In Hashimoto’s thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines’ response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.

Graves’ disease (GD) is an autoimmune thyroiditis often associated with Graves’ orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.

Caveolin-1 (cav1) is a member of the thyroxisome multiprotein complex required for thyroid hormones synthesis which is decreased in Hashimoto’s thyroiditis (HT) and excessive in Graves’ disease (GD). Both pathologies are characterized by oxidative stress (OS), the production of reactive oxygen species (ROS) exceeding the antioxidant defenses. The aim of this study is to correlate cav1 expression and OS in HT and GD. Primary cultures of human thyrocytes were treated or not with Th1 (Interleukin-1α and Interferonγ) or Th2 (Interleukin-4) cytokines. Thyroid samples from HT and GD patients were compared to paranodular tissues of patients with multinodular goiter (controls). Orbital fats were obtained from controls or patients with Graves’ orbitopathy (GO). Samples were processed for RT-PCR, Western blots or immunohistochemistry to analyze the expression of T4, cav1, 4-hydroxynonenal (HNE), caspase-6, catalase, peroxiredoxin 1 (PRDX1), sirtuin-1 (sirt1), NADPH oxidase (NOX)4 and NOX2. In HT, the glands comprised a mix of normal and altered follicles. The altered follicles, located within inflammatory zones, did not express cav1 and were unable to form and store T4 in the follicular lumen, T4 being detected inside the cytoplasm. They also highly expressed HNE, an OS marker, and presented numerous dead cells labelled with caspase-6. In primary cultures, Th1 cytokines decreased cav1 but also catalase and PRDX1 which detoxify H2O2 and sirt1 known to positively regulate antioxidants expression. At the opposite, in GD thyroids, all the follicles were able to form and store T4. Cav1 protein was increased as compared to controls and properly located at the apical pole. The high HNE expression indicative of OS correlated with an increase of NOX4 continually generating H2O2. However, antioxidants like catalase were upregulated to cope with ROS production so that there were few dead thyrocytes. Interleukin-4 which could mimic GD did not influence cav1 expression. Of interest, in GO orbital adipocytes, NOX4 was also increased, as well as NOX2, inducing OS further aggravated in these cells by a reduction of catalase. In conclusion, cav1 expression is diametrically opposed in HT and GD. In HT, the downregulation of cav1 by Th1 cytokines induces thyroxisome disruption, hypothyroidism and intracellular H202 production responsible for OS still aggravated by a Th1-induced decrease of sirt1 and antioxidant defenses. In GD, cav1 overexpression could be correlated with hyperthyroidism and NOX4 could be the prime target to prevent OS.