ABSTRACT
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.
Subject(s)
Anion Transport Proteins/metabolism , Dual Oxidases/metabolism , Interleukin-4/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Thyroid Gland/metabolism , Up-Regulation , Absorption, Physiological , Animals , Anion Transport Proteins/genetics , Cells, Cultured , Down-Regulation , Dual Oxidases/genetics , Gene Expression Profiling , Hydrogen Peroxide/metabolism , Interleukin-4/genetics , Iodides/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Sulfate Transporters , Symporters/genetics , Symporters/metabolism , Thyroid Gland/cytology , Thyroid Gland/immunology , Thyrotropin/blood , Thyrotropin/metabolism , Thyroxine/blood , Thyroxine/metabolismABSTRACT
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.
