in pancreatic cancer via activation of the cGAS-STING signaling pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3348.

DUOX2 promotes the progression of colorectal cancer cells by regulating the AKT pathway and interacting with RPL3.

Dual oxidase 2 (DUOX2) is an important regulatory protein in the organic process of thyroid hormone iodine. Mounting evidence suggests that DUOX2 plays a crucial role in the occurrence and development of cancers. However, the function and mechanism of DUOX2 in colorectal cancer (CRC) have not been fully clarified. In the present study, the relationship between the expression of DUOX2 and the clinicopathological features and prognosis of CRC patients was analyzed. Furthermore, the effects of DUOX2 on proliferation and invasion in vitro and in vivo were examined. DUOX2-associated proteins were identified by immunoprecipitation (IP). Next-generation sequencing detection was performed to illustrate the mechanism of DUOX2 in CRC cells. It was found that the expression levels of DUOX2 in metastatic sites were significantly higher than those in primary tumor tissues, and this was demonstrated to be associated with poor prognosis. The knockdown of DUOX2 inhibited the invasion and migration of CRC cells. Furthermore, DUOX2 regulated the stability of ribosomal protein uL3 (RPL3) by affecting the ubiquitination status of RPL3, and the invasion and migration ability of DUOX2 can be reversed by the overexpression of RPL3. The downregulation of DUOX2 can affect the expression level of a large number of genes, and a number of these are enriched in the PI3K–AKT pathway. Some of the changes caused by DUOX2 can be reversed by RPL3. In summary, DUOX2 exhibits a significantly higher expression in CRC tumor samples, and facilitates the invasion and metastasis ability of CRC cells by interacting with RPL3.

Dual oxidase 1 limits the IFNγ-associated antitumor effect of macrophages.

BackgroundMacrophages play pivotal roles in tumor progression and the response to anticancer therapies, including radiotherapy (RT). Dual oxidase (DUOX) 1 is a transmembrane enzyme that plays a critical role in oxidant generation.MethodsSince we found DUOX1 expression in macrophages from human lung samples exposed to ionizing radiation, we aimed to assess the involvement of DUOX1 in macrophage activation and the role of these macrophages in tumor development.ResultsUsing Duox1−/− mice, we demonstrated that the lack of DUOX1 in proinflammatory macrophages improved the antitumor effect of these cells. Furthermore, intratumoral injection of Duox1−/− proinflammatory macrophages significantly enhanced the antitumor effect of RT. Mechanistically, DUOX1 deficiency increased the production of proinflammatory cytokines (IFNγ, CXCL9, CCL3 and TNFα) by activated macrophages in vitro and the expression of major histocompatibility complex class II in the membranes of macrophages. We also demonstrated that DUOX1 was involved in the phagocytotic function of macrophages in vitro and in vivo. The antitumor effect of Duox1−/− macrophages was associated with a significant increase in IFNγ production by both lymphoid and myeloid immune cells.ConclusionsOur data indicate that DUOX1 is a new target for macrophage reprogramming and suggest that DUOX1 inhibition in macrophages combined with RT is a new therapeutic strategy for the management of cancers.

Effect of beta- and alpha-glucans on immune modulating factors expression in enterocyte-like Caco-2 and goblet-like LS 174T cells

Glucans are complex polysaccharides consisting of repeated units of d-glucose linked by glycosidic bonds. The nutritional contribution in α-glucans is mainly given by starch and glycogen while in β-glucans by mushrooms, yeasts and whole grains, such as barley and spelt well represented in the Mediterranean Diet. Numerous and extensive studies performed on glucans highlighted their marked anti-tumor, antioxidant and immunomodulatory activity. It has recently been shown that rather than merely being a passive barrier, the intestinal epithelium is an essential modulator of immunity. Indeed, epithelial absorptive enterocytes and mucin secreting goblet cells can produce specific immune modulating factors, driving innate immunity to pathogens as well as preventing autoimmunity. Despite the clear evidence of the effects of glucans on immune system cells, there are only limited data about their effects on immune activity of mucosal intestinal cells strictly related to intestinal barrier integrity. The aim of the study was to evaluate the effects of α and β glucans, alone or in combination with other substances with antioxidant properties, on reactive oxygen species (ROS) levels, on the expression of ROS-generating enzyme DUOX-2 and of the immune modulating factors Tumor Necrosis Factor (TNF-α), Interleukin 1 β (IL-1β) and cyclooxygenase-2 (COX-2) in two intestinal epithelial cells, the enterocyte-like Caco-2 cells and goblet cell-like LS174T.In our research, the experiments were carried out incubating the cells with glucans for 18 h in culture medium containing 0.2% FBS and measuring ROS levels fluorimetrically as dihydrodichlorofluoresce diacetate (DCF-DA) fluorescence, protein levels of DUOX-2 by Western blotting and mRNA levels of, TNF-α, IL-1β and COX-2 by qRT-PCR. α and β glucans decreased ROS levels in Caco-2 and LS 174T cells. The expression levels of COX-2, TNF-α, and IL-1β were also reduced by α- and β-glucans. Additive effects on the expression of these immune modulating factors were exerted by vitamin C. In Caco-2 cells, the dual oxidase DUOX-2 expression is positively modulated by ROS. Accordingly, in Caco-2 or LS174T cells treated with α and β-glucans alone or in combination with Vitamin C, the decrease of ROS levels was associated with a reduced expression of DUOX-2. The treatment of cells with the NADPH oxidase (NOX) inhibitor apocynin decrease ROS, DUOX-2, COX-2, TNF-α and IL-1β levels indicating that NOX dependent ROS regulate the expression of immune modulating factors of intestinal cells. However, the combination of vitamin C, α and β-glucans with apocynin did not exert an additive effect on COX-2, TNF-α and IL-1β levels when compared with α-, β-glucans and Vitamin C alone. The present study showing a modulatory effect of α and β-glucans on ROS and on the expression of immune modulating factors in intestinal epithelial cells suggests that the assumption of food containing high levels of these substances or dietary supplementation can contribute to normal immunomodulatory function of intestinal barrier.

SFPQ is involved in regulating arsenic-induced oxidative stress by interacting with the miRNA-induced silencing complexes

Arsenic exposure contributed to the development of human diseases. Arsenic exerted multiple organ toxicities mainly by triggering oxidative stress. However, the signaling pathway underlying oxidative stress is unclear. We previously found that the expression of SFPQ, a splicing factor, was positively associated with urinary arsenic concentration in an arsenic-exposed population, suggesting an oxidative stress regulatory role for SFPQ. To test this hypothesis, we established cell models of oxidative stress in human hepatocyte cells (L02) treated with NaAsO2. Reactive oxygen species (ROS) synthesis displayed a time- and dose-dependent increase with NaAsO2 treatment. SFPQ suppression resulted in a 36%–53% decrease in ROS generation, leading to enhanced cellular damage determined by 8-OHdG, comet tail moment, and micronucleus analysis. Particularly, SFPQ deficiency attenuated expression of the oxidase genes DUOX1, DUOX2, NCF2, and NOX2. A fluorescent-based RNA electrophoretic mobility shift assay (FREMSA) and dual-luciferase reporter system revealed that miR-92b-5p targeted DUOX2 mRNA degradation. An RNA immunoprecipitation assay showed an interaction between SFPQ and miR-92b-5p of the miRNA-induced silencing complex (miRISC). Notably, NaAsO2 treatment diminished the interaction between SFPQ and miR92b-5p, accompanied by decreased binding between miR-92b-5p and 3′-UTR of DUOX2. However, SFPQ deficiency suppressed the dissociation of miR-92b-5p from 3′-UTR of DUOX2, indicating that miR-92b-5p regulated the SFPQ-dependent DUOX2 expression. Taken together, we reveal that SFPQ responds to arsenic-induced oxidative stress by interacting with the miRISC. These findings offer new insight into the potential role of SFPQ in regulating cellular stress response.

Particulate Matters Induce Apoptosis in Human Hair Follicular Keratinocytes.

BackgroundParticulate matters (PM) comprise a heterogeneous mixture of particles suspended in air. A recent study found that urban PMs may penetrate into hair follicles via transfollicular and transdermal routes in dorsal skin.ObjectiveTo investigate the effects of PM on ex vivo cultured human scalp hair follicles and hair follicular keratinocytes in vitro.MethodsTUNEL staining was employed to check cells undergoing apoptosis in cultured hair follicles after PM treatment. MTT assay was employed to check cell viability after PM treatment. Quantitative real-time PCR analysis was employed to quantitate the expression of inflammatory genes, matrix metalloproteinases (MMPs), and Duox1. Inflammatory cytokine levels were measured by ELISA after PM treatment. The level of reactive oxygen species (ROS) production was measured using a chemical fluorescent probe by a fluorescence plate reader.ResultsAbundant TUNEL-positive cells were observed in the keratinocyte region of hair including the epidermis, sebaceous gland, outer root sheath (ORS), inner root sheath (IRS), and bulb region. The viability of follicular cells, including the ORS, was found to be decreased upon PM exposure. mRNA expression and protein levels of inflammatory response genes and MMPs were upregulated in a dose-dependent manner by PM treatment. ROS levels were also increased by PM.ConclusionThese data strongly suggest that penetrated PMs from air pollution may cause apoptotic cell death to follicular keratinocytes by increased production of ROS and inflammatory cytokines, which could impair hair growth.

Active vitamin D supplementation alleviates initiation and progression of nonalcoholic fatty liver disease by repressing the p53 pathway

Background/aimsRecent studies have found vitamin D deficiency promotes fat deposition into the hepatocytes, thus contributing to the development of nonalcoholic fatty liver disease (NAFLD), which is a hepatic manifestation of metabolic syndrome. This study aimed to investigate the potential effects of vitamin D on NAFLD with the involvement of the p53 pathway. MethodsInitially, an in vivo high-fat diet (HFD)-induced NAFLD mouse model was established. Then the HFD-induced NAFLD mice were treated with vitamin D. Next, the serum levels of TNF-α, GSH-px and malondialdehyde (MDA) were assessed using ELISA and ROS content was evaluated by flow cytometry, followed by the measurement of expression of Duox1, Duox2, SOD1, SOD2, PRDX1 I, ACC, SREBP1c, MTTP, PPARα, p53, p21 and p16 using RT-qPCR and Western blot analysis. Positive expression of FAS and FASL proteins was measured using immunohistochemistry. TUNEL and Senescence-associated β-galactosidase (SA-β-Gal) staining were subsequently conducted to assess the senescence and apoptosis of hepatocytes. ResultsHFD-induced mice treated with vitamin D presented with significantly increased GSH-px levels, as well as protein expression of SOD1, SOD2, PRDX1, MTTP and PPARα, but decreased MDA and ROS levels, expression of Duox1, Duox2, ACC, SREBP1c, p53, p21 and p16, positive expression of FAS and FASL proteins as well as impaired senescence and apoptosis of hepatocytes. ConclusionActive vitamin D supplementation could potentially impede hepatocyte senescence and apoptosis via suppression of the p53 pathway, thus preventing the progression of NAFLD. Our study provides available evidence on the potential clinical utility of vitamin D supplementation in NAFLD.

Dual oxidase 1 and NADPH oxidase 2 exert favorable effects in cervical cancer patients by activating immune response

BackgroundNicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) not only can promote cancer progression, but also they have recently emerged as mediators of the mucosal immune system. However, the roles and clinical relevance of the collective or individual NADPH oxidase (NOX) family genes in cervical cancer have not been studied.MethodsWe investigated the clinical significance of the NOX family genes using data from 307 patients with cervical cancer obtained from The Cancer Genome Atlas. Bioinformatics and experimental analyses were performed to examine NOX family genes in cervical cancer patients.ResultsDual Oxidase1 (DUOX1) and Dual Oxidase 2 (DUOX2) mRNA levels were upregulated 57.9- and 67.5-fold, respectively, in cervical cancer patients. The protein expression of DUOX1, DUOX2, and NOX2 also identified in cervical squamous cell carcinoma tissues. Especially, DUOX1 and DUOX2 mRNA levels were significantly increased in patients infected with human papillomavirus (HPV) 16. Moreover, high DUOX1 mRNA levels were significantly associated with both favorable overall survival and disease-free survival in cervical cancer patients. High NOX2 mRNA levels was significantly associated with favorable overall survival. Gene set enrichment analyses revealed that high DUOX1 and NOX2 expression was significantly correlated with the enrichment of immune pathways related to interferon (IFN)-alpha, IFN-gamma, and natural killer (NK) cell signaling. Cell-type identification by estimating relative subsets of known RNA transcript analyses indicated that the fraction of innate immune cells, including NK cells, monocytes, dendritic cells, and mast cells, was elevated in patients with high DUOX1 expression.ConclusionsDUOX1 and NOX2 expression are associated with mucosal immunity activated in cervical squamous cell carcinoma and predicts a favorable prognosis in cervical cancer patients.

Identification of Two Missense Mutations in DUOX1 (p.R1307Q) and DUOXA1 (p.R56W) That Can Cause Congenital Hypothyroidism Through Impairing H2O2 Generation.

Context: The DUOX/DUOXA systems play a key role in H2O2 generation in thyroid cells, which is required for iodine organification and thyroid hormone synthesis. DUOX2/DUOXA2 defects can cause congenital hypothyroidism (CH), but it is unknown whether DUOX1/DUOXA1 mutations can also cause CH.Objective: We aimed to identify DUOX1/DUOXA1 mutations and explore their role in the development of CH by investigating their functional impacts on H2O2 generation.Patients and Methods: Forty-three children with CH with goiter were enrolled, in whom all exons and flanking intronic regions of DUOX1/DUOXA1 were directly sequenced. We characterized the functional effects of identified mutations on the expression of DUOX1 and DUOXA1 and H2O2 generation.Results: We identified a heterozygous DUOX1 missense mutation (G > A base substitution at nucleotide 3920 in exon 31) that changed a highly conserved arginine to glutamine at residual 1307 (p.R1307Q) in patient 1. A heterozygous-missense mutation (c.166 C>T; p.R56W) was identified in DUOXA1 in patient 2. Functional studies demonstrated that both p.R1307Q mutant or p.R56W mutant decreased the DUOX1 expression at mRNA and protein levels, with a corresponding impairment in H2O2 generation (P < 0.01). The results also showed that intact DUOXA1 was required for full activity of DUOX1 and H2O2 generation.Conclusions: We have identified two heterozygous missense mutations in DUOX1 and DUOXA1 in two patients that can cause CH through disrupting the coordination of DUOX1 and DUOXA1 in the generation of H2O2. This study for the first time demonstrates that the DUOX1/DUOXA1 system, if genetically defective, can cause CH.

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Abstract 5240: Long-term treatment with IL-4, IL-6, and/or IL-17 increases the expression of the NADPH oxidase DUOX2 in human colon cancer cells in vitro

Abstract Colorectal cancer (CRC) develops through a multistep process that is enhanced by chronic inflammation and includes complex interactions between the colonic epithelium and pro-inflammatory cytokines capable of stimulating cancer initiation and progression due, in part, to enhanced production of reactive oxygen species (ROS). Expression of colonic DUOX2, a H2O2 generator, and NOX1, a superoxide producer, is increased in patients with inflammatory bowel disease and adenomatous polyps. For these experiments, we investigated the effect of prolonged treatment with pro-inflammatory cytokines known to be associated with CRC (IL-4, IL-6, and IL-17A alone and in combination) on NADPH oxidase (NOX) expression in human colon cancer lines, and early passage, two-dimensional cultures of conditionally-reprogrammed patient-derived colon cancer cells (PDCs). HT-29 colon cancer cells were treated with IL-4, IL-6, IL-17A, IL-4+IL-17A, and IL-6+IL-17A at a concentration of 25ng/ml each for 4, 6, 8, 15, and 30 days. We measured the effect of these cytokines on expression of NOX family members NOX1, DUOX2, and DUOXA2 by quantitative RT-PCR, and by Western analysis. The production of ROS by NOX proteins was measured with chemiluminescence (superoxide) and Amplex Red assays (H2O2), respectively. DUOX2 protein, which is absent at baseline in HT-29 cells, was easily detectable after 4 days, and increased as a function of the duration of cytokine exposure and for cytokine combinations. In addition to HT-29 cells, similar results were obtained using Ls513 and DLD-1 colon cancer lines. Six PDC lines were also studied: T 280-R, F725, F1126, CK7962, CK7354 and 027-R1. Modest increases in NOX1 expression were observed for 2 lines (T 280-R, CK7962) following 6 days of treatment with IL-6+IL-17A or IL-4; however, NOX1 was highly expressed in 4 cell lines following exposure to the combination of IL-4+IL-17A. Moreover, DUOX2 mRNA and protein were highly expressed after IL-6+IL-17A treatment for 6 days in the T 280-R and F725 cell lines, and very highly expressed after IL-4+IL-17A treatment in all 6 PDCs. Enhanced NOX1 and/or DUOX2 expression in CRC cells was accompanied by concomitant enhancement of NOX-specific ROS. RNA-Seq analysis of cytokine-modulated NOX expression in HT-29 cells and a NOX1 shRNA inhibited subline of HT-29 cells, suggests that both NF-κB and Stat signaling pathways are involved in the control of DUOX2 expression in both established and early passage human CRC cells. These experiments suggest that multiple pro-inflammatory cytokines known to be present in the CRC microenvironment may contribute to the development of a hyperoxidant milieu through the up-regulation of the two NOX species, NOX1 and DUOX2, expressed in the colon. Citation Format: Agnes N. Juhasz, Mariam Konate Monnard, Guojian Jiang, YongZhong Wu, Abdalla Amr Abdelmaksoud, Jinqiu Chen, Xialong Luo, Noemi Kedei, Smitha Antony, Jennifer L. Meitzler, Jiamo Lu, Iris M. Dahan, Krishnendu Roy, James H. Doroshow. Long-term treatment with IL-4, IL-6, and/or IL-17 increases the expression of the NADPH oxidase DUOX2 in human colon cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5240.

IL-17 receptor and IL-22 receptor signaling in Citrrobacter rodentium infection

Abstract Th17 cells producing cytokines, IL-17A and IL-22 are important key player in host defense. We have reported that intestinal epithelial expression of IL-17 receptors in gut epithelial cells control the development of microbiota. To study the roles of these receptors in response to an attaching and effacing pathogen we challenged Citobacter rodenium (C. rodentium) to intestinal epithelial specific IL-17RA or IL-22RA1 KO mice. Conditional deletion of Il17ra or Il22ra1 resulted in increased bacterial growth in the colon as well as enhanced dissemination to the spleen. It suggested both cytokine receptors are required for control of this infection. In addition this was supported by unbiased RNAseq analysis of the colonic epithelium that showed diminished expression of Pigr, Duox2 and Duoxa2 in the absence of IL-17RA signaling and reduced Bcl3 expression in the absence of IL-22ra1 expression. To elucidate whether production from epithelial cells effect bacteria killing we cultured crypt and stimulated IL-17A. It resulted in increasing Pigr, Duoxa2 and Duox2 were associated with increasing bacteria killing. Pigr binds dimeric IgA and translocation into lumen from lamina propria, while Duox2 and Duoxa2 make complex and produce hydrogen peroxide into lumen from apical epithelial cell membrane. These result suggested mucosal immunity to C. rodentium requires both IL-17RA and IL-22ra1 signaling and that IL-17RA regulates transcytosis of C. rodentium specific IgA as well as luminal hydrogen peroxide concentrations to control bacterial growth. In contrast, Il-22Ra1 regulates epithelial Bcl3 expression, which restrains TNFα and NF-κB signaling in the colonic epithelium.

Identification of novel factors associated with severe respiratory syncytial virus disease in infants

Background Almost all infants are infected with RSV by 2 years. 1–3 % of RSV-infected infants are hospitalised with severe disease. Reasons for susceptibility to severe disease remain obscure. We aimed to identify factors that might explain such susceptibility. Methods We generated well-differentiated primary nasal epithelial cell (WD-PNEC) cultures from infants with histories of severe or mild RSV disease. Following infection with RSV BT2a (clinical isolate), virus growth kinetics, cytopathogenesis, chemokines, and IL-29/IFNλ1 responses and differential gene expression were determined. In an exciting development, 1 differentially expressed gene, ptn, encodes pleiotrophin (PTN), which interacts with nucleolin (NCL), an RSV entry co-factor. The PTN antiviral activity was determined in BEAS-2B cells and WD-PBECs (where B=bronchial). Results Viral growth kinetics, cell tropism, IP-10/CXCL10, TRAIL and RANTES/CCL5 responses did not differ significantly between cohorts. However, apical cell sloughing and IL-29/IFNλ1 were diminished in severe WD-PNECs following infection, while expression of isg15, ifi6, irf9, duox2 and tap1 was also reduced. Interestingly, diminished ptn expression was evident in severe WD-PNECs, irrespective of RSV infection. Furthermore, pre-treatment with PTN blocked RSV infection in BEAS-2B cells and WD-PBECs, while neutralisation of PTN with either antibodies or siRNAs resulted in increased RSV replication. Conclusions Diminished apical cell sloughing and expression/secretion of IL-29/IFNλ1 and specific interferon stimulated genes in WD-PNECs were associated with severe RSV. Importantly, PTN was identified as a novel endogenously expressed RSV antiviral protein in human airway epithelium. Lower expression of PTN in paediatric airway epithelium may explain, in part, increased susceptibility to severe disease.

Early life innate immune responses to RSV in cystic fibrosis airway epithelium

Respiratory syncytial virus (RSV) infection is the major cause of severe lower respiratory tract disease in young infants. Evidence suggests that cystic fibrosis (CF) is associated with significant morbidity following RSV infection. We and others previously demonstrated that airway epithelium is the primary target of RSV infection. However, little is known about the impact of RSV infection on CF airway epithelium. To address this, we established and characterised well-differentiated primary nasal epithelial cell cultures (WD-PNECs) from recently diagnosed CF infants to study RSV cytopathogenesis in CF airway epithelium. CF WD-PNECs were successfully generated from 11 infants and characterised by light and fluorescent microscopy. CF WD-PNECs secreted thick dry apical mucous, consistent with in vivo observations. Extensive cilia coverage was also evident, although cilia beat frequencies appeared lower than those evident in WD-PNECs from healthy neonates. Quantification of goblet and ciliated cells in the CF WD-PNEC cultures were similar to those of healthy cultures. Viral growth kinetics were similar for CF WD-PNECs and healthy WD- PNECs, with peak virus titres evident at 72–96 hpi. CXCL8/IL-8 and CXCL10/IP-10 secretions were upregulated following RSV infection of CF WD-PNECs, while IL-6 secretions did not change. Interestingly, our data suggested that duoxa2 and duox2 expression post-infection were reduced in WD-PNECs from CF compared to healthy infants. Our data suggest that this model provides an exciting opportunity to elucidate the cytopathogenic, inflammatory and molecular consequences of RSV infection of airway epithelium derived from very young CF infants.

P122 EPITHELIAL TLR4 PROMOTES OXIDATIVE STRESS VIA NADPH OXIDASE ACTIVATION AND INDUCES REDOX-SENSITIVE PATHWAYS DURING TUMORIGENESIS

Dysbiosis and accumulation of reactive oxygen species (ROS) are hallmarks of ulcerative colitis (UC) and have been associated with tumor progression. We have previously shown that UC patient biopsies and colorectal adenomas overexpress toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs). A potential mechanism of TLR4-mediated tumorigenesis is by providing cancer stem cells with redox signals that they efficiently use to outgrow their non-transformed counterparts. We test the hypothesis that epithelial signaling via TLR4 induces the production of ROS through the NADPH oxidases Nox1 and Duox2. We also explore the main redox-sensitive cascades that participate in TLR4-driven tumorigenesis. Colonoids from wild-type (WT), TLR4-KO, and Nox1-KO mice were stimulated with LPS and tested for H2O2 production in the presence of the NADPH oxidase inhibitor, diphenyleneiodonium. IECs isolated from villin-TLR4 mice (which have constitutive activation of epithelial TLR4) and their WT littermates were analyzed for H2O2 production (Amplex Red) and expression of the NADPH oxidases Nox1 and Duox2 (qPCR). Villin-TLR4 and their littermates underwent a model of CAC by azoxymethane injection followed by 2 cycles of 3% dextran sulfate sodium (1.5% in villin-TLR4 mice). Cancer stem cells (aldehyde dehydrogenase positive) were sorted for RNA sequencing. LPS incubation enhanced the expression of Nox1 and Duox2 in WT but not TLR4-KO colonoids, demonstrating that these oxidative enzymes are regulated by TLR4. Consistently, LPS stimulation elicited H2O2 release by WT organoids in a NADPH oxidase-dependent manner. Nox1-KO colonoids showed attenuated production of H2O2 upon LPS stimulation, confirming the participation of this NADPH oxidase in TLR4-mediated production of ROS. Constitutive activation of TLR4 induced the production of H2O2 in IECs, which was accompanied by a marked upregulation of Nox1, Duox2, and DuoxA2. RNAseq done on cancer stem cells shows that cancer stem cells of villin-TLR4 tumors have increased expression of NADPH oxidases, as well as enrichment of redox-sensitive pathways, including PI3K, myc, Rac1, and NF-κB. All of these pathways have been implicated in the promotion of proliferative and antiapoptotic signals. TLR4 controls the expression and function of oxidative enzymes in IECs, enhancing redox signals in cancer stem cells upon bacterial challenge. In the context of inflammation-associated dysbiosis, we speculate that overexpression of TLR4 provides additional activation of proliferative and antiapoptotic pathways, accelerating tumor development and growth. The results of our studies highlight how we might target critical signaling pathways in inflammatory-driven cancer stem cells.

Duox1 Regulates Primary B Cell Function under the Influence of IL-4 through BCR-Mediated Generation of Hydrogen Peroxide.

Engagement of the BCR with Ags triggers signaling pathways for commitment of B lymphocyte responses that can be regulated, in part, by reactive oxygen species. To investigate the functional relevance of reactive oxygen species produced in primary B cells, we focused on the role of the hydrogen peroxide generator Duox1 in stimulated splenic B cells under the influence of the TH2 cytokine IL-4. We found that H2O2 production in wild type (WT) and Nox2-deficient CD19+ B cells was boosted concomitantly with enhanced expression of Duox1 following costimulation with BCR agonists together with IL-4, whereas stimulated Duox1-/- cells showed attenuated H2O2 release. We examined whether Duox1-derived H2O2 contributes to proliferative activity and Ig isotype production in CD19+ cells upon BCR stimulation. Duox1-/- CD19+ B cells showed normal responses of Ig production but a higher rate of proliferation than WT or Nox2-deficient cells. Furthermore, we demonstrated that the H2O2 scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of WT CD19+ B cells in vitro. Results from immunized mice reflected the in vitro observations: T cell-independent Ag induced increased B cell expansion in germinal centers from Duox1-/- mice relative to WT and Nox2-/- mice, whereas immunization with T cell-dependent or -independent Ag elicited normal Ig isotype secretion in the Duox1 mutant mice. These observations, obtained both by in vitro and in vivo approaches, strongly suggest that Duox1-derived hydrogen peroxide negatively regulates proliferative activity but not Ig isotype production in primary splenic CD19+ B cells.