Reactive oxygen species, key regulators for thyroid cell function : physiological and pathological aspects

Dans la glande thyroïde, l'H2O2, qui appartient au groupe des espèces réactives de l'oxygène (ERO) est indispensable à la synthèse des hormones thyroïdiennes. Partant de ce constat, notre travail repose sur l'hypothèse que dans des conditions physiologiques, la production d'ERO est contrôlée de telle manière, qu'il n'en résulte aucune lésion cellulaire. En revanche, la quantité d'ERO et la modification de leur nature peuvent les rendre toxiques au cours de processus pathologiques. Nous avons donc étudié l'influence des ERO sur la fonction thyroïdienne dans des conditions physiologiques et leur implication dans des phénomènes d'adaptation, comme la croissance du goitre, ou pathologiques, comme l'involution goitreuse induite par de fortes doses d'iode et la thyroïdite auto-immune de type Hashimoto. Dans la première partie de ce travail, nous montrons que lorsque la charge oxydative de la cellule descend sous un certain seuil, l'expression de protéines impliquées dans la fonction des thyrocytes est altérée. Ces données suggèrent qu'il est indispensable que la charge oxydative soit maintenue à un niveau minimal dans le thyrocyte afin que sa fonction soit préservée. Dans la seconde partie de ce travail, nous montrons que lors de la formation du goitre, un stress oxydatif (SO) survient, cependant strictement limité au thyrocyte et sans qu'il ne soit dommageable pour les cellules. Ce SO, qui est correctement compensé par les défenses anti-oxydantes des cellules folliculaires, apparaît même nécessaire à la prolifération cellulaire et au développement du goitre puisqu'un traitement par la N-acétylcystéine (NAC), une substance puissamment anti-oxydante, freine la division des cellules. Dans la troisième partie de ce travail, nous nous intéressons aux modèles expérimentaux d'inflammation induits par l'administration de fortes doses d'iode à des animaux goitreux. Ici, le SO, est élevé dans les thyrocytes, mais aussi dans le tissu environnant. Il est toxique pour les thyrocytes et s'accompagne d'une réaction inflammatoire transitoire. L'administration d'une molécule anti-inflammatoire, la 15 déoxy-Δ12,14-prostaglandine J2 (15dPGJ2) permet effectivement d'en réduire l'importance, ainsi que les lésions cellulaires. Chez la souris NOD, la réaction inflammatoire de type transitoire devient permanente et peut engendrer une thyroïdite auto-immune destructive, proche de la thyroïdite d'Hashimoto où les cytokines Th1 jouent un rôle important. Le rôle possible des ERO dans l'effet des cytokines Th1 a été analysé en utilisant le NAC et la 15dPGJ2 dans un modèle in vitro de thyrocytes incubés en présence de cytokines Th1, et dans un modèle in vivo de thyroïdite obtenue chez la souris NOD. In vitro, les cytokines Th1 stimulent fortement la production intracellulaire d'ERO, tout en diminuant l'expression des protéines impliquées dans la fonction thyroïdienne. En présence de NAC, malgré la quasi disparition des ERO, la fonction du thyrocyte s'effondre, ce qui suggère que d'autres médiateurs que les ERO doivent être impliqués dans l'effet inhibiteur des cytokines Th1. A l'inverse du NAC, la 15dPGJ2 n'influence d'aucune manière, ni la charge en ERO, ni l'expression des protéines de différenciation. Dans le modèle de la souris NOD, la réaction inflammatoire est typiquement associée à des zones étendues de destruction cellulaire et à un SO intense et toxique pour la cellule thyroïdienne. Le NAC et la 15dPGJ2 exercent tout deux un effet protecteur sur la glande en agissant tant sur le SO que sur la réaction inflammatoire en les inhibant tous les deux. Ces résultats suggèrent que le NAC et la 15dPGJ2 exercent un effet protecteur sur la glande en agissant davantage sur les cellules inflammatoires de l'interstitium que sur les thyrocytes eux-mêmes. Nous nous intéressons dans la dernière partie de ce travail à l'effet des cytokines anti-inflammatoires de type Th2 (IL-4) et Th3 (IL-10 et TGF-β) sur l'inhibition de la fonction cellulaire induite par les cytokines pro-inflammatoires de type Th1. Nous montrons que l'IL-4, qui seule, n'influence en rien la fonction cellulaire thyroïdienne, diminue partiellement la production de ROS induite par les cytokines Th1, mais surtout s'oppose à leur effet inhibiteur. A l'inverse, le TGF-β et l'IL-10, qui par eux-mêmes inhibent la fonction des cellules, n'influencent par contre en rien l'effet inhibiteur des cytokines Th1. Ces données obtenues in vitro confirment, en les écl...H2O2 that belongs to reactive oxygen species(ROS) is continuously produced in the thyroid gland where it is involved in the thyroid hormone synthesis. We therefore hypothesized that the production of ROS is controlled in such a way that cell injury is prevented in physiological conditions. When ROS are overproduced, they may become deleterious as in pathological situations. Thus, we evaluated the role played by ROS in physiological conditions, as well as in adaptation and pathological conditions such as iodine deficiency-induced goiter, iodine-induced thyroid involution, and thyroid autoimmunity. We first show that when basal oxidative load drops below a threshold level, the thyroid cell function is severely hampered. This suggests that the maintenance of OL at a minimal level is essential to safeguard thyroid cell functions. In the second part of this work, we demonstrate the existence of an oxidative stress (OS) which is exclusively present in thyrocytes during goiter formation. Here, OS is harmless for cells because their antioxidant defenses are likely strong enough to buffer OS. Likewise, this harmless OS appears to be required for thyroid cell proliferation and in turn for goiter expansion, as the administration of N-acetylcysteine (NAC), a potent antioxidant, hinders cell division. In the third part of the work, we show that OS is toxic in experimental models of inflammation and autoimmunity. OS, located this time both in thyrocytes and in their surrounding tissue, is accompanied by a strong inflammatory reaction, as well as cell injuries and cell death. Tissue damage is significantly reduced when 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2), a potent anti-inflammatory substance which acts mainly on infiltrating inflammatory cells, is used. In NOD mice, the acute inflammation which occurs after iodide administration may evolve toward destructive autoimmune thyroiditis (Hashimoto's-like) characterized by an ongoing inflammatory reaction where Th1 cytokines play a major role. To determine if Th1 cytokine deleterious effects are mediated by ROS and toxic OS, we analyzed NAC or 15dPGJ2-induced effects in an in vitro model of thyrocytes treated with Th1 cytokines, and in an in vivo model of destructive thyroiditis in NOD mice. In the in vitro model, Th1 cytokines strongly stimulate ROS production while thyroid cell function is severely hampered. NAC, although blocking ROS production, actually aggravates Th1-induced downregulation of the thyroid cell function, suggesting that other mediators than ROS must be involved in Th1 cytokines-induced effects. In the NOD mouse model, the inflammatory reaction associated with tissue destruction goes together with toxic OS. Both NAC and 15dPGJ2 reduce OS and inflammatory reaction. Their protective effect likely results from their action on infiltrating inflammatory cells rather than on thyrocytes themselves. In the last part of this work, we look at possible effects of IL-4, a Th2 cytokine, and of TGF-β or IL-10, two Th3 cytokines, on pro-inflammatory Th1 cytokine-induced inhibition of thyroid cell function. Using again an in vitro model, we show that although IL-4 has no effect by its own on thyroid cell function, it reduces only partially Th1 cytokine-induced ROS production, but fully reverses Th1 cytokine inhibitory effects. By contrast, both TGF-β and IL-10, while having inhibitory effects on thyroid cell function, do not affect Th1 cytokine inhibitory effects. In conclusion, this work shows that a minimal OL is essential for thyroid cell function and when OS is increased only in thyrocytes, as observed in goiter, it is harmless and actually required for cell proliferation. By contrast, in inflammatory contexts, while being localized both in thyrocytes and in their neighbouring environment, OS is toxic. While OS is likely not the sole intracellular mediator of Th1 cytokine-induced inhibitory effects, it may be a key element as a trigger and for the perpetuation of the inflammatory reaction in autoimmune thyroiditis. Having then shown how anti-inflammatory Th2 and Th3 cytokines influence pro-inflammatory Th1 cytokine actions, this thesis contributes to make the medical community better understanding why and how the clinical and biological outcomes of patients with destructive thyroiditis may sometimes be so variable and unpredictable.Thèse de doctorat en sciences biomédicales et pharmaceutiques (SBIM3) -- UCL, 200

Interactions of apamin with pore mutated SK3 channels

In the present work, we have tested the impact of the replacement of valine residues in the pore region of SK3 (520) by either an alanine or a phenylalanine residue in terms of the interactions of apamin with these mutants in comparison with the corresponding native channels. Replacing valine residue at position 520 of the SK3 channel by a phenylalanine significantly increased the sensitivity of the channel to be blocked by tetraethylammonium (TEA) as previously reported. Indeed, an aromatic residue, such as a phenylalanine or a tyrosine, is frequently found in the pore region of several potassium channels more sensitive to TEA than SK channels. We measured the affinity (Kd) of apamin in saturation experiments and studied SK currents in transfected cells using patch clamp techniques. In parallel, molecular modelling techniques were used to examine the impact of these local modifications on the interaction of apamin with the corresponding channels. The presence of a phenylalanine in the pore region of potassium channels led to a higher sensitivity for TEA by creating more hydrophobic interactions as found by the docking procedure. In the in vitro binding experiments, the phenylalanine mutant (SK3VF) displayed a very low affinity for apamin. In patch clamp experiments, the SK current was only very partially blocked by apamin in the SK3VF mutant. Furthermore, apamin displayed an affinity and a blocking activity for the alanine mutant close to that for the corresponding native channels. In conclusion, the presence of a bulky and hydrophobic residue at a position near the pore mouth of SK3 channels has a negative impact on their interactions with apamin.SK channel

Minimal oxidative load: a prerequisite for thyroid cell function

In addition to reactive oxygen species (ROS) produced by mitochondria during aerobic respiration, thyrocytes are continuously producing H2O2, a key element for hormonogenesis. Because nothing is known about ROS implication in normal non stimulated cells, we studied their possible involvement in thyrocytes incubated with a potent antioxidant, N-acetylcysteine (NAC). NAC, which blocked the production of intracellular ROS, also decreased dual oxidases, thyroperoxidase, pendrin and thyroglobulin protein and/or gene expression. By contrast, Na+/I- symporter mRNA expression was unaffected. Among antioxidant systems, peroxiredoxin5 expression was reduced by NAC, whereas peroxiredoxin3 increased and catalase remained unchanged. In vivo, the expression of both dual oxidases and peroxiredoxin5 proteins was also decreased by NAC. In conclusion, when intracellular ROS levels drop below a basal threshold, the expression of proteins involved in thyroid cell function is hampered. This suggests that keeping ROS at a minimal level is required for safeguarding thyrocyte function

Differential Interactions between Th1/Th2, Th1/Th3, and Th2/Th3 Cytokines in the Regulation of Thyroperoxidase and Dual Oxidase Expression, and of Thyroglobulin Secretion in Thyrocytes in Vitro

Hypothyroidism, together with glandular atrophy, is the usual outcome of destructive autoimmune thyroiditis. The impairment in the thyroid function results either from cell destruction or from Th1 cytokine-induced alteration in hormonogenesis. Here, we investigated the impact of the local immune context on the thyroid function. We used two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes incubated with IL-1alpha/interferon (IFN) gamma together with IL-4, a Th2 cytokine, or with TGF-beta, or IL-10, two Th3 cytokines. We first observed that IL-4 totally blocked IL-1alpha/interferon gamma-induced alteration in dual oxidase and thyroperoxidase expression, and in thyroglobulin secretion. By contrast, TGF-beta and IL-10 had no such effect. They rather repressed thyrocyte function as do Th1 cytokines. In addition, IL-4 blocked IL-10-induced repression of thyrocyte function, but not that induced by TGF-beta. In conclusion, Th1 cytokine- and IL-10-induced local inhibitory actions on thyroid function can be totally overturned by Th2 cytokines. These data provide new clues about the influence of the immune context on thyrocyte function

Differential Regulation of the Production of Reactive Oxygen Species in Th1 Cytokine–Treated Thyroid Cells

BACKGROUND: Th1 cytokines exert pleiotropic effects in Hashimoto's thyroiditis. Previous studies reported a downregulation of thyroperoxidase and dual oxidase (DUOX) protein and mRNA expression in thyroid cells treated with Th1 cytokines. Although this effect is partially mediated by intracellular reactive oxygen species (ROS) and reactive nitrogen species, the nature and the source of the ROS involved are currently unknown. The aim of this study was to examine further the nature and source of the ROS produced in response to Th1 cytokines. METHODS: Two rat thyroid cell lines (PCCL3 and FRTL-5) and human thyrocytes were incubated with Th1 cytokines (interleukin [IL]-1α and interferon-γ) in the presence or absence of the Th2 cytokine IL-4, the nitric oxide synthase inhibitor N-nitroso-L-arginine methyl ester (L-NAME), or the synthetic antioxidant N-acetylcysteine. The nature and source of the intracellular and extracellular ROS produced were determined. RESULTS: A rapid increase in intracellular ROS was observed in cells incubated with Th1 cytokines. This increase was not caused by extracellular hydrogen peroxide (H2O2) produced by DUOX because both DUOX expression and extracellular H2O2 synthesis were decreased by Th1 cytokines. Confocal colocalization experiments showed that the Th1 cytokine-triggered ROS were not produced from mitochondria. Electron paramagnetic resonance investigations of PCCL3 cells indicated that the highly reactive hydroxyl radical was not involved in the response to Th1 cytokines. NOX2 mRNA expression was significantly increased in PCCL3 cells incubated with Th1 cytokines, as was the expression of the protein in the thyroid of Hashimoto's thyroiditis patients. NOX4 expression was by contrast unaffected. These results suggest that at least superoxide could be produced after exposure of thyroid cells to Th1 cytokines. The effects of L-NAME and IL-4, both of which partially or totally reverse Th1 cytokine-induced effects, on ROS release were also analyzed. L-NAME and IL-4 significantly reduced the Th1 cytokine-induced surge of intracellular ROS in PCCL3 and human thyroid cells. CONCLUSION: The data presented here reinforce the idea that ROS, other than extracellular H2O2 produced by DUOX, are released from NOX2 after exposure of thyroid cells to Th1 cytokines. ROS/reactive nitrogen species act as important, but as further explained, not exclusive intracellular mediators of Th1 cytokine-induced effects in thyroid cells

Proteomic profiling of cold thyroid nodules

Cold thyroid nodules ( CTNs) represent a frequent endocrine disorder accounting for up to 85% of thyroid nodules in a population living in an iodine-deficient area. Benign CTNs need to be distinguished from thyroid cancer, which is relatively rare. The molecular etiology of benign CTNs is unresolved. To obtain novel insights into their pathogenesis, protein expression profiling was performed in a series of 27 solitary CTNs ( 10 follicular adenoma and 20 adenomatous nodules) and surrounding normal thyroid tissues using two-dimensional gel electrophoresis combined with mass spectrometry analysis, Western blotting, and immunohistochemistry. The proteome analysis revealed a specific fingerprint of CTNs with up-regulation of three functional systems: 1) thyroid cell proliferation, 2) turnover of thyroglobulin, and 3) H2O2 detoxification. Western blot analysis and immunohistochemistry confirmed the proteome data and showed that CTNs exhibit significant up-regulation of proteins involved in thyroid hormone synthesis yet are deficient in T-4-containing thyroglobulin. This is consequential to intranodular iodide deficiency, mainly due to cytoplasmic sodium iodide symporter localization, and portrays the CTN as an activated proliferating lesion with an intranodular hypothyroid milieu. Furthermore, we provide preliminary evidence that up-regulation of H2O2 generation in CTNs could override the antioxidative system resulting in oxidative stress, which is suggested by the finding of raised 8-oxo-guanidine DNA adduct formation in CTNs

Iodide deficiency-induced angiogenic stimulus in the thyroid occurs via HIF- and ROS-dependent VEGF-A secretion from thyrocytes

Vascular supply is an obvious requirement for all organs. In addition to oxygen and nutrients, blood flow also transports essential trace elements. Iodine, which is a key element in thyroid hormone synthesis, is one of them. An inverse relationship exists between the expansion of the thyroid microvasculature and the local availability of iodine. This microvascular trace element-dependent regulation is unique and contributes to keep steady the iodide delivery to the thyroid. Signals involved in this regulation, such as VEGF-A, originate from thyrocytes as early TSH-independent responses to iodide scarcity. The question raised in this paper is how thyrocytes, facing an acute drop in intracellular stores of iodine; generate angiogenic signals acting on adjacent capillaries. Using in vitro models of rat and human thyroid cells, we show for the first time that the deficit in iodine, is related to the release of VEGF-A via a reactive oxygen species/hypoxia inducible factor-1-dependent pathway. Key words: thyroid function, angiogenesis - VEGF, ROS, iodine, HIF

Les stratégies volitionnelles dans l'enseignement supérieur: se mettre au travail et s'y maintenir

La métacognition ?L’apprentissage autorégulé ?Quelles différences ?Quelles similitudes ?Comment les définir ?Comment les utiliser en contexte scolaire, académique, professionnel ?Voici les questions auxquelles cet ouvrage tente de répondre.Dans l’introduction, les coordinatrices dressent un historique des concepts et des travaux de recherche construits autour de cette problématique, depuis Flavell à nos jours. Les contributions de ce livre couvrent les différents niveaux de l’enseignement, de l’élémentaire au supérieur, en passant par le secondaire et l’éducation continue. Elles sont rédigées par des chercheurs enseignants francophones issus de Belgique, de France, de Suisse et du Canada.Certains chapitres, plus théoriques, intéresseront surtout les chercheurs et spécialistes du domaine, d’autres, plus proches des pratiques de classe sur le terrain, interpelleront davantage les praticiens-didacticiens qui souhaiteraient intégrer ces approches constructives et dynamiques dans leurs pratiques pédagogiques.info:eu-repo/semantics/publishe