Duox2 Expression Research Articles

Eosinophil-Airway Epithelial Cells crosstalk reveals the eosinophils-mediated DUOX1 up-regulation in a murine allergic inflammation setting.

Blood and airway eosinophilia represent markers for the endotype-driven treatment of allergic asthma. Little is known on mechanisms that link eosinophils and airway epithelial cells before and after these cells are infiltrated by eosinophils during allergic response. Given that innate immune mechanisms, mainly mediated by epithelial-derived cytokines (IL-33, IL-25, TSLP), induce eosinophil-maturing/attractive substances, we thought to evaluate the crosstalk between eosinophils and airway epithelial cells in the context of IL-33-mediated allergic inflammation. DUOX1 was previously described in clinically relevant aspects of allergic inflammation in a HDM -induced allergic asthma mice model, and in patients with chronic sinusitis or allergic asthma. Thus, we evaluated the involvement of HDM and eosinophils in the regulation of DUOX1 in airway epithelial cells. To recapitulate the lung environment present at the allergen challenge time in acute asthma, we set up an in vitro model based on murine bone marrow-derived eosinophils differentiated with IL-5 and then activated with IL-33 (EOs33) and TC1 or C57 airway epithelial cells. We found that treatment of epithelial cells with HDM induced an eosinophil-attractive environment and increased DUOX1 expression. Importantly, we found that the co-culture of airway epithelial cells with EOs33 or with conditioned medium from EOs33 enhanced the expression of DUOX1, which was further increased by combined stimulation (HDM plus EOs33). Our results suggest that lung recruited EOs once activated by IL-33 could be involved in a crosstalk loop with airway epithelial cells by DUOX1-mediated IL-33 secretion.

Identification and multimodal characterization of a specialized epithelial cell type associated with Crohn’s disease

Crohn’s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as ‘LND’) with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn’s ileitis and colitis.

Beneficial effects of oxymatrine from Sophora flavescens on alleviating Ulcerative colitis by improving inflammation and ferroptosis

Ethnopharmacological relevanceSophora flavescens is often used in traditional Chinese medicine for skin issues, diarrhea, and vaginal itching (Plant names have been checked with http://www.the/plant/list.org on Feb 22nd, 2024). Oxymatrine (OY), a major bioactive compound from Sophora flavescens, is commonly used in China to treat ulcerative colitis, but its mechanisms are still unclear. Aim of the studyRecent studies have found that the crosstalk between ferroptosis and inflammation is an important mechanism in the pathogenesis of UC. The aim of this study was to investigate the potential underlying mechanisms of OY treatment on DSS-induced ulcerative colitis, specifically focusing on the processes of ferroptosis and inflammation. Materials and methodsBioinformatics methods were used to identify key targets of OY for ferroptosis and inflammation in ulcerative colitis, based on GEO data and FerrDb database. Then, 4% DSS solution was used to induce UC model. OY’s impact on morphological changes was assessed using colon views, Hematoxylin and eosin (HE) staining, and transmission electron microscopy (TEM). Ferroptosis phenotype index and inflammations factors were detected by ELISA or chem-bio detection kits.The screen out hub related genes about ferroptosis and inflammation were verified by RT-PCR, immunohistochemistry (IHC), and western blotting (WB) respectively. ResultsBioinformatics results show that there are 16 key target genes involved in ferroptosis and inflammation interaction of OY treatment for UC, such as IL6, NOS2, IDO1, SOCS1, and DUOX. The results of animal experiments show that OY could depress inflammatory factors (IL-1β, IL-6, TNF-α, HMGB1, and NLRP3) and reduce iron deposition (Fe2+, GSH). Additionally, OY suppressed the hub genes or proteins expression involved in ferroptosis and inflammation, including IL-1β, IL-6, NOS2, HIF1A, IDO1, TIMP1, and DUOX2. ConclusionThis present study combines bioinformatics, molecular biology, and animal experimental research evidently demonstrated that OY attenuates UC by improving ferroptosis and inflammation, mainly target to the expression of IL-1β, IL-6, NOS2, HIF1A, IDO1, TIMP1, and DUOX2.

Contactin 6, A Novel Causative Gene for Congenital Hypothyroidism, Mediates Thyroid Hormone Biosynthesis Through Notch Signaling.

Background: Congenital hypothyroidism (CH) is the most common neonatal metabolic disorder. In patients with CH in China, thyroid dyshormonogenesis is more common than thyroid dysgenesis; however, the genetic causes of CH due to thyroid dyshormonogenesis remain largely unknown. Therefore, we aimed at identifying novel candidate causative genes for CH. Methods: To identify novel CH candidate genes, a total of 599 patients with CH were enrolled and next-generation sequencing was performed. The functions of the identified variants were confirmed using HEK293T and FTC-133 cell lines in vitro and in a mouse model organism in vivo. Results: Three pathogenic contactin 6 (CNTN6) variants were identified in two patients with CH. Pedigree analysis showed that CH caused by CNTN6 variants was inherited in an autosomal recessive pattern. The CNTN6 gene was highly expressed in the thyroid in humans and mice. Cntn6 knockout mice presented with thyroid dyshormonogenesis and CH due to the decreased expression of crucial genes for thyroid hormone biosynthesis (Slc5a5, Tpo, and Duox2). All three CNTN6 variants resulted in the blocking of the release of the Notch intracellular domain, which could not translocate into the nucleus, impaired NOTCH1 transcriptional activity, and decreased expression of SLC5A5, TPO, and DUOX2. Further, we found that DTX1 was required for CNTN6 to promote thyroid hormone biosynthesis through Notch signaling. Conclusions: This study demonstrated that CNTN6 is a novel causative gene for CH through the mediation of thyroid hormone biosynthesis via Notch signaling, which provides new insights into the genetic background and mechanisms involved in CH and thyroid dyshormonogenesis.

Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

Screening and experimental validation of diagnostic gene in ulcerative colitis with anti-TNF-α therapy.

In clinical practice, the diagnosis of ulcerative colitis (UC) mainly relies on a comprehensive analysis of a series of signs and symptoms of patients. The current biomarkers for diagnosis of UC and prognostic prediction of anti-TNF-α therapy are inaccurate. The present study aimed to perform an integrative analysis of gene expression profiles in patients with UC. A total of seven datasets from the GEO database that met our strict inclusion criteria were included. After identifying differentially expressed genes (DEGs) between UC patients and healthy individuals, the diagnostic and prognostic utility of the DEGs were then analyzed via least absolute shrinkage and selection operator and support-vector machine recursive feature elimination. Subgroup analyses of the treated and untreated groups, as well as the treatment-response group and non-response group, were also performed. Furthermore, the relationship between the expressions of UC-related genes and infiltration of immune cells in the course of treatment was also investigated. Immunohistochemical (IHC) assay was used to verify the gene expression in inflamed UC tissues. When considering all the applied methods, DUOX2, PI3, S100P, MMP7, and S100A8 had priority to be defined as the characteristic genes among DEGs. The area under curve (AUC) of the five genes, which were all consistently over-expressed, based on an external validation dataset, were all above 0.94 for UC diagnosis. Four of the five genes (DUOX2, PI3, MMP7, and S100A8) were down-regulated between treatment-responsive and nonresponsive patients. A significant difference was also observed concerning the infiltration of immune cells, including macrophage and neutrophil, between the two groups (treatment responsive and nonresponsive). The changes in the expression of DUOX2 and MMP7 based on the IHC assay were highly consistent with the results obtained in the current study. This confirmed the mild to moderate diagnostic and predictive value of DUOX2 and MMP7 in patients with UC. The conducted analyses showed that the expression profile of the five identified biomarkers accurately detects UC, whereas four of the five genes evidently predicted the response to anti-TNF-α therapy.

Dual Oxidase 1, 2 Gene Expression in Women With Polycystic Ovary Syndrome (PCOS).

Dual oxidases (DUOX1, DUOX2) belong to the NADPH oxidase (NOX) family, which produce H2O2 necessary for thyroid hormone biosynthesis. This study aims to evaluate gene expression for DUOX1, DUOX2 in PCOS patients and its relation with thyroid hormone and magnesium levels. Totally 88 cases were studied including 24 people with PCOS and hypothyroidism, 44 people with PCOS and normal thyroid function, and 20 hypothyroid patients without PCOS. In comparison 40 healthy controls in the age group of 16-35 years matched for age group and BMI were evaluated. Using Vegaro syringe 5 cc of blood was sampled from all 128 people and after RNA extraction and cDNA synthesis using Real-Time PCR technique, the expression level of DUOX1 and DUOX2 genes was investigated. The results of hormonal tests showed that there is a significant difference between the level of T4, T3, and TSH hormones in hypothyroid patients with or without PCOS in comparison to the control group. Regarding the level of Mg, the results showed that there is a significant difference between the levels of Mg in PCOS group with or without hypothyroidism in comparison to the control group. Gene expression results showed that the relative changes of DUOX1 gene expression in different groups compared to the control group were significantly reduced P<0.05. In the polycystic group with hypothyroidism, the gene expression level showed a decrease compared to the normo-thyroid polycystic group and the hypothyroid non-PCO group, which was statistically significant P<0.05. According to the results of the present study and the previous studies that have been published in the field of Duox1, it can be assumed that the reduction of Duox1 expression can interfere with the oxidative stress system. Further studies with other molecular techniques may help to understand the exact action mechanism of these genes.

Salvianolic acid A attenuates arsenic-induced ferroptosis and kidney injury via HIF-2α/DUOX1/GPX4 and iron homeostasis

Arsenic (As) is a prevalent pollutant in the environment and causes a high frequency of kidney disease in areas of high arsenic contamination, but its pathogenic mechanisms have yet to be completely clarified. Ferroptosis is a new form of cell death mainly dependent on lipid peroxidation and iron accumulation. Several reports have suggested that ferroptosis is operative in a spectrum of diseases caused by arsenic exposure, including in the lungs, pancreas, and testis. However, the mechanism and exact role of ferroptosis in arsenic-induced kidney injury is not known. Firstly, by constructing in vivo and in vitro arsenic exposure models, we confirmed the occurrence of ferroptosis based on the identification of the ability of NaASO2 to cause kidney injury. In addition, we found that arsenic exposure could upregulate DUOX1 expression in kidney and HK-2 cells, and after knocking down DUOX1 using siRNA was able to significantly upregulate GPX4 expression and attenuate ferroptosis. Subsequently using bioinformatics, we identified and proved the involvement of HIF-2α in the course of ferroptosis, and further confirmed by dual luciferase assay that HIF-2α promotes DUOX1 transcription to increase its expression. Finally, intervention with the natural ingredient SAA significantly attenuated arsenic-induced ferroptosis and kidney injury. These results showed that arsenic could cause ferroptosis and kidney injury by affecting HIF-2α/DUOX1/GPX4 and iron homeostasis and that SAA was an effective intervention component. This study not only discovered the molecular mechanism of sodium arsenite-induced kidney injury but also explored an active ingredient with intervention potential, providing a scientific basis for the prevention and treatment of sodium arsenite-induced kidney injury.

Abstract 1219: Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines

Abstract Some types of thyroid cancers are known to arise from thyroid adenomas, as a malignant transition from benign adenoma, as proposed in the multi-step carcinogenesis model. Thyroid cancer studies using human thyroid cancer cell lines have been intensely performed by many researchers. However, mouse thyroid adenoma cell lines have not been well established and characterized. In order to obtain further insights into thyroid carcinogenesis process, we established five cell lines from thyroid adenomas developed in mice treated with goitrogen amitrole. Among them, two cell lines, CAT458/458s (CAT: cells from amitrole treated thyroids; CAT458s: a sub-line from CAT458) and CAT459 were found to be positive for E-cadherin and Claudin-1, representative epithelial markers, and negative for Vimentin, a mesenchymal marker, suggesting that they originated from thyroid epithelial cells. We found higher expression of DUOX1, a member of NADPH oxidase involved in thyroid hormone synthesis, in both CAT458s and 459 cells than normal mouse thyroids, presumably due to an unknown mechanism caused by amitrole treatment. NKX2-1, a master regulator of thyroid development, is not expressed in CAT458s cells while NKX2-1 is abundantly expressed in CAT459 cells. Consistent with this finding, NKX2-1-positive CAT459 cells showed higher mRNA expression of thyroid differentiation markers including Tg, Duox1, and Duox2. NKX2-1 overexpression in NKX2-1-negative CAT458s cells using a lentiviral infection system induced mRNA expressions of these thyroid differentiation marker genes, suggesting that partial thyroid differentiation of the adenoma cells is induced by NKX2-1. TSH and IGF-I are known to work synergistically for thyroid growth. However, Tshr mRNA expression is not detected in either CAT458s or 459 cells, suggesting that TSH signal is not responsible for their growth. On the other hand, IGF-IR protein is expressed and IGF-I induces AKT and S6 phosphorylation in both cells. We further tried to assess somatic gene mutations involved in adenoma development by whole-genome sequencing (WGS). WGS analysis revealed that somatic missense mutations are not found in genes known to be involved in thyroid carcinogenesis and cancer progression, such as Braf, Trp53, Ret, Tert, Hras, Kras, and Nras. This finding suggests that prominent oncogenic mutations are not required for thyroid adenoma development that may later progress to carcinoma. Currently we are validating several missense mutations, including mutations in tumor suppressor genes Atm and Tgfb1, found in these cell lines. In conclusion, we established mouse thyroid adenoma cell lines with different differentiation and molecular profiles. CAT458s and 459 cells would provide a tool to further clarify the process of thyroid adenoma development and thyroid differentiation. Citation Format: Yo-Taro Shirai, Nobuo Hoshi, Huaitian Liu, Maxwell P. Lee, Shioko Kimura. Establishment and characterization of amitrole-induced mouse thyroid adenoma-derived cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1219.

PcEiger links the Imd/Relish pathway to ROSproduction in the intestine of the red swampcrayfish.

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

Sulfasalazine and Chromotrope 2B reduce oxidative stress in murine bone marrow-derived mesenchymal stem cells

With advancing age of stem cells, dysregulation of various processes at the cellular level occurs, thereby decreasing their regeneration potential. One of the changes that occurs during the aging process is the accumulation of reactive oxygen species (ROS), which accelerates the processes of cellular senescence and cell death. The aim of this study is to evaluate two antioxidant compounds; Chromotrope 2B and Sulfasalazine, for their antioxidant effects on young and old rat bone marrow mesenchymal stem cells (MSCs). Oxidative stress was induced in MSCs by 5 µM dexamethasone for 96h and the cells were treated with Chromotrope 2B or Sulfasalazine, 50 µM each. The effects of antioxidant treatment following oxidative stress induction was evaluated by transcriptional profiling of genes involved in the oxidative stress and telomere maintenance. Expression levels of Cat, Gpx7, Sod1, Dhcr24, Idh1, and Txnrd2 were found to be increased in young MSCs (yMSCs) as a result of oxidative stress, while Duox2, Parp1, and Tert1 expression were found to be decreased as compared to the control. In old MSCs (oMSCs), the expressions of Dhcr24, Txnrd2, and Parp1 increased, while that of Duox2, Gpx7, Idh1, and Sod1 decreased following oxidative stress. In both MSC groups, Chromotrope 2B prompted decrease in the ROS generation before and after the induction of oxidative stress. In oMSCs, ROS content was significantly reduced in the Sulfasalazine treated group. Our findings suggest that both Chromotrope 2B and Sulfasalazine possess the potential to reduce the ROS content in both age groups, though the latter was found to be more potent. These compounds can be used to precondition MSCs to enhance their regenerative potential for future cell-based therapeutics.

Diet-induced obesity worsens allergen-induced type 2/type 17 inflammation in airways by enhancing DUOX1 activation.

More than 50% of people with asthma in the United States are obese, and obesity often worsens symptoms of allergic asthma and impairs response to treatment. Based on previously established roles of the epithelial NADPH oxidase DUOX1 in allergic airway inflammation, we addressed the potential involvement of DUOX1 in altered allergic inflammation in the context of obesity. Intranasal house dust mite (HDM) allergen challenge of subjects with allergic asthma induced rapid secretion of IL-33, then IL-13, into the nasal lumen, responses that were significantly enhanced in obese asthmatic subjects (BMI >30). Induction of diet-induced obesity (DIO) in mice by high-fat diet (HFD) feeding similarly enhanced acute airway responses to intranasal HDM challenge, particularly with respect to secretion of IL-33 and type 2/type 3 cytokines, and this was associated with enhanced epithelial DUOX1 expression and was avoided in DUOX1-deficient mice. DIO also enhanced DUOX1-dependent features of chronic HDM-induced allergic inflammation. Although DUOX1 did not affect overall weight gain by HFD feeding, it contributed to glucose intolerance, suggesting a role in glucose metabolism. However, glucose intolerance induced by short-term HFD feeding, in the absence of adiposity, was not sufficient to alter HDM-induced acute airway responses. DIO was associated with enhanced presence of the adipokine leptin in the airways, and leptin enhanced DUOX1-dependent IL-13 and mucin production in airway epithelial cells. In conclusion, augmented inflammatory airway responses to HDM in obesity are associated with increases in airway epithelial DUOX1, and by increased airway epithelial leptin signaling.

Retracted: High Expression of PDE8B and DUOX2 Associated with Ability of Metastasis in Thyroid Carcinoma.

[This retracts the article DOI: 10.1155/2021/2362195.].

Плазменная кислота моделирует окислительный и нитрозативный стресс в ткани мочевого пузыря лабораторных животных in vitro

To analyze some effects of plasma acid in vitro on the bladder tissue obtained from laboratory animals and to evaluate the possibility of its application for in vitro modeling of IC/BPS.The tissue samples of the bladder wall were obtained from female Wistar rats aged 3 months (n=16, weighing 180-200 g). The tissues were processed for 1 hour in the plasma acid prepared by spark discharge of water for injection in air. The immunohistochemical study of obtained samples was performed.The changes in the expression profile of bladder epithelial cells under the action of plasma acid in vitro were found indicating the development of oxidative, nitrosative and dicarbonyl stress, impaired expression of NADPH oxidase DUOX2 and VEGF, and a decrease in cell proliferative activity, which, in general, corresponds to the main mechanisms of urothelial alterations specific for the IC/BPS.The revealed effects of plasma acid on bladder epithelial cells confirm the possibility of using it as an inducer of urothelial cell damage typical for IC/BPS in the in vitro models.

Increased NOX1 and DUOX2 expression in the colonic mucosa of patients with chronic functional constipation.

To determine whether oxidative stress and inflammation are associated with constipation by examining the expression of the main producers of reactive oxygen species, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, and pro-inflammatory cytokines in the colon of patients with chronic functional constipation.The colonic biopsies were collected from 32 patients with chronic functional constipation and 30 healthy subjects who underwent colonoscopy. Colonic mucosal histology was observed. Interleukin (IL)-1β, IL-6, IL-8 messenger RNA (mRNA), and 4 members of NADPH oxidase (NOX1, NOX2, DUOX2, and NOX4) protein and mRNA were assessed by immunohistochemistry, western blotting, and reverse transcription polymerase chain reaction.The tissues from both patients and healthy subjects showed normal histological structure without increase of inflammatory cells. NOX1 protein and mRNA levels were significantly increased compared to controls (P < .05). DUOX2 protein, but not mRNA, was increased by 2-fold compared to controls (P < .05). The levels of NOX2 and NOX4 protein and mRNA demonstrated no significant difference between patients and control subjects. The levels of IL-1β and IL-6 mRNA were significantly higher in constipation patients (P < .05), while IL-8 mRNA level was no different between the 2 groups.NADPH oxidase and pro-inflammatory cytokine might be involved in the pathogeneses of chronic functional constipation.

Telocytes reduce oxidative stress by downregulating DUOX2 expression in inflamed lungs of mice.

Telocytes (TCs), a novel type of interstitial cells, have been found to participate in tissue protection and repair. In this study, we investigated the antioxidative effects of TCs in inflamed lungs of mice. Acute respiratory distress syndrome (ARDS) mice were used as models of inflamed lungs of mice. Gene sequencing was used to screen the differentially expressed miRNAs in TCs after lipopolysaccharide (LPS) stimulation. AntagomiR-146a-5p-pretreated TCs were first injected into mice, and antioxidant activity of TCs was estimated. TCs, RAW264.7 cells, and MLE-12 cells were collected for the detection of expressions of NOX1-4, DUOX1-2, SOD1-3, GPX1-2, CAT, Nrf2, miR-146a-5p, and miR-21a-3p after LPS stimulation. Silencing miRNAs were delivered to examine the involved signaling pathways. Oxidative stress was examined by measuring malondialdehyde (MDA) levels. We found that microRNA-146a-5p and microRNA-21a-3p were upregulated in TCs after LPS stimulation. ARDS mice that were preinfused with TCs had lower lung tissue injury scores, lung wet-dry ratios, white blood cell counts in alveolar lavage fluid and lower MDA concentrations in lung tissue. However, in antagomiR-146a-5p-pretreated ARDS mice, the infusion of TCs caused no corresponding changes. After LPS stimulation, DUOX2 and MDA concentrations were downregulated in TCs, while DUOX2 was restored by antagomiR-146a-5p in TCs. Dual-luciferase reporter assay confirmed that CREB1 was downregulated by miR-146a-5p, while DUOX2 was downregulated by CREB1, which was confirmed by treating TCs with a specific CREB1 inhibitor. This study demonstrates that LPS stimulation upregulates miR-146a-5p in TCs, which downregulates the CREB1/DUOX2 pathway, resulting in a decrease in oxidative stress in cultured TCs. TCs reduce LPS-induced oxidative stress by decreasing DUOX2 in inflamed lungs of mice.

EPITHELIAL DUOX2 ACTIVATION INCREASES GUT PERMEABILITY AND BACTERIAL TRANSLOCATION THAT IS RESCUED WITH BUTYRATE SUPPLEMENTATION

Abstract BACKGROUND Inflammatory bowel diseases (IBD) are chronic pathologies characterized by dysbiosis, defects in epithelial barrier function, and increased redox stress. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, which produce metabolites important in maintaining gut homeostasis. The epithelial NADPH oxidase dual oxidase 2 (DUOX2) catalyzes the production of hydrogen peroxide (H2O2) and is the only gene consistently altered in IBD patients in association with dysbiosis and before the onset of disease. However, the functional consequence of DUOX2 activation in IBD is not understood. Using models of dysbiosis leading to DUOX2 activation, we aimed to dissect the contribution of DUOX2 activity to the defects in epithelial barrier function observed in IBD. METHODS villin-TLR4 (vTLR4) mice, which express a constitutively active form of TLR4 in colonic epithelial cells (CECs) and have increased activation of DUOX2, vTLR4-DUOX2-KO mice, and their wild-type (WT) littermates were euthanized. Livers were collected for permeability assessment by bacterial translocation. Colonic tissue was collected for RNA-sequencing and isolation of CECs for gene expression determinations by qPCR. Separately, vTLR4 mice and littermates were treated with butyrate in drinking water. Tissue was harvested for the same determinations and for measurement of epithelial H2O2 production via Amplex Red. Colonoids from WT mice were treated with butyrate and stimulated with IFNγ or heat-killed adherent invasive Escherichia coli. H2O2 production and gene expression of Duox2 were determined. RESULTS RNA-sequencing revealed that abrogation of DUOX2 activity downregulated cytokine-mediated signaling and cytokine production pathways, including tumor necrosis factors and key chemotactic proteins such as CXCL1 and CXCL2. Similarly, expression of Tnfa, Cxcl1, and Cxcl2 was significantly downregulated to WT levels in vTLR4-DUOX2-KO mice. vTLR4 mice had increased bacterial translocation, which was rescued by deletion of DUOX2. Butyrate treatment inhibited epithelial H2O2 response to stimuli in vitro, which was accompanied by a reduction in Duox2 transcripts. In vivo, butyrate treatment of vTLR4 mice significantly reduced epithelial production of H2O2, bacterial translocation to the liver, and led to a downregulation of Tnfa, Cxcl1, and Cxcl2 transcripts. CONCLUSIONS Chronic DUOX2 activity leads to defects in epithelial barrier function resulting in increased bacterial translocation. This leads to an increase in proinflammatory cytokines and chemokines. The microbial metabolite butyrate blocked DUOX2 activity, rescuing intestinal permeability and reducing bacterial translocation and inflammatory cytokine gene expression. We posit that therapies aimed at controlling DUOX2 activity through microbiome or metabolome-targeted approaches may be beneficial in IBD.

OP31 Dietary and Multi-Omic characterization of new onset treatment naive Crohn Disease identifies factors that may contribute to disease pathogenesis

Abstract Background Crohn Disease (CD) prevalence is rising worldwide. As altered genetics are improbable, this phenomenon likely relates to environmental-dietary changes linked with gut microbiome. We aimed to define host and microbial factors at CD diagnosis. Methods Multiomics analyses of SOURCE cohort including clinical, biomarkers (CRP, fecal calprotectin), food frequency questionnaire (FFQ), serum metabolomics, mucosal terminal ileum (TI) transcriptomics, and fecal and mucosal biopsy samples for 16S microbial amplicon sequencing. Results 25 newly-diagnosed CD and 33 controls (median age 28 years, 50% males). Gender, age, and BMI did not differ between groups, but CRP (p=0.001) and calprotectin (p=E-10) were significantly higher in CD. FFQ results showed that compared to controls, pre-diagnosis CD patients consumed significantly more added sugar (g/day), starch (g/day) nitrite (mg/day), and significantly less vitamin K, D, vegetables, and olive oil (Fig 1). Microbial analyses highlighted significant differences (FDR&amp;lt;0.1) in amplicon sequence variants (ASVs) abundance between stool and biopsies samples (73 ASVs) and between CD and Controls samples (82 ASVs). Biopsy vs. stool samples were enriched for Veillonella, Fusobacterium, Neisseria, and Ruminococcus gnavus. CD showed higher abundance of Enterobacteriaceae and Ruminococcus gnavus with reduction of several Ruminococcaceae and Lachnospiraceae taxa (Fig. 1). Ileal transcriptomics differential expression (FC&amp;gt;1.5, FDR&amp;lt;0.05) replicated previous results with significant induction in CD of DUOX2, CXCL9, and DEFB4A and pathways linked to innate and adaptive immunity, and to extracellular matrix. CD down regulated genes included GUCA2B, SLC10A2, and GSTA1, and pathways linked with epithelial transporters. Serum metabolomics highlighted significant variations (FDR&amp;lt;0.25) in Linoleic acid, aKG, Tryptophan, nicotinamide, Docosahexaenoic acid, oxalate, and GABA between CD and controls. We next tested for significant association (FDR&amp;lt;0.25) between diet and multi-OMICs (Fig. 2). Associations between gut microbiome and TI transcriptomics, and serum metabolomics showed that Erysipelotrichacea taxa positivity correlated with serum oxalate, and TI expression of CEACAM6 and DUOX2. Associations between diet and TI transcriptomics and serum metabolomics indicated that B12, tryptophan and riboflavin consumptions were negatively associated with the bile acid transporter SLC10A2 in the TI, and vegetables consumption was positively associated with oxalate degrading Oxalobacter. Conclusion Conclusions: FFQ identifies difference in diet at the onset of CD that may contribute to pathogenesis. Integration between dietary and OMICs layers disclosed novel correlations warranting further exploration.

TLR4-Dependent DUOX2 Activation Triggered Oxidative Stress and Promoted HMGB1 Release in Dry Eye.

Dry eye disease (DED) is one of the most common ocular surface diseases worldwide. DED has been characterized by excessive accumulation of reactive oxygen species (ROS), following significant corneal epithelial cell death and ocular surface inflammation. However, the key regulatory factor remains unclear. In this study, we tended to explore whether DUOX2 contributed to DED development and the underlying mechanism. Human corneal epithelial (HCE) cells were treated with hyperosmolarity, C57BL/6 mice were injected of subcutaneous scopolamine to imitate DED. Expression of mRNA was investigated by RNA sequencing (RNA-seq) and quantitative real-time PCR (qPCR). Protein changes and distribution of DUOX2, high mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), and 4-hydroxynonenal (4-HNE) were evaluated by western blot assays and immunofluorescence. Cell death was assessed by Cell Counting Kit-8 (CCK8), lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Cellular ROS levels and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. RNA-seq and western blot assay indicated a significant increase of DUOX2 dependent of TLR4 activation in DED both in vitro and in vivo. Immunofluorescence revealed significant translocation of HMGB1 within corneal epithelial cells under hyperosmolar stress. Interestingly, after ablated DUOX2 expression by siRNA, we found a remarkable decrease of ROS level and recovered MMP in HCE cells. Moreover, knockdown of DUOX2 greatly inhibited HMGB1 release, protected cell viability and abolished inflammatory activation. Taken together, our data here suggest that upregulation of DUOX2 plays a crucial role in ROS production, thereafter, induce HMGB1 release and cell death, which triggers ocular surface inflammation in DED.

Fibrin(ogen) Promotes Immune Cell Infiltration, Dysbiosis and ROS Production in Experimental Colitis

Elevated stool fibrinogen has recently been shown to predict disease course in ulcerative colitis (UC), suggesting that fibrin(ogen) in the colitis microenvironment promotes UC pathogenesis. This conclusion is consistent with previous studies from our laboratory showing that fibrin(ogen)/leukocyte interactions mediated by the integrin receptor α Mβ 2 promote experimental colitis and colitis-associated cancer, but the mechanisms are not well defined. To delineate the mechanism coupling fibrinogen to colitis pathogenesis, we induced colitis in Fib WT and mice carrying a mutant fibrinogen lacking the α Mβ 2 binding motif (Fibg 390-396A) with Dextran Sulfate Sodium (DSS). We performed flow cytometric, protein analyses of colons and fecal microbiome and metabolomics analyses after DSS exposure. Five days after DSS challenge, a timepoint prior to significant epithelial damage, we observed significantly diminished infiltration of natural killer cells, T cells, dendritic cells, macrophages and neutrophils in colons harvested from DSS-challenged Fibg 390-396A mice relative to Fib WT mice. We also observed significantly diminished proinflammatory cytokine production by NK cells, macrophages and dendritic cells isolated from DSS-challenged Fibg 390-396A colons. It is well-established that the microbiome composition is a major determinant of colitis in humans and mice. One mechanism by which microbiome contents alter the course of colitis is by the elaboration of certain fecal metabolites. NMR-based fecal metabolomics analyses demonstrated no significant changes in short-chain fatty acids (acetate, propionate, and butyrate) and various amino acids (valine, proline, alanine) however we found significantly less uracil in fecal from Fibg 390-296A relative to Fib WT mice following DSS exposure. Uracil is a key ligand for Duox2 (dual NADPH oxidase), a gut epithelial specific enzyme, that drives reactive oxygen species production by gut epithelial in response to dysbiosis. Notably, Duox2 is the highest induced gene in inflammatory bowel disease (IBD) and has been identified as a risk gene. Our molecular analyses showed no difference in colonic Duox2 expression between genotypes at baseline, but Duox2 expression significantly increased in Fib WT relative to Fibg 390-296A after just 5 days of DSS exposure. Further western blot analyses revealed that Duox2 expression only in the colonic epithelial cells and not in the lamina propria cells. To determine if fibrinogen cause dysbiosis in DSS-induced colitis model, we performed shotgun sequencing on fecal samples from Fib WT and Fibg 390-396A mice at baseline and after 7 days of DSS challenge. Interesting shotgun sequencing analyses revealed major fibrin genotype dependent significant differences in the microbiomes of Fib WT and Fibg 390-396A mice at baseline as well as following DSS challenge. Our studies are the first to show that fibrin(ogen) is a major determinant of the gut-microbiome in the context of experimental colitis. Altogether, these studies demonstrate that fibrin(ogen) in the colitis microenvironment promotes the infiltration and activation of multiple leukocyte subsets that drive colitis pathogenesis. These results also suggest that fibrin(ogen) promotes colitis-associated dysbiosis and Duox2 expression, leading to ROS production that contributes to colitis pathogenesis and possibley tumorigenesis. Overall, these studies suggest that fibrin(ogen)-α Mβ 2 interactions represent an attractive therapeutic target for IBD without incurring the potential bleeding risks associated with anticoagulants or other modalities targeting fibrin deposition. DisclosuresNo relevant conflicts of interest to declare.