Severe Autoimmune Thyroiditis in Transgenic NOD.H2h4 Mice Expressing Interleukin-4 in the Thyroid.

Background: Hashimoto's thyroiditis is a common autoimmune thyroid disorder characterized by thyroid lymphocytic infiltrates and autoreactive antibodies against thyroglobulin (TgAbs) and thyroperoxidase. Final evolution of the disease can lead to hypothyroidism with destruction of the thyroid architecture. Interleukin-4 (IL-4) is involved in the humoral immune response and B cell activation required in autoimmune thyroiditis (AT) progression. We used our mouse model overexpressing IL-4 by thyrocytes (Thyr-IL4) to study the impact of a local IL-4 expression in AT using transgenic nonobese diabetic (NOD.H2h4) derived animals treated with iodide-supplemented water to increase the incidence of spontaneous AT (SAT). Methods: Thyr-IL4 NOD.H2h4 and nonpathogenic C57BL/6 animals aged 8 weeks were exposed to 0.05% sodium iodide (NaI) in their drinking water for 8 and 16 weeks. Circulating TgAbs and expression of intrathyroidal cytokines were quantified. Thyroid inflammation was assessed by classical histological analyses, including identification of some immune cell populations. The most sensitive parameter to evaluate the thyroid function, serum thyrotropin (TSH), was also measured at the end of the treatment. Results: Relative to wild-type (WT) animals, Thyr-IL4 NOD.H2h4 mice developed severe accelerated SAT with elevated serum TgAbs and numerous thyroid infiltrates mainly composed of CD4+/CD8+ T cells, B lymphocytes, and monocytes/macrophages. Thyroid expression of T helper (Th) Th1/Th2 cytokines was also enhanced, as well as IL-17. In contrast, excessive iodide supply did not induce TgAbs in WT and Thyr-IL4 SAT-resistant C57BL/6 animals. However, moderate leukocyte infiltrations in transgenic thyroids were evident compared to WT, but associated with a limited number of T and B cells and a different cytokine profile from Thyr-IL4 NOD.H2h4 mice. Finally, and despite their diverse immune responses, both transgenic strains presented marked thyroid enlargement and elevated serum TSH at the end of the treatment in contrast to their WT littermates. Conclusions: These findings demonstrated that ectopic expression of IL-4 from thyrocytes enhanced the severity of accelerated SAT in disease-prone Thyr-IL4 NOD.H2h4 animals and promoted thyroid leukocyte infiltration in SAT-resistant transgenic C57BL/6 mice. Moreover, impaired thyroid function emerged in both transgenic strains during the progression of the disease.

Murine Thyroid IL-4 Expression Worsens Hypothyroidism on Iodine Restriction and Mitigates Graves Disease Development.

Cytokines are known to perturb thyroid function and the role of interleukin-4 (IL-4) in the pathogenesis of Graves disease (GD) remains controversial. In our mouse model overexpressing IL-4 in thyrocytes (Thyr-IL4), we have reported that adult mice preserved normal serum thyroxine despite an iodide uptake defect. In the present work, we evaluated if iodine restriction could uncover the thyroid deficiency in Thyr-IL4 animals as well as the role of pendrin overexpression as a compensatory mechanism. Moreover, using an experimental model of GD we investigated the effect of a local expression of IL-4 on the incidence of hyperthyroidism. Thyr-IL4 mice developed more rapidly elevated serum thyrotropin under low-iodine supply with thyroid enlargement and classical histological modifications. These hallmarks of hypothyroidism were all enhanced in Thyr-IL4 mice with complete pendrin invalidation. Following immunization, a lower proportion of Thyr-IL4 animals developed hyperthyroidism. Surprisingly, immunized Thyr-IL4 animals presented numerous leukocyte infiltrates, associated with increased intrathyroidal expression of IFN-γ. We have demonstrated that thyroid deficiency in Thyr-IL4 mice is partially compensated for by the excessive iodide content of the standard chow and the overexpression of pendrin in these animals. Furthermore, we have shown that the local expression of IL-4 in the thyroid attenuates GD progression, which was associated with enhanced thyroid infiltration by immune cells that could negatively affect thyroid function.

Functional model of rat thyroid follicles cultured in Matrigel.

BACKGROUND: Long-term maintenance of functional activity of thyroid cells is an essential requirement for basic in vitro studies on the physiology and pathology of the thyroid. An important prerequisite of thyrocytes' functional activity in vivo and in vitro is their follicle organization. AIM: This study aimed at developing a method of cultivation of functionally active rat thyroid follicles in Matrigel under three-dimensional conditions. METHODS: Undamaged rat thyroid follicles were isolated by enzymatic digestion with collagenase/dispase, then embedded into Matrigel, and cultivated for 2 weeks. Thyroglobulin, thyroxine and zonula occludens-1 (ZO-1) localization were revealed by immunofluorescence analysis. Iodide organification was tested by protein-bound 125I (PBI) measurement. RESULTS: Integrity of the follicles was preserved during the whole period of cultivation and was confirmed by 3D reconstruction of ZO-1 localization. Thyroglobulin was detected in the thyrocyte cytoplasm, as well as in the intrafollicular lumen. Thyroxine was observed predominantly at the apical side of thyrocytes. Also, generated cultures were characterized by a high level of iodide organification: PB125I represented 39% of the total radioactivity in the Matrigel drop embedding the follicles; at the same time, methimazole almost totally inhibited this process (0.2% of total radioactivity). CONCLUSION: The method of rat thyrocyte cultivation in Matrigel, as described here allows to maintain the structural integrity and the functional activity of thyroid follicles in vitro and could be used for wide ranges of basic and applied researches in thyroidology.

Dissecting the Role of Thyrotropin in the DNA Damage Response in Human Thyrocytes after 131I, γ Radiation and H2O2.

BACKGROUND: The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. METHODS: Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (ß radiation), γ radiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. RESULTS: Overall, the thyrocyte responses following exposure to ß or γ radiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα q cascade and a rise in intracellular reactive oxygen species (ROS) levels. ß and γ radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to ß/γ radiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. CONCLUSIONS: TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I.

The Dual Oxidase Duox2 stabilized with DuoxA2 in an enzymatic complex at the surface of the cell produces extracellular H2O2 able to induce DNA damage in an inducible cellular model.

Thyroid hormone synthesis requires H2O2, produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H2O2 generation. By normalizing H2O2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H2O2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

DUOX Defects and Their Roles in Congenital Hypothyroidism.

Extracellular hydrogen peroxide is required for thyroperoxidase-mediated thyroid hormone synthesis in the follicular lumen of the thyroid gland. Among the NADPH oxidases, dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially identified as thyroid oxidases, based on their level of expression in the thyroid. Despite their high sequence similarity, the two isoforms present distinct regulations, tissue expression, and catalytic functions. Inactivating mutations in many of the genes involved in thyroid hormone synthesis cause thyroid dyshormonogenesis associated with iodide organification defect. This chapter provides an overview of the genetic alterations in DUOX2 and its maturation factor, DUOXA2, causing inherited severe hypothyroidism that clearly demonstrate the physiological implication of this oxidase in thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in permanent but also in transient forms of congenital hypothyroidism. Moreover, accumulating evidence demonstrates that the high phenotypic variability associated with altered DUOX2 function is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors. The presence of two DUOX isoforms and their corresponding maturation factors in the same organ could certainly constitute an efficient redundant mechanism to maintain sufficient H2O2 supply for iodide organification. Many of the reported DUOX2 missense variants have not been functionally characterized, their clinical impact in the observed phenotype remaining unresolved, especially in mild transient congenital hypothyroidism. DUOX2 function should be carefully evaluated using an in vitro assay wherein (1) DUOXA2 is co-expressed, (2) H2O2 production is activated, (3) and DUOX2 membrane expression is precisely analyzed.

Factors contributing to the resistance of the thyrocyte to hydrogen peroxide.

We studied the mechanism that may explain the relative resistance of thyrocytes to H2O2 compared to other cell types. Ability to degrade H2O2, glutathione peroxidase (GPx) activity, heme oxygenase-1 (HO-1) expression, cell survival and capacity to repair DNA damage after H2O2 exposure or irradiation were measured in human thyrocytes in primary culture and compared to the values obtained in human T-cells and different cell lines. Compared to other cell types, thyrocytes presented a low mortality rate after H2O2 exposure, rapidly degraded extracellular H2O2 and presented a high basal seleno-dependent GPx activity. Only in thyrocytes, H2O2 up-regulated GPx activity and expression of HO-1 mRNA. These effects were not reproduced by irradiation. DNA damage caused by H2O2 was more slowly repaired than that caused by irradiation and not repaired at all in T-cells. Our study demonstrates that the thyrocyte has specific protective mechanisms against H2O2 and its mutagenic effects.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

BACKGROUND: The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. METHODS: Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. RESULTS: Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. CONCLUSIONS: This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.

Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling.

SIGNIFICANCE: 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. RECENT ADVANCES: 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. CRITICAL ISSUES: 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. FUTURE DIRECTIONS: 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.

Comparative analysis of the thyrocytes and T cells: responses to H2O2 and radiation reveals an H2O2-induced antioxidant transcriptional program in thyrocytes.

CONTEXT: Radiation is an established cause of thyroid cancer, and growing evidence supports a role for hydrogen peroxide (H2O2) in spontaneous thyroid carcinogenesis. Little is known about the molecular programs activated by these agents in thyrocytes. OBJECTIVE: The purpose of this study was to compare the responses of thyrocytes and T cells to H2O2 and radiation. METHODS: We profiled the DNA damage and cell death induced by γ-radiation (0.1-5 Gy) and H2O2 (0.0025-0.3 mM) in primary human thyrocytes and T cells. We next prepared thyroid and T-cell primary cultures from 8 donors operated for noncancerous thyroid pathological conditions and profiled their genome-wide transcriptional response 4 hours after (1) exposure to 1-Gy radiation, (2) treatment with H2O2 and (3) no treatment. Two H2O2 concentrations were investigated, calibrated in each cell type to elicit levels of single- and double-strand breaks equivalent to 1-Gy γ-radiation. RESULTS: Although thyrocytes and T cells had comparable radiation responses, 3- to 10-fold more H2O2 was needed to induce detectable DNA damage in thyrocytes. At H2O2 and radiation doses inducing double-strand breaks, cell death occurred after 24 hours in T cells but not in thyrocytes. The transcriptional responses of thyrocytes and T cells to radiation were similar, involving DNA repair and cell death genes. In addition to this transcriptional program, H2O2 also up-regulated antioxidant genes in thyrocytes, including glutathione peroxidases and heme oxygenase at the double-strand breaks-inducing concentration. In contrast, a transcriptional storm involving thousands of genes was raised in T cells. Finally, we showed that inhibiting glutathione peroxidases activity increased the DNA damaging effect of H2O2 in thyrocytes. CONCLUSION: We propose that high H2O2 production in thyrocytes is matched with specific transcriptionally regulated antioxidant protection.

IFNß/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response.

Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNß secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNß and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction.

Stroma cell-derived factor-1α signaling enhances calcium transients and beating frequency in rat neonatal cardiomyocytes.

Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP3R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP3R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP3-gated fashion leading to a positive chronotropic and inotropic effect.

Thyroid hydrogen peroxide production is enhanced by the Th2 cytokines, IL-4 and IL-13, through increased expression of the dual oxidase 2 and its maturation factor DUOXA2.

The dual oxidases (DUOX) 1 and 2 constitute the major components of the thyroid H(2)O(2)-generating system required for thyroid hormone synthesis. With their maturation factor, DUOXA1 or DUOXA2, they share the same bidirectional promoter allowing coexpression of DUOX/DUOXA in the same tissue. However, the molecular mechanisms regulating their transcription in the human thyroid gland are not well characterized yet. Inflammatory molecules associated with autoimmune thyroid diseases have been shown to repress the thyroid function by down-regulating the expression of the major thyroid differentiation markers. These findings led us to investigate the effects of the main cytokines involved in Hashimoto thyroiditis (IFN-γ) and Graves' diseases (IL-4/IL-13) on the transcriptional regulation of DUOX and their corresponding DUOXA genes in thyroid cells. Human thyrocytes exposed to the Th2 cytokines IL-4 and IL-13 showed up-regulation of DUOX2 and DUOXA2 genes but not DUOX1/DUOXA1. The DUOX2/DUOXA2 induction was rapid and associated with a significant increase of calcium-stimulated extracellular H(2)O(2) generation. IFN-γ treatment inhibited DUOX gene expression and repressed the Th2 cytokine-dependent DUOX2/DUOXA2 expression. In another DUOX-expressing model, the human intestinal Caco-2 cell line, expression of DUOX2 and DUOXA2 mRNA was also positively modulated by IL-4 and IL-13. Analysis of the IL-4 signaling pathway revealed that the JAK1-STAT6 cascade activated by the IL-4 type 2 receptor is required for DUOX2/DUOXA2 induction. The present data open new perspectives for a better understanding of the pathophysiology of thyroid autoimmune diseases considering DUOX2-mediated oxidative damages.

The type of DUOX-dependent ROS production is dictated by defined sequences in DUOXA.

A deliberate generation of ROS is now recognized to be achieved by specific NADPH oxidases (NOX). Dual oxidases (DUOXs) are Ca(2+)-activated NOXs and operate as H(2)O(2)-generators in various tissues. A tight regulation is however required to avoid ROS overproduction that can rapidly be harmful to biological systems. DUOX activator (DUOXA) proteins act as organizing elements for surface expression and activity of the DUOX enzymes. To study DUOX activation by the maturation factors, chimeric DUOXA proteins were generated by replacing particular domains between DUOXA1 and DUOXA2. Their impact on DUOX function and membrane expression were explored in a reconstituted heterologous cell system composed of COS-7 cells. We have shown that the COOH-terminal end of DUOXA1 is responsible for DUOX1-dependent H(2)O(2) generation. The NH(2)-terminal tail of DUOXA2 is critical to specify the type of ROS released by DUOX2, hydrogen peroxide or superoxide. Native DUOXA2 would constrain DUOX2 to produce H(2)O(2). However, alterations of the DUOXA2 NH(2)-terminal domain modify DUOX2 activity triggering superoxide leaking. Our results demonstrate that specific domains of the DUOX maturation factors promote the activation of DUOXs as well as the type of ROS generated by the oxidases.

Reactive oxygen species regulate the levels of dual oxidase (Duox1-2) in human neuroblastoma cells.

Dual Oxidases (DUOX) 1 and 2 are efficiently expressed in thyroid, gut, lung and immune system. The function and the regulation of these enzymes in mammals are still largely unknown. We report here that DUOX 1 and 2 are expressed in human neuroblastoma SK-N-BE cells as well as in a human oligodendrocyte cell line (MO3-13) and in rat brain and they are induced by platelet derived growth factor (PDGF). The levels of DUOX 1 and 2 proteins and mRNAs are induced by reactive oxygen species (ROS) produced by the membrane NADPH oxidase. As to the mechanism, we find that PDGF stimulates membrane NADPH oxidase to produce ROS, which stabilize DUOX1 and 2 mRNAs and increases the levels of the proteins. Silencing of gp91(phox) (NOX2), or of the other membrane subunit of NADPH oxidase, p22(phox), blocks PDGF induction of DUOX1 and 2. These data unravel a novel mechanism of regulation of DUOX enzymes by ROS and identify a circuitry linking NADPH oxidase activity to DUOX1 and 2 levels in neuroblastoma cells.

The nonphagocytic NADPH oxidase Duox1 mediates a positive feedback loop during T cell receptor signaling.

Production of reactive oxygen species, often by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, plays a role in the signaling responses of cells to many receptor stimuli. Here, we describe the function of the calcium-dependent, nonphagocytic NADPH oxidase Duox1 in primary human CD4(+) T cells and cultured T cell lines. Duox1 bound to inositol 1,4,5-trisphosphate receptor 1 and was required for early T cell receptor (TCR)-stimulated production of hydrogen peroxide (H(2)O(2)) through a pathway that was dependent on TCR-proximal kinases. Transient or stable knockdown of Duox1 inhibited TCR signaling, especially phosphorylation of tyrosine-319 of zeta chain-associated protein kinase of 70 kilodaltons (ZAP-70), store-operated entry of calcium ions (Ca(2+)), and activation of extracellular signal-regulated kinase. The production of cytokines was also inhibited by knockdown of Duox1. Duox1-mediated inactivation of Src homology 2 domain-containing protein tyrosine phosphatase 2 promoted the phosphorylation of ZAP-70 and its association with the Src family tyrosine kinase Lck and the CD3zeta chain of the TCR complex. Thus, we suggest that activation of Duox1, downstream of proximal TCR signals, generates H(2)O(2) that acts in a positive feedback loop to enhance and sustain further TCR signaling.

Compound heterozygosity for a novel hemizygous missense mutation and a partial deletion affecting the catalytic core of the H2O2-generating enzyme DUOX2 associated with transient congenital hypothyroidism.

Dual oxidases (DUOX) 1 and 2 are components of the thyroid H(2)O(2)-generating system. H(2)O(2) is used by thyroperoxidase to oxidize iodide for thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in transient and permanent congenital thyroid dyshormonogenesis. We report here a novel genetic defect causing congenital hypothyroidism in a French-Canadian patient. At neonatal screening, the patient had high TSH and low total T(4) levels. (99m)Tc scan showed a normally shaped orthotopic but mildly enlarged thyroid gland, suggesting dyshormonogenesis. Thyroxine treatment was given from 1 month to 17 years, after which it was stopped for re-evaluation and the patient remained euthyroid. The transient congenital hypothyroidism phenotype prompted us to screen for mutations in DUOX2 and DUOXA2 genes using the PCR-amplified direct sequencing method. We found complete inactivation of DUOX2 caused by a partial genomic deletion of one allele inherited from the mother associated with a paternally inherited missense mutation (c.4552G>A, p.Gly1518Ser). The deleted fragment encompasses the entire COOH-terminal end which is responsible for the NADPH-oxidase activity. The Gly1518Ser DUOX2 protein is expressed at the cell surface of transfected cells albeit at low level, but it is non-functional. This study provides further evidence that the permanent or transient nature of congenital hypothyroidism is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors.