Background: Under sufficient iodine supply, dual oxidase (DUOX)-dependent H2O2 production constitutes the limiting factor for thyroid hormone (TH) synthesis. Inherited loss-of-function mutations in related genes can trigger congenital hypothyroidism (CH). TH supplementation is not always well-tolerated and requires dose adjustments throughout life. Regenerative medicine directed at thyroid follicular cells (TFCs) could offer an alternative therapy; however, the minimal number of TFCs to be corrected remains unknown. Methods: Thyroid dyshormonogenesis in Duoxa-/- deficient mice was rescued by conditional thyroid-specific expression of DUOXA2/DUOX2 subunits. In order to restrict reactivation in a subset of TFCs, low doses of tamoxifen (0.1-2 mg) were injected. Thyroid function was assessed by immunostaining of iodinated-thyroglobulin (iTG). Circulating serum thyrotropin (TSH) and total thyroxin (T4) were quantified, and thyroidal expression of TSH-responsive genes (Nis, Tpo, Tshr) and hepatic deiodinase type-1 (Dio1) was measured. Last, combining iTG immunostaining with Duox2 in situ hybridization, we estimated the fraction of rescued TFCs required to restore follicular TG iodination. Results: Colloidal iTG+ staining in more than 90% of follicles demonstrated the successful rescue of TH biogenesis in doxycycline-induced Tet:Da2D2+/-;mTg:CreERT2+/-;Duoxa-/- animals (3TA). In contrast, reducing tamoxifen doses to 0.1 mg resulted in unresolved primary CH with thyroid enlargement, induction of Nis, Tpo, and Tshr, decrease of Dio1, and growth delay. Corresponding thyroid sections revealed scattered iTG+ colloidal lumens dispersed in histologically altered parenchyma. Nevertheless, we determined that only 11-15% of TFCs need to be reactivated within the follicle to functionally restore iodide organification. Goiter involution was also studied in 3TA following functional oxidase recovery or levothyroxine supplementation. Although thyroid enlargement was similarly reduced in both groups, expression of Nis, Tpo, Tshr, and Dio1 more rapidly normalized in genetically rescued 3TA. In these pathological conditions, around 50-70% of iTG+ follicles would seem sufficient to recover a healthy thyroid function until two weeks. Conclusions: Our data in Duoxa-/- mice suggest that the percentage of TFCs to be corrected is limited to 10-15% per follicle, which could be compatible with future somatic gene therapies. Furthermore, the reconstitution of hormonogenic-competent TFCs successfully results in goiter resolution, to an extent comparable to that of gold-standard TH replacement therapy.

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.H2(h4)) derived animals treated with iodide-supplemented water to increase the incidence of spontaneous AT (SAT).Methods: Thyr-IL4 NOD.H2(h4) 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.H2(h4) 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.H2(h4) 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.H2(h4) 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.

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.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

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.

Background: The early molecular events in human thyrocytes after I-131 exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the I-131-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 I-131 (beta radiation), gamma 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 beta or gamma radiation and to H2O2 were similar. However, TSH increased I-131-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 I-131 exposure. This effect most likely occurred via G alpha(q) cascade and a rise in intracellular reactive oxygen species (ROS) levels. beta and gamma radiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to beta/gamma 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 I-131 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 I-131.

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.

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.

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.

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.

GENERAL COMMENTARY article Front. Endocrinol., 25 August 2017Sec. Thyroid Endocrinology Volume 8 - 2017 | https://doi.org/10.3389/fendo.2017.00214

Introduction: Ionizing radiation (IR) causes not only acute tissue damage but also late effects in several cell generations after the initial exposure. The mechanism by which the radiation exposure is memorized and lead to delayed DNA breakage remains to be determined. Objectives: We investigated the mechanism by which IR induced ROS generation several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Methodology: Human thyroid cell line, primary human thyroid cells and 35 frozen tissues (20 radio-induced thyroid tumors, 9 sporadic tumors and 6 normal tissues) were included in this study. Results: IR (10 Gy) induces a delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which was sustained for several days. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 downregulation by siRNA abrogate IR-induced DNA-damage. Analysis of human thyroid tissues showed that DUOX1 is elevated in human radio-induced thyroid tumors. Conclusion: 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 generation of neoplastic cells through its mutagenic effect.

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.

The main function of the thyroid gland is to make hormones, T4 and T3, which are essential for the regulation of metabolic processes throughout the body. As at any factory, effective production depends on three key components - adequate raw material, efficient machinery, and appropriate controls. Iodine is the critical raw material, because 65% of T4 weight is iodine. Ingested iodine is absorbed and carried in the circulation as iodide. The thyroid actively concentrates the iodide across the basolateral plasma membrane of thyrocytes by the sodium/iodide symporter, NIS. Intracellular iodide is then transported in the lumen of thyroid follicles. Meanwhile, the thyrocyte endoplasmic reticulum synthesizes two key proteins, TPO and Tg. Tg is a 660kDa glycoprotein secreted into the lumen of follicles, whose tyrosyls serve as substrate for iodination and hormone formation. TPO sits at the apical plasma membrane, where it reduces H2O2, elevating the oxidation state of iodide to an iodinating species, and attaches the iodine to tyrosyls in Tg. H2O2 is generated at the apex of the thyrocyte by Duox, a NADPH oxydase. Initial iodination of Tg produces MIT and DIT. Further iodination couples two residues of DIT, both still in peptide linkage, to produce T4, principally at residues 5 in the Tg polypeptide chain. When thyroid hormone is needed, Tg is internalized at the apical pole of thyrocytes, conveyed to endosomes and lysosomes and digested by proteases, particularly the endopeptidases cathepsins B, L, D and exopeptidases. After Tg digestion, T4 and T3 are released into the circulation. Nonhormonal iodine, about 70% of Tg iodine, is retrieved intrathyroidally by DEHAL1, an iodotyrosine deiodinase and made available for recycling within the gland. TSH is the stimulator that affects virtually every stage of thyroid hormone synthesis and release. Early control involves the direct activation of the cellular and enzymatic machineries while delayed and chronic controls are on gene expression of key proteins. Iodine supply, either too much or too little, impairs adequate synthesis. Antithyroid drugs act by interfering with iodide oxidation. Genetic abnormalities in any of the key proteins, particularly NIS, TPO, Duox and Tg, can produce goiter and hypothyroidism. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org .

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.