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Stimulation of type I Interferon system by double-stranded RNA in cultured human thyroid follicles

ABSTRACT: Although viral infection is thought to be associated with subacute thyroiditis and probably with autoimmune thyroid disease, possible changes in thyroid function during the prodromal period of infection or subclinical infection remain largely unknown. Recently, it was shown that pathogen-associated molecular patterns stimulate Toll-like receptors (TLR) and activate innate immune responses by producing type I interferons (IFN). Using a human thyroid follicle culture system, in which de novo synthesized thyroid hormones are released into the culture medium under physiological concentrations of hTSH, we studied the effects of polyinosinic-polycytidylic acid (Poly(I:C)), a chemical analog of viral double-stranded RNA (dsRNA), on TSH-induced thyroid function. Thyrocytes expressed ligands for dsRNA (TLR 3, CD14, and RIG-1) comparable to the TSH receptor. DNA microarray and real-time PCR analyses revealed that dsRNA increased the expression of mRNA for TLR3, IFN-g, interferon-regulating factors, proinflammatory cytokines, and class I MHC, whereas genes associated with thyroid hormonogenesis (NIS, peroxidase, deiodinases) were suppressed. In accordance to these data, Poly(I:C) suppressed TSH-induced 125I uptake and hormone synthesis dose-dependently, accompanied by a decrease in the ratio of 125I-T3/125I-T4 released into the culture medium, whereas peptidoglycan, lipopolysaccharides, or unmethylated CpG DNA, ligands for TLR2, TLR4, and TLR9, respectively, had no significant effect. These inhibitory effects of Poly(I:C) were not blocked by a neutralizing antibody against TLR3 and an anti-IFN alpha/beta receptor antibody. These in vitro findings suggest that when thyrocytes are infected with certain viruses, dsRNA formed intracellularly in thyrocytes may be a cause for thyroid dysfunction, leading to development of autoimmune thyroiditis. This data was published in Endocrinology, vol.148, 3226-3235, 2007. Keywords: Cultured human thyroid follicles

ORGANISM(S): Homo sapiens

PROVIDER: GSE12154 | GEO | 2008/07/18

SECONDARY ACCESSION(S): PRJNA113771

REPOSITORIES: GEO

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Project description:Although viral infection is thought to be associated with subacute thyroiditis and probably with autoimmune thyroid disease, possible changes in thyroid function during the prodromal period of infection or subclinical infection remain largely unknown. Recently, it was shown that pathogen-associated molecular patterns stimulate Toll-like receptors (TLR) and activate innate immune responses by producing type I interferons (IFN). Using a human thyroid follicle culture system, in which de novo synthesized thyroid hormones are released into the culture medium under physiological concentrations of hTSH, we studied the effects of polyinosinic-polycytidylic acid (Poly(I:C)), a chemical analog of viral double-stranded RNA (dsRNA), on TSH-induced thyroid function. Thyrocytes expressed ligands for dsRNA (TLR 3, CD14, and RIG-1) comparable to the TSH receptor. DNA microarray and real-time PCR analyses revealed that dsRNA increased the expression of mRNA for TLR3, IFN-g, interferon-regulating factors, proinflammatory cytokines, and class I MHC, whereas genes associated with thyroid hormonogenesis (NIS, peroxidase, deiodinases) were suppressed. In accordance to these data, Poly(I:C) suppressed TSH-induced 125I uptake and hormone synthesis dose-dependently, accompanied by a decrease in the ratio of 125I-T3/125I-T4 released into the culture medium, whereas peptidoglycan, lipopolysaccharides, or unmethylated CpG DNA, ligands for TLR2, TLR4, and TLR9, respectively, had no significant effect. These inhibitory effects of Poly(I:C) were not blocked by a neutralizing antibody against TLR3 and an anti-IFN alpha/beta receptor antibody. These in vitro findings suggest that when thyrocytes are infected with certain viruses, dsRNA formed intracellularly in thyrocytes may be a cause for thyroid dysfunction, leading to development of autoimmune thyroiditis. This data was published in Endocrinology, vol.148, 3226-3235, 2007. Experiment Overall Design: Two conditioned experimets, control vs. double-stranded RNA (polyI-C), cultured for 6

Project description:Pro-inflammation triggered by microbial lipopolysaccharide (LPS) through Toll-like receptor (TLR) 4 in the presence of interferon (IFN)-g induces cytokine secretion in dendritic cells (DCs) tightly regulated by a defined differentiation program. This DC differentiation is characterized by a dynamic immune activating but also tolerance inducing phenotype associated with irreversible down-modulation of cytokines. CD40L on activated T cells further modifies DC differentiation. Using DNA micro arrays we showed down-regulated mRNA levels of TLR signaling molecules while CD40/CD40L signaling molecules were up-regulated at a time when LPS/IFN-g activated DCs have ceased cytokine expression. Accordingly we demonstrated that CD40/CD40L but not TLR4 or TLR3 signaling mediated by LPS or poly (cytidylic-inosinic) acid (poly I:C) and dsRNA re-established the capacity to secret interleukin (IL)-12 in LPS/IFN-g activated DCs, which have exhausted their potential for cytokine secretion. This resulting TH1 polarizing DC phenotype â which lacked accompanying secretion of the crucial immune suppressive IL-10 - enhanced activation of cytotoxic T lymphocytes (CTLs). We therefore conclude that immune modulation is restricted to a secondary T-cell mediated stimulus at an exhausted DC state which prevents an immune tolerant DC phenotype. These findings impacts on the rational design of TLR activated DC-based cancer vaccines for the induction of anti-tumoral CTL responses. Experiment Overall Design: Monocyte derived DCs were matured for 6 hours with 30 ng/ml lipopolysaccharide (LPS, E. coli strain O111:B4, Calbiochem, San Diego, CA) and 1.000 U/ml IFN-g (Imukin, Boehringer Ingelheim, Austria). RNA of DCs was isolated after 6, 12, 24, or 48 hours of maturation using the RNeasy kit (Qiagen, Hilden, Germany) and analyzed by the RZPD (German Resource Centre for Genome Research, Berlin) using the Affymetrix DNA micro array platform (U133 plus 2.0). DNA array data from different maturation-time experiments were compared to controls that were kept in culture for the same time without stimulation.

Project description:Amiodarone reversibly decreases sodium-iodide symporter mRNA expression at therapeutic concentrations and induces antioxidant responses at supraphysiological concentrations in cultured human thyroid follicles Amiodarone, a potent antiarrhythmic agent, is a highly active oxidant, exerting cytotoxic effects on thyrocytes at pharmacological concentrations. Patients receiving amiodarone usually remain euthyroid, but occasionally develop thyroid dysfunction, such as amiodarone-associated hypothyroidism or amiodarone-induced thyrotoxicosis. To elucidate the mechanism by which amiodarone elicits thyroid dysfunction, human thyroid follicles were cultured with TSH and amiodarone at therapeutic (1-2 microM) and pharmacological (10-20microM) concentrations, and the drug-induced effect on whole human gene expression was analyzed by cDNA microarray. Amiodarone at 1-2microM decreased the expression level of the sodium-iodide symporter (NIS) to nearly half, but did not affect genes participating in thyroid hormonogenesis (thyroid peroxidase, thyroglobulin, pendrin, NADPH oxidase). Higher concentrations (10-20 microM) decreased the expression of all these genes, accompanied by increased expression of antioxidant proteins such as heme oxygenase 1 and ferritin. When thyroid follicles obtained from a patient with Graves’ disease who had been treated with amiodarone for 2 months before thyroidectomy were cultured in amiodarone-free medium, TSH-induced thyroid function was intact, suggesting that amiodarone at a maintenance dose did not elicit any cytotoxic effect on thyrocytes. The ultrastructural features of cultured thyroid follicles were compatible with these in vitro findings. These in vitro and ex vivo findings suggest that patients taking maintenance doses of amiodarone usually remain euthyroid, probably due to escape from the Wolff-Chaikoff effect mediated by decreased expression of NIS mRNA. Furthermore, amiodarone is not cytotoxic for thyrocytes at therapeutic concentrations but elicits cytotoxicity through oxidant activity at supra-physiological concentrations. Keywords: Cultured human thyroid follicles

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Stimulation of type I Interferon system by double-stranded RNA in cultured human thyroid follicles

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