Hypothyroidism-associated immunosuppression involves induction of galectin-1-producing regulatory T cells.

Hypothyroidism exerts deleterious effects on immunity, but the precise role of the hypothalamic-pituitary-thyroid (HPT) axis in immunoregulatory and tolerogenic programs is barely understood. Here, we investigated the mechanisms underlying hypothyroid-related immunosuppression by examining the regulatory role of components of the HPT axis. We first analyzed lymphocyte activity in mice overexpressing the TRH gene (Tg-Trh). T cells from Tg-Trh showed increased proliferation than wild-type (WT) euthyroid mice in response to polyclonal activation. The release of Th1 pro-inflammatory cytokines was also increased in Tg-Trh and TSH levels correlated with T-cell proliferation. To gain further mechanistic insights into hypothyroidism-related immunosuppression, we evaluated T-cell subpopulations in lymphoid tissues of hypothyroid and control mice. No differences were observed in CD3/CD19 or CD4/CD8 ratios between these strains. However, the frequency of regulatory T cells (Tregs) was significantly increased in hypothyroid mice, and not in Tg-Trh mice. Accordingly, in vitro Tregs differentiation was more pronounced in naïve T cells isolated from hypothyroid mice. Since Tregs overexpress galectin-1 (Gal-1) and mice lacking this lectin (Lgals1-/- ) show reduced Treg function, we investigated the involvement of this immunoregulatory lectin in the control of Tregs in settings of hypothyroidism. Increased T lymphocyte reactivity and reduced frequency of Tregs were found in hypothyroid Lgals1-/- mice when compared to hypothyroid WT animals. This effect was rescued by the addition of recombinant Gal-1. Finally, increased expression of Gal-1 was found in Tregs purified from hypothyroid WT mice compared with their euthyroid counterpart. Thus, a substantial increase in the frequency and activity of Gal-1-expressing Tregs underlies immunosuppression associated with hypothyroid conditions, with critical implications in immunopathology, metabolic disorders, and cancer.

Regulation of the cellular redox state and the expression of DNA methyltransferase-1 in peripheral blood mononuclear cells from patients with Graves' disease.

BACKGROUND: Graves' disease is an autoimmune disorder characterised by excessive production of thyroid hormones, which induces increased cellular metabolism in most tissues and increased production of reactive oxygen species (ROS). The aim of this work was to analyse the effect of ROS on cell viability and the expression of catalase (CAT), glutathione peroxidase-1 (GPx-1), superoxide dismutase (SOD-1) and DNA methyltransferase-1 (DNMT-1) in peripheral blood mononuclear cells (PBMC) from patients with newly diagnosed Graves' disease or treated with methimazole. PATIENTS AND METHODS: For this study, women patients with newly diagnosed Graves' disease (n=18), treated with methimazole (n=6) and healthy subjects (n=15) were recruited. ROS were evaluated by flow cytometry, and the viability/apoptosis of PBMC was analysed by flow cytometry and fluorescence microscopy. Genomic expression of CAT, GPx-1, SOD-1 and DNMT-1 was quantified by real-time PCR. RESULTS: We found high levels of ROS and increased expression of CAT, GPx-1, SOD-1 and DNMT-1 in PBMC from patients with newly diagnosed Graves' disease. Methimazole treatment reversed these parameters. Cell viability was similar in all study groups. CONCLUSIONS: ROS induces the expression of CAT, GPx-1, and SOD-1. The activity of these enzymes may contribute to the protection of PBMC from the harmful effect of free radicals on cell viability. Increased expression of DNMT-1 may be associated with aberrant methylation patterns in immunoregulatory genes contributing to autoimmunity in Graves' disease.

Thyroid status regulates the tumor microenvironment delineating breast cancer fate.

The patient's hormonal context plays a crucial role in the outcome of cancer. However, the association between thyroid disease and breast cancer risk remains unclear. We evaluated the effect of thyroid status on breast cancer growth and dissemination in an immunocompetent mouse model. For this, hyperthyroid and hypothyroid Balb/c mice were orthotopically inoculated with triple-negative breast cancer 4T1 cells. Tumors from hyperthyroid mice showed an increased growth rate and an immunosuppressive tumor microenvironment, characterized by increased IL-10 levels and decreased percentage of activated cytotoxic T cells. On the other hand, delayed tumor growth in hypothyroid animals was associated with increased tumor infiltration of activated CD8+ cells and a high IFNγ/IL-10 ratio. Paradoxically, hypothyroid mice developed a higher number of lung metastasis than hyperthyroid animals. This was related to an increased secretion of tumor CCL2 and an immunosuppressive systemic environment, with increased proportion of regulatory T cells and IL-10 levels in spleens. A lower number of lung metastasis in hyperthyroid mice was related to the reduced presence of mesenchymal stem cells in tumors and metastatic sites. These animals also exhibited decreased percentages of regulatory T lymphocytes and myeloid-derived suppressor cells in spleens but increased activated CD8+ cells and the IFNγ/IL-10 ratio. Therefore, thyroid hormones modulate the cellular and cytokine content of the breast tumor microenvironment. A better understanding of the mechanisms involved in these effects could be a starting point for the discovery of new therapeutic targets for breast cancer.

Oxidative Stress Produced by Hyperthyroidism Status Induces the Antioxidant Enzyme Transcription through the Activation of the Nrf-2 Factor in Lymphoid Tissues of Balb/c Mice.

Hyperthyroidism is an endocrine disorder characterized by excessive secretion of thyroid hormones T3 and T4. Thyroid hormones exert pleiotropic actions on numerous tissues and induce an overall increase in metabolism, with an increase in energy demand and oxygen consumption. Therefore, the purpose of this study was to investigate the effects of hyperthyroidism on the production of reactive oxygen species (ROS) in lymph node and spleen cells of euthyroid and hyperthyroid mice, analyzing antioxidant mechanisms involved in the restitution of the cellular redox state. For this, thirty female Balb/c (H-2d) mice were randomly divided into two groups: euthyroid (by treatment with placebo) and hyperthyroid (by treatment with 12 mg/l of T4 in drinking water for 30 days). We found a significant increase in ROS and an increase in the genomic and protein expression of the antioxidant enzymes catalase (CAT) and glutathione peroxidase-1 (GPx-1) in lymph node and spleen cells of hyperthyroid mice. In vitro treatment with H2O2 (250 µM) of the lymphoid cells of euthyroid mice increased the expression levels of CAT and GPx-1. The hyperthyroidism increased the phosphorylation levels of Nrf2 (nuclear factor erythroid 2-related factor) and the kinase activity of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK). Additionally, we found an increase in the expression of the classic isoenzymes of PKCα, ß and γ. In conclusion, these results indicated that the increase in ROS found in the hyperthyroid state induces the antioxidant enzyme transcription through the activation of the Nrf-2 factor in lymphoid tissues. This shows the influence of hyperthyroidism on the regulation of the cellular antioxidant system.

Hypothyroidism-related zinc deficiency leads to suppression of T lymphocyte activity.

PURPOSE: Hypothyroidism has been shown to induce immunosuppression and both the thyroid status and immunity are affected by zinc deficiency. However, the impact of hypothyroidism on zinc metabolism and its possible relationship with the immune status has not yet been deeply explored. Here, our aim was to study whether hypothyroidism may alter zinc metabolism and thus lead to the impairment of T lymphocyte activity. METHODS: Variations in the distribution of zinc in the body were evaluated in PTU-treated hypothyroid mice. The effects of hypothyroidism and zinc deficiency were studied on T lymphocyte proliferation after stimulation both in vitro and in vivo. For in vitro assays, thyroid hormone-free or zinc chelator (TPEN or DTPA)-supplemented media were used. For in vivo assays, lymphocyte activity was evaluated in cells from hypothyroid, T3-treated, and zinc-supplemented mice. RESULTS: Hypothyroid mice showed lower levels of zinc in femur and lymph nodes than controls. T3 and zinc supplementation reversed these effects. In vitro, both thyroid hormone and zinc deficiency led to a decreased response to mitogen stimulation. However, only zinc deficiency was able to induce lymphocyte apoptosis. Mitogen-stimulated T cells from hypothyroid mice showed impaired proliferation, accompanied by decreased activation of PKC and lower levels of p-ERK, effects that were reversed by T3 replacement or zinc supplementation. CONCLUSIONS: Our results show an important role of zinc deficiency in hypothyroid-mediated T-cell suppression and suggest the importance of evaluating zinc levels and restoring them when necessary to maintain an efficient immune response in hypothyroid patients.

Cooperative nongenomic and genomic actions on thyroid hormone mediated-modulation of T cell proliferation involve up-regulation of thyroid hormone receptor and inducible nitric oxide synthase expression.

Thyroid hormones (THs) exert a broad range of actions on development, growth, and cell differentiation by both genomic and nongenomic mechanisms. THs regulate lymphocyte function, but the participation of nongenomic actions is still unknown. Here the contribution of both genomic and nongenomic effects on TH-induced division of T cells was studied by using free and noncell permeable THs coupled to agarose (TH-ag). THs-ag led to cell division, but to a lesser extent than free hormones. THs induced nongenomically the rapid translocation of protein kinase C (PKC) ζ isoform to cell membranes, extracellular-signal-regulated kinases (ERK1/2) phosphorylation and nuclear factor-κB (NF-κB) activation. The signaling cascade include sphingomyelinases acting up-stream the activation of PKCζ isoform, while ERK and NF-κB are activated downstream this PKC isoenzyme. Both free and THs-ag increased the protein and mRNA levels of TH nuclear receptor TRα1, while only free hormones incremented the inducible NOS gene and protein levels as well as a calcium independent NOS activity. Both effects were blunted by PKCζ inhibition. These results indicate that THs, by triggering a nongenomic signaling cascade that involves Smases-mediated activation of PKCζ, lead to ERK 1/2 and NF-κB activation and to the genomic increase of TRs and the inducible nitric oxide synthase protein and mRNA levels, improving T lymphocyte proliferation. These finding not only contribute to the understanding of the mechanisms involved in TH modulation of lymphocyte physiology, but would also point out for the first time the interplay between genomic and nongenomic TH actions in T cells.

Involvement of thyroid hormones in the alterations of T-cell immunity and tumor progression induced by chronic stress.

BACKGROUND: Stress alters the neuroendocrine system, immunity, and cancer. Although the classic stress hormones are glucocorticoids and catecholamines, thyroid hormones have also been related to stress. We recently reported that chronic restraint stress impairs T-cell mediated immunity and enhances tumor growth in mice. METHODS: To study the participation of these hormones on the stress-induced alterations of the immune function and lymphoma growth, mice were subjected to acute or chronic stress, with or without thyroxin supplementation. Hormone levels, immune status, and cancer progression were evaluated. RESULTS: Differential endocrine alterations were observed in response to acute and chronic stress. Although corticosterone and noradrenaline levels were increased by acute stress, they were restored after prolonged exposure to the stressor. Instead, thyroid hormone levels were only reduced in chronically stressed animals in comparison with control subjects. Correlating, chronic but not acute stress impaired T-cell reactivity. Thyroxin replacement treatment of chronic restraint stress-exposed mice, which restored the euthyroid status, reversed the observed reduction of T-cell lymphoproliferative responses. Moreover, therapeutic thyroid replacement also reversed the alterations of lymphoma growth induced by chronic stress in syngeneic mice bearing tumors as well as Interleukin-2 production and specific cytotoxic response against tumor cells. Finally, we found that the isoforms theta and alpha of the protein kinase C are involved in these events. CONCLUSIONS: These results show for the first time that thyroid hormones are important neuroendocrine regulators of tumor evolution, most probably acting through the modulation of T-cell mediated immunity affected by chronic stress.

Immune-endocrine interactions in autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITD) are the most common organ-specific autoimmune disorders affecting approximately 5% of the overall population. An aberrant interaction between abnormal thyrocytes, abnormal antigen-presenting cells and abnormal T cells forms the basis for the atypical autoimmune reaction targeting thyroid antigens. It was proposed that nongenetic (environmental and hormonal) factors play a crucial etiological role in AITD development, through altering immune-endocrine interactions. The most outstanding fact is that in genetically predisposed individuals, the disruption of these neuroendocrine-immune interactions by environmental factors results in thyroid autoimmune dysfunction. These interactions are able to incline the balance between Th1-Th2 immune response toward one side, resulting in a Th1-cell-mediated autoimmune reaction with thyrocyte destruction and hypothyroidism in Hashimoto's thyroiditis but to a hyperreactive Th2-mediated humoral response against TSH receptor with stimulatory antibodies leading to Graves' disease hyperthyroidism. In this review the main mechanisms involved are summarized. In this sense, the participation of stress-mediated activation of the sympathoadrenal system and hypothalamic-pituitary-adrenal axis, the hormonal changes occurring during pregnancy and postpartum acting on antigen-presenting cells and influencing, in this way, the balance of the immune status are shown to participate in AITD etiology. The possibility that altered levels of thyroid hormones during the course of the AITD may alter immune function is also discussed.

Integrative study of hypothalamus-pituitary-thyroid-immune system interaction: thyroid hormone-mediated modulation of lymphocyte activity through the protein kinase C signaling pathway.

Thyroid hormones play critical roles in differentiation, growth and metabolism, but their participation in immune system regulation has not been completely elucidated. Modulation of in vivo thyroid status was used to carry out an integrative analysis of the role of the hypothalamus-pituitary-thyroid (HPT) axis in T and B lymphocyte activity. The participation of the protein kinase C (PKC) signaling pathway and the release of some cytokines upon antigenic stimulation were analyzed. Lymphocytes from hyperthyroid mice displayed higher T-and B-cell mitogen-induced proliferation, and those from hypothyroid mice displayed lower T- and B-cell mitogen-induced proliferation, compared with euthyroid animals. Reversion of hypothyroid state by triiodothyronine (T3) administration recovered the proliferative responses. No differences were found in lymphoid subset balance. Both total PKC content and mitogen-induced PKC translocation were higher in T and B cells from hyperthyroid mice, and lower in cells from hypothyroid mice, compared with controls. Levels of thyroid-stimulating (TSH) and TSH-releasing (TRH) hormones were not directly related to lymphocyte proliferative responses. After immunization with sheep red blood cells (SRBCs) and re-stimulation, in vitro spleen cells from hyper- or hypothyroid mice showed, respectively, increased or decreased production of interleukin (IL)-2 and interferon (IFN)-gamma cytokines. Additionally, an increase in IL-6 and IFN-gamma levels was found in hyperthyroid cells after in vivo injection and in vitro re-stimulation with lipopolysaccharide (LPS). Our results show for the first time a thyroid hormone-mediated regulation of PKC content and of cytokine production in lymphocytes; this regulation could be involved in the altered responsiveness to mitogen-induced proliferation of T and B cells. The results also confirm the important role that these hormones play in regulating lymphocyte reactivity.

Thyroid hormones increase inducible nitric oxide synthase gene expression downstream from PKC-zeta in murine tumor T lymphocytes.

Regulation of cell proliferation by thyroid hormone (TH) has been demonstrated, but the effect of THs and the mechanisms involved in lymphocyte activity have not been elucidated. Differential expression of PKC isoenzymes and high nitric oxide synthase (NOS) activity have been described in tumor T lymphocytes. We have analyzed the direct actions of TH on normal T lymphocytes and BW5147 T lymphoma cells in relation to PKC and NOS activities. THs increased tumor and mitogen-induced normal T lymphocyte proliferation. PKC isoenzyme-selective blockers impaired these effects in both cell types, indicating the participation of Ca2+-dependent and -independent isoenzymes in normal and tumor cells, respectively. TH actions were blunted by extra- and intracellular Ca2+ blockers only in normal T lymphocytes, whereas NOS blockers impaired TH-induced proliferation in T lymphoma cells. Incubation for 24 h with TH induced a rise in total and membrane-associated PKC activities in both cell types and led to a rapid and transient effect only in tumor cells. THs increased atypical PKC-zeta expression in BW5147 cells and classical PKC isoenzymes in mitogen-stimulated normal T cells. TH augmented NOS activity and inducible NOS protein and gene expression only in tumor cells. Blockade of PKC and the atypical PKC-zeta isoform inhibited TH-mediated stimulation of inducible NOS and cell proliferation. These results show, for the first time, that differential intracellular signals are involved in TH modulation of lymphocyte physiology and pathophysiology.

Different mitogen-mediated Beta-adrenergic receptor modulation in murine T lymphocytes depending on the thyroid status.

OBJECTIVE: The aim of this work was to analyze beta-adrenergic receptor (betaAR) regulation of T-lymphocyte proliferation in mice according to different thyroid hormone statuses. METHODS: T cells from eu-, hypo- (by propylthiouracil treatment) and hyperthyroid (by thyroxine, T4 administration) mice were purified and specific radioligand binding assays were performed. The effects of the beta-agonist isoproterenol (ISO) on intracellular levels of cyclic AMP (cAMP) were determined. Mitogen-induced T-cell proliferation was measured by [(3)H]-thymidine incorporation. Finally, protein kinase C (PKC) activity in cytosol and membrane fractions were determined using radiolabelled enzymatic substrates. RESULTS: Adecrease or a non-significant increase in betaAR number was found on T lymphocytes from hypo- and hyperthyroid mice compared to euthyroid controls. ISO stimulation of cAMP levels was lower in hypothyroid and higher in hyperthyroid T lymphocytes compared to controls. T-selective mitogen-induced proliferation was increased in T4-treated animals, but decreased in hypothyroid mice. During the peak of proliferation, downregulation of betaAR was observed in all animals. However, a higher or a lower decrease was observed in hyper- and hypothyroid T cells, respectively. In parallel, a higher translocation of PKC activity was observed in hyperthyroid cells, and a lower one was found in hypothyroid lymphocytes with respect to controls. CONCLUSIONS: These results indicate that intracellular signals triggered by mitogen activation, namely PKC, would be related to differential betaAR downregulation in T lymphocytes depending on the thyroid hormone status, contributing to the distinct proliferative responses found in hypo- or hyperthyroidism compared to the euthyroid state.

Intra-assay total uncertainty of results in immunoassay techniques.

Uncertainty is a parameter associated with the result of a measurement; this parameter characterizes the dispersion of the values that could reasonably be attributed to the sample. Data processing methods do not take into account the influence of the imprecision and deviation of the experimental points of the calibration system and their impact on the final result of a sample analysis. The aim of this work is: (a) to propose, for each run, a simple method to calculate the uncertainty due to the calibration system (Uc); and (b) to present a method to determine the "intra-assay total uncertainty" (Ut) and evaluate its impact on the final result for an analyte. Ten replicates of standards, controls, and two serum-male and female samples were measured in the same run with a manual kit for determination of testosterone. To calculate Ut, random duplicate responses were selected. For controls and samples, Ut was affected by Uc (2.91% to 6.59%) and by the uncertainty of the measurement of the sample (Us) (1.01 to 8.73%); this allowed us to determine that Ut had values from 3.73% to 9.87%. While Us affects the result of a given sample, Uc affects the result of all the samples with a similar response (cpm). In the method proposed, Ut involves Us and Uc, both factors that introduce variations into the result of a sample by random causes. Intraassay total uncertainty includes the most probable result for the analytical methodology selected.

Inducible nitric oxide synthase-mediated proliferation of a T lymphoma cell line.

Nitric oxide (NO)-derived from T lymphocytes in an autocrine fashion can modulate events in the cell. However, the exact role of NO on the control of lymphocyte growth is controversial since both stimulation and inhibition have been demonstrated. Nitric oxide synthase (NOS) activity in normal and tumor T lymphocyte proliferation was studied here. Resting normal T lymphocytes displayed low levels of NOS activity that were slightly increased upon mitogenic stimulation. In contrast, BW5147 T lymphoma cells displayed higher basal levels than normal T lymphocytes that were significantly augmented when induced to proliferate. This activity was slightly modified in the presence of the calcium chelator EGTA and was blocked by competitive and irreversible NOS inhibitors, as well as by selective blockers of iNOS. Furthermore, tumor but not normal cell proliferation was impaired by NOS and iNOS blockers, while a calcium blocker only affected normal cell growth. iNOS expression, both at the protein and at the mRNA levels, was demonstrated on growing BW5147 cells but not on arrested tumor or normal lymphocytes. The contribution of iNOS to sustained proliferation of tumor cells is discussed.

Differential expression of protein kinase C isoenzymes related to high nitric oxide synthase activity in a T lymphoma cell line.

Protein kinase C (PKC) is critical for T lymphocyte activation and proliferation, while nitric oxide synthase (NOS) may function both as an activator or inhibitor of T cell apoptosis. Both enzymatic activities were studied in T lymphoma cells in comparison to normal and activated T lymphocytes. Here we show a higher translocation of PKC in BW5147 lymphoma cells than in mitogen-stimulated T lymphocytes. Tumor cells overexpressed PKC zeta isoform, while high levels of the PKC beta isotype were found in mitogen-stimulated T lymphocytes. Moreover, tumoral T cells showed high NOS activity, almost undetectable in normal or stimulated T lymphocytes. PKC and NOS inhibitors or the intracellular delivery of an anti-PKC zeta antibody diminished both NO production and proliferation in tumor cells. These results suggest that atypical PKC zeta isoform expression and its association with NOS activity regulation would participate in the multistep process leading to BW5147 cell malignant transformation.

Importancia del empleo de un patrón de 129I para la medición de 125I / Usefulness of a 129I for 125I measurement

En el presente trabajo se muestran las ventajas de la utilización de un patrón de 129I para comprobar diariamente que la respuesta del equipo de detección es constante para el 125I y poder determinar además la eficiencia para dicho nucleido. Una alternativa para conocer ese valor consiste en la aplicación de un método de coincidencia. La comparación de los resultados logrados con uno y otro método, demuestra que ambos valores de eficiencia son iguales. Por otra parte se analizan algunas cuestiones relacionadas con las ecuaciones teóricas utilizadas

Importancia del empleo de un patrón de 129I para la medición de 125I / Usefulness of a 129I for 125I measurement

En el presente trabajo se muestran las ventajas de la utilización de un patrón de 129I para comprobar diariamente que la respuesta del equipo de detección es constante para el 125I y poder determinar además la eficiencia para dicho nucleido. Una alternativa para conocer ese valor consiste en la aplicación de un método de coincidencia. La comparación de los resultados logrados con uno y otro método, demuestra que ambos valores de eficiencia son iguales. Por otra parte se analizan algunas cuestiones relacionadas con las ecuaciones teóricas utilizadas (AU)