Hormonal Regulation of the MHC Class I Gene in Thyroid Cells: Role of the Promoter "Tissue-Specific" Region.

In previous studies we have demonstrated that the expression of the Major Histocompatibility Complex (MHC) class I gene in thyrocytes is controlled by several hormones, growth factors, and drugs. These substances mainly act on two regions of the MHC class I promoter a "tissue-specific" region (-800 to -676 bp) and a "hormone/cytokines-sensitive" region (-500 to -68 bp). In a previous study, we have shown that the role of the "tissue-specific" region in the MHC class I gene expression is dominant compared to that of the "hormone/cytokines-sensitive" region. In the present report we further investigate the dominant role of the "tissue-specific" region evaluating the effect of thyroid stimulating hormone (TSH), methimazole (MMI), phenylmethimazole (C10), glucose and thymosin-α1. By performing experiments of electrophoretic mobility shift assays (EMSAs) we show that TSH, MMI and C10, which inhibit MHC class I expression, act on the "tissue-specific" region increasing the formation of a silencer complex. Glucose and thymosin-α1, which stimulate MHC class I expression, act decreasing the formation of this complex. We further show that the silencer complex is formed by two distinct members of the transcription factors families activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), c-jun and p65, respectively. These observations are important in order to understand the regulation of MHC class I gene expression in thyroid cells and its involvement in the development of thyroid autoimmunity.

Phenylmethimazole is a candidate drug for the treatment of severe forms of coronavirus disease 2019 (COVID-19) as well as other virus-induced "cytokines storm".

Severe forms of the Coronavirus disease 2019 (COVID-19) are characterized by an enhanced inflammatory syndrome called "cytokine storm" that produces an aberrant release of high amounts of cytokines, chemokines, and other proinflammatory mediators. The pathogenetic role of the "cytokine storm" has been confirmed by the efficacy of immunosuppressive drugs such as corticosteroids along with antiviral drugs in the treatment of the severe forms of this disease. Phenylmethimazole (C10) is a derivative of methimazole with anti-inflammatory properties. Studies performed both in vitro and in vivo have shown that C10 is able to block the production of multiple cytokines, chemokines, and other proinflammatory molecules involved in the pathogenesis of inflammation. Particularly, C10 is effective in reducing the increased secretion of cytokines in animal models of endotoxic shock. We hypothesize that these effects are not limited to the endotoxic shock, but can also be applied to any disease characterized by the presence of a "cytokine storm". Therefore, C10 may be a potential drug to be used alternatively or in association with the corticosteroids or other immunosuppressive agents in the severe forms of COVID-19 as well as other viral diseases that induce a "cytokine storm". Preclinical and clinical studies have to be performed to confirm this hypothesis.

Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of both metabolic and inflammatory diseases and has become the leading chronic liver disease worldwide. High-fat (HF) diets promote an increased uptake and storage of free fatty acids (FFAs) and triglycerides (TGs) in hepatocytes, which initiates steatosis and induces lipotoxicity, inflammation and insulin resistance. Activation and signaling of Toll-like receptor 4 (TLR4) by FFAs induces inflammation evident in NAFLD and insulin resistance. Currently, there are no effective treatments to specifically target inflammation associated with this disease. We have established the efficacy of phenylmethimazole (C10) to prevent lipopolysaccharide and palmitate-induced TLR4 signaling. Because TLR4 is a key mediator in pro-inflammatory responses, it is a potential therapeutic target for NAFLD. Here, we show that treatment with C10 inhibits HF diet-induced inflammation in both liver and mesenteric adipose tissue measured by a decrease in mRNA levels of pro-inflammatory cytokines. Additionally, C10 treatment improves glucose tolerance and hepatic steatosis despite the development of obesity due to HF diet feeding. Administration of C10 after 16 weeks of HF diet feeding reversed glucose intolerance, hepatic inflammation, and improved hepatic steatosis. Thus, our findings establish C10 as a potential therapeutic for the treatment of NAFLD.

Phenylmethimazole and a thiazole derivative of phenylmethimazole inhibit IL-6 expression by triple negative breast cancer cells.

Inhibition of interleukin-6 (IL-6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL-6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB-141. In the present work, we tested the hypothesis that C10 and COB-141 inhibit TNBC cell expressed IL-6 and investigated the potential for classical IL-6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB-141 inhibit MDA-MB-231 cell IL-6 secretion, with COB-141 being ~6.5 times more potent than C10. Therefore, the remainder of the study focused on COB-141 which inhibited IL-6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT-PCR), to inhibit IL-6 mRNA in the TNBC panel. COB-141 had little, if any, effect on metabolic activity indicating that the IL-6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT-PCR revealed that the TNBC cell lines do not express the IL-6 receptor (IL-6Rα). Trans-AM assays suggested that COB-141 exerts its inhibitory effect, at least in part, by reducing NF-κB (p65/p50) DNA binding. In summary, COB-141 is a potent inhibitor of TNBC cell expressed IL-6 and the inhibition does not appear to be due to non-specific toxicity. The TNBC cell lines do not have an intact classical IL-6 signaling pathway. COB-141's inhibitory effect may be due, at least in part, to reducing NF-κB (p65/p50) DNA binding.

Inhibition of LPS-induced TLR4 signaling products in murine macrophages by phenylmethimazole: an assay methodology for screening potential phenylmethimazole analogs.

Preclinical Research Phenylmethimazole (C10) is an inhibitor of Toll-like receptor (TLR3 and TLR4) expression and signaling. In this study, we carried out a detailed investigation of the effect of C10 on TLR4 and its molecular signaling products in RAW 264.7 macrophages using quantitative real-time polymerase chain reaction (PCR), ELISA and cell toxicity assays, a set of in vitro assays that may be used to screen future C10 analogs. C10 exhibited an inhibitory effect on TLR4 MyD88-dependent and MyD88-independent pathways. Within the TLR4 pathway, C10 inhibited the expression of cytokines, cytokine receptors, kinases, adapter molecules and transcription factors, suggesting a pathway-wide inhibitory effect. We also found that C10 dose-dependently inhibited the expression of TLR4 signaling products, specifically IL-6, inducible nitric oxide (NO) synthase and IFNß. Additionally, pre-treatment of RAW 264.7 cells with C10 resulted in protection from lipopolysaccharide (LPS) insults, suggesting C10 may be bound to the target thus exhibiting activity during/following LPS stimulation. Also, dimethyl sulfoxide, the solvent for C10 exhibited inhibitory effect on TLR4 signaling products independent from the effects of C10. Combined, this study enhances understanding of the actions of C10 on the TLR4 signaling pathway providing a path for the development of new C10 analogs for inhibiting TLR expression and signaling [corrected].

Phenylmethimazole suppresses dsRNA-induced cytotoxicity and inflammatory cytokines in murine pancreatic beta cells and blocks viral acceleration of type 1 diabetes in NOD mice.

Accumulating evidence supports a role for viruses in the pathogenesis of type 1 diabetes mellitus (T1DM). Activation of dsRNA-sensing pathways by viral dsRNA induces the production of inflammatory cytokines and chemokines that trigger beta cell apoptosis, insulitis, and autoimmune-mediated beta cell destruction. This study was designed to evaluate and describe potential protective effects of phenylmethimazole (C10), a small molecule which blocks dsRNA-mediated signaling, on preventing dsRNA activation of beta cell apoptosis and the inflammatory pathways important in the pathogenesis of T1DM. We first investigated the biological effects of C10, on dsRNA-treated pancreatic beta cells in culture. Cell viability assays, quantitative real-time PCR, and ELISAs were utilized to evaluate the effects of C10 on dsRNA-induced beta cell cytotoxicity and cytokine/chemokine production in murine pancreatic beta cells in culture. We found that C10 significantly impairs dsRNA-induced beta cell cytotoxicity and up-regulation of cytokines and chemokines involved in the pathogenesis of T1DM, which prompted us to evaluate C10 effects on viral acceleration of T1DM in NOD mice. C10 significantly inhibited viral acceleration of T1DM in NOD mice. These findings demonstrate that C10 (1) possesses novel beta cell protective activity which may have potential clinical relevance in T1DM and (2) may be a useful tool in achieving a better understanding of the role that dsRNA-mediated responses play in the pathogenesis of T1DM.

Phenylmethimazole blocks dsRNA-induced IRF3 nuclear translocation and homodimerization.

Previous studies revealed that phenylmethimazole (C10) inhibits IRF3 signaling, preventing dsRNA-induction of type 1 interferon gene expression, production, and downstream signaling. In the present study, we investigated the molecular basis for C10 inhibition of dsRNA-stimulated IRF3 signaling. IRF-3 Trans-AM assays were used to measure C10 effects on dsRNA induction of IRF3 DNA binding. Green fluorescent protein-labeled IRF3 was used to measure C10 effects on dsRNA-induced IRF3 nuclear translocation. Native PAGE, SDS PAGE, and western blotting were used to identify effects of C10 on IRF3 homodimer formation and phosphorylation, respectively. There was a significant impairment of dsRNA-induced IRF3 DNA binding activity in human embryonic kidney and pancreatic cancer cells with C10 treatment. C10 also blocked dsRNA-induced IRF3 nuclear translocation and homodimer formation without blocking serine 396 phosphorylation of IRF3. Together, these results indicate that C10 interferes with IRF3 signaling by blocking dsRNA-induced IRF3 homodimer formation, a prerequisite for nuclear translocation and DNA binding activities.

Phenylmethimazole inhibits production of proinflammatory mediators and is protective in an experimental model of endotoxic shock*.

BACKGROUND: One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. OBJECTIVE: In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. DESIGN: Experimental animal model. SETTING: University laboratory. SUBJECTS: Male C57BL/6J mice weighing 18-22 g. INTERVENTIONS: Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: : Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. CONCLUSIONS: Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.

Phenylmethimazole blocks palmitate-mediated induction of inflammatory cytokine pathways in 3T3L1 adipocytes and RAW 264.7 macrophages.

Visceral adipocytes and associated macrophages produce and release excessive amounts of biologically active inflammatory cytokines via the portal and systemic vascular system, which induce insulin resistance in insulin target tissues such as fat, liver, and muscle. Free fatty acids (FFAs) absorbed via the portal system or released from adipocytes also induce insulin resistance. In this report, we show that phenylmethimazole (C10) blocks basal IL6 and leptin production as well as basal Socs-3 expression in fully differentiated 3T3L1 cells (3T3L1 adipocytes) without affecting insulin-stimulated AKT signaling. In addition, C10 inhibits palmitate-induced IL6 and iNos up-regulation in both 3T3L1 adipocytes and RAW 264.7 macrophages, LPS-induced NF-κB and IFN-ß activation in 3T3L1 cells, and LPS-induced iNos, Ifn-ß, Il1ß, Cxcl10, and Il6 expression in RAW 264.7 macrophages. C10 also blocks palmitate-induced Socs-3 up-regulation and insulin receptor substrate-1 (IRS-1) serine 307 phosphorylation in 3T3L1 adipocytes. Additionally, we show for the first time that although palmitate increases IRS-1 serine 307 phosphorylation in 3T3L1 adipocytes, AKT serine 473 phosphorylation is enhanced, not reduced, by palmitate. These results suggest that through inhibition of FFA-mediated signaling in adipocytes and associated macrophages, as well as possibly other insulin target cells/tissues (i.e. non-immune cells), C10 might be efficacious to prevent or reverse cytokine-induced insulin resistance seen in obesity-related insulin resistance and type 2 diabetes mellitus.

Phenyl methimazole suppresses dextran sulfate sodium-induced murine colitis.

Ulcerative colitis is an autoimmune-inflammatory disease characterized by abnormally increased expression of Toll-like receptor-4 (TLR4) in colonic epithelial cells, increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, IL-12), chemokines (e.g., IP-10), and endothelial cell adhesion molecules (e.g., VCAM-1), plus enhanced leukocyte infiltration into colonic interstitium. Previously, we have shown that phenyl methimazole (C10) markedly decreases virally-induced TLR-3 expression and signaling and potently inhibits both TNF-alpha-induced VCAM-1 expression and the resultant leukocyte-endothelial cell adhesion. In this study we probed the hypothesis that C10 is efficacious in a TLR-4- and VCAM-1-associated murine model [the dextran sulfate sodium (DSS) model] of human colitis. C10 was administered intraperitoneally coincident with or after DSS treatment was initiated. Macroscopic colon observations revealed that C10 significantly reversed DSS-induced shortening of the colon (P<0.05) and reduced the presence of blood in the colon. Histological analyses of colonic tissues revealed that C10 distinctly attenuated both DSS-induced edema as well as leukocyte infiltration in the colonic mucosa and resulted in pronounced protection against DSS-induced crypt damage (P<0.001). Northern blot analyses and immunohistochemistry of colonic tissue revealed that C10 markedly diminished DSS-induced expression of pertinent inflammatory mediators: TNF-alpha, IL-1beta, IL-6, IL-12, IP-10, TLR-4 and VCAM-1. Most importantly, C10 significantly improved survival and protected mice against DSS-induced colitic-death: 75% by comparison to 12.5% with identical treatment with DMSO-control (log rank test: P=0.005). These results provide direct evidence that C10 suppresses DSS-induced colitis by inhibiting expression of key inflammatory mediators and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.

Regulation of major histocompatibility complex gene expression in thyroid epithelial cells by methimazole and phenylmethimazole.

Increased expression of major histocompatibility complex (MHC) class-I genes and aberrant expression of MHC class-II genes in thyroid epithelial cells (TECs) are associated with autoimmune thyroid diseases. Previous studies have shown that methimazole (MMI) reduces MHC class-I expression and inhibits interferon-gamma (IFN-gamma or IFNG as listed in the MGI Database)-induced expression of the MHC class-II genes in TECs. The action of MMI on the MHC class-I genes is transcriptional, but its mechanism has not been investigated previously. In the present study, we show that in Fisher rat thyroid cell line 5 cells, the ability of MMI and its novel derivative phenylmethimazole (C10) to decrease MHC class-I promoter activity is similar to TSH/cAMP suppression of MHC class-I and TSH receptor genes, and involves a 39 bp silencer containing a cAMP response element (CRE)-like site. Furthermore, we show that C10 decreases MHC class-I gene expression to a greater extent than MMI and at 10- to 50-fold lower concentrations. C10 also reduces the IFN-gamma-induced increase in the expression of MHC class-I and MHC class-II genes more effectively than MMI. Finally, we show that in comparison to MMI, C10 is a better inhibitor of specific protein-DNA complexes that are formed with a CRE-like element on the MHC class-II promoter. These data support the conclusion that the immunosuppressive mechanism by which MMI and C10 inhibit MHC gene expression mimics 'normal' hormonal suppression by TSH/cAMP.

Phenylmethimazole decreases Toll-like receptor 3 and noncanonical Wnt5a expression in pancreatic cancer and melanoma together with tumor cell growth and migration.

PURPOSE: To evaluate whether (a) Wnt5a expression in pancreatic cancer and malignant melanoma cells might be associated with constitutive levels of Toll-like receptor 3 (TLR3) and/or TLR3 signaling; (b) phenylmethimazole (C10), a novel TLR signaling inhibitor, could decrease constitutive Wnt5a and TLR3 levels together with cell growth and migration; and (c) the efficacy of C10 as a potential inhibitor of pancreatic cancer and malignant melanoma cell growth in vivo. EXPERIMENTAL DESIGN: We used a variety of molecular biology techniques including but not limited to PCR, Western blotting, and ELISA to evaluate the presence of constitutively activated TLR3/Wnt5a expression and signaling. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based technology and scratch assays were used to evaluate inhibition of cell growth and migration, respectively. TLR3 regulation of cell growth was confirmed using small interfering RNA technology. Nude and severe combined immunodeficient mice were implanted with human pancreatic cancer and/or melanoma cells and the effects of C10 on tumor growth were evaluated. RESULTS: We show that constitutive TLR3 expression is associated with constitutive Wnt5a in human pancreatic cancer and malignant melanoma cell lines, that C10 can decrease constitutive TLR3/Wnt5a expression and signaling, suggesting that they are interrelated signal systems, and that C10 inhibits growth and migration in both of these cancer cell lines. We also report that C10 is effective at inhibiting human pancreatic cancer and malignant melanoma tumor growth in vivo in nude or severe combined immunodeficient mice and associate this with inhibition of signal transducers and activators of transcription 3 activation. CONCLUSIONS: C10 may have potential therapeutic applicability in pancreatic cancer and malignant melanoma.

Selenium analogues of antithyroid drugs--recent developments.

Thyroxine (T4), the main secretory hormone of the thyroid gland, is produced on thyroglobulin by thyroid peroxidase (TPO)/H(2)O(2)/iodide system and deiodinated to its active form (T3) by a selenocysteine-containing enzyme, iodothyronine deiodinase (ID). The activation of thyroid-stimulating hormone (TSH) receptor by auto-antibodies leads to 'hyperthyroidism', a life-threatening disease which is treated by antithyroid drugs such as 6-propyl-2-thiouracil (PTU) and methimazole (MMI). The present review describes the biological activities of a number of S/Se derivatives bearing the methimazole pharmacophore. It is shown that the isosteric substitutions in the existing drugs lead to compounds that can effectively and reversibly inhibit the heme-containing lactoperoxidase (LPO). In contrast to methimazole, the selenium analogue, MSeI, does not interfere with the enzyme directly, but it inhibits LPO by reducing the H(2)O(2) that is required for the oxidation of the Fe-center in LPO. These studies reveal that the degradation of the intracellular H(2)O(2) by the Se analogues of antithyroid drugs may be beneficial to the thyroid gland, as these compounds may act as antioxidants and protect thyroid cells from oxidative damage. Because the drugs with an action essentially on H(2)O(2) can reversibly inhibit the thyroid peroxidase, such drugs could be of great importance in the treatment of hyperthyroidism.

High basal levels of functional toll-like receptor 3 (TLR3) and noncanonical Wnt5a are expressed in papillary thyroid cancer and are coordinately decreased by phenylmethimazole together with cell proliferation and migration.

High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation.

Anti-thyroid drugs and thyroid hormone synthesis: effect of methimazole derivatives on peroxidase-catalyzed reactions.

Syntheses and characterization of the selenium analogue (MSeI) of anti-thyroid drug methimazole and a series of organoselenium compounds bearing N-methylimidazole pharmacophore are described. In contrast to the sulfur compound that exists predominantly in its thione form, the selenium analogue exists in a selenol form, which spontaneously oxidizes in air to produce the corresponding diselenide. The reduction of the diselenide by GSH or NaBH(4) affords the biologically active selenol, which effectively inhibits the lactoperoxidase (LPO) activity in vitro. The monoselenides having N-methylimidazole moiety are found to be much less active than the selenol, suggesting that the presence of a selenol moiety is important for the LPO inhibition. The kinetic and mechanistic studies reveal that MSeI inhibits the LPO activity by reducing the H(2)O(2), providing a novel method to reversibly inhibit the enzyme. Although MSeI strongly inhibits LPO, the enzyme's activity can be completely recovered by increasing the H(2)O(2) concentration. On the other hand, the inhibition by methimazole (MMI), the sulfur analogue, cannot be reversed by increasing the H(2)O(2) concentration, leading to a complete inactivation of the enzyme. The reversible inhibition of LPO by some of the selenium derivatives is correlated with their glutathione peroxidase (GPx) activity, and the high GPx activity of the selenium compounds as compared with their sulfur analogues suggests that the selenium derivatives may protect the thyroid gland from oxidative damage.

Thyrocytes express a functional toll-like receptor 3: overexpression can be induced by viral infection and reversed by phenylmethimazole and is associated with Hashimoto's autoimmune thyroiditis.

Toll-like receptors (TLRs) initiate an innate immune response. TLR3 on dendritic cells recognize double-stranded (ds) RNA and then signal increases in cytokines and recognition molecules important for immune cell interactions. In this report, we demonstrate TLR3 mRNA and protein are expressed on Fisher rat thyroid cell line-5 (FRTL-5) thyroid cells and are functional because incubating cells with polyinosine-polycytidylic acid causes 1) transcriptional activation of both the nuclear factor kappaB (NF-kappaB)/Elk1 and interferon (IFN) regulatory factor-3/IFN-beta signal paths, 2) posttranscriptional activation of NF-kappaB and ERK1/2, and 3) increased IFN-beta mRNA. TLR3 can be overexpressed, along with dsRNA-dependent protein kinase, major histocompatibility complex-I or II, and IFN regulatory factor-1, by transfecting dsRNA into the cells, infection with Influenza A virus, or incubation with IFN-beta, but not by incubation with dsRNA or IFNgamma, or by dsDNA transfection. A methimazole (MMI) derivative, phenylmethimazole, to a significantly greater degree than MMI, prevents overexpression by inhibiting increased transcriptional activation of IRF-3 and of IFN-stimulated response elements, phosphorylation of signal transducers and activation of transcription (STAT-1), but not NF-kappaB activation. TLR3 can be functionally overexpressed in cultured human thyrocytes by dsRNA transfection or IFN-beta treatment. Immunohistochemical studies show that TLR3 protein is overexpressed in human thyrocytes surrounded by immune cells in 100% of patients with Hashimoto's thyroiditis examined, but not in normal or Graves' thyrocytes. We conclude that functional TLR3 are present on thyrocytes; TLR3 downstream signals can be overexpressed by pathogen-related stimuli; overexpression can be reversed by phenylmethimazole to a significantly greater extent than MMI by inhibiting only the IFN regulatory factor-3/IFN-beta/signal transducers and activation of transcription arm of the TLR3 signal system; and TLR3 overexpression can induce an innate immune response in thyrocytes, which may be important in the pathogenesis of Hashimoto's thyroiditis and in the immune cell infiltrates.

Phenyl methimazole inhibits TNF-alpha-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and reduces monocytic cell adhesion to endothelial cells.

Proinflammatory cytokine (e.g., TNF-alpha)-induced expression of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of the vascular endothelium and a consequent increase in leukocyte adhesion are key aspects of pathological inflammation. A promising therapeutic approach to diminish aberrant leukocyte adhesion is, therefore, to inhibit cytokine-induced ECAM expression at the transcription level. Several studies suggest that methimazole, a compound used clinically to treat autoimmune diseases, such as Graves' disease, may also diminish pathological inflammation by suppressing ECAM expression. In this study we probed the hypothesis that a derivative of methimazole, phenyl methimazole (compound 10), can reduce cytokine-induced ECAM expression and consequent leukocyte adhesion. We found that compound 10 1) dramatically inhibits TNF-alpha-induced VCAM-1 mRNA and protein expression in human aortic endothelial cells (HAEC), has a relatively modest inhibitory effect on TNF-alpha induced E-selectin expression and has no effect on ICAM-1 expression; 2) significantly reduces TNF-alpha-induced monocytic (U937) cell adhesion to HAEC under in vitro flow conditions similar to that present in vivo; 3) inhibits TNF-alpha-induced IFN regulatory factor-1 binding to VCAM-1 promoter; and 4) reduces TNF-alpha-induced IRF-1 expression in HAEC. Combined, the results indicate that phenyl methimazole can reduce TNF-alpha-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and that this contributes significantly to reduced monocytic cell adhesion to TNF-alpha-activated HAEC.

Biomimetic studies on anti-thyroid drugs and thyroid hormone synthesis.

The selenium analogues of anti-thyroid drugs exhibit their anti-thyroid action by a mechanism different from that of MMI. The selenium analogue of MMI and related selenium compounds exhibit high GPx activity, providing a novel method for the reversible inhibition of thyroid hormone biosynthesis.

Subchronic toxicity study in rats with 1-methyl-3-propylimidazole-2-thione (PTI): effects on the thyroid.

A 90-day gavage study was performed to evaluate the subchronic toxicity of 1-methyl-3-propylimidazole-2-thione (PTI) when administered to Crl:CD BR rats. PTI is a chemical catalyst and is structurally similar to the thioureas, which are known to adversely affect the thyroid. Therefore, this study was designed to investigate the effects of PTI on the thyroid. Male and female rats were dosed with 0, 5, 10, 25, or 75 mgPTI/kg/day for 13 weeks. Clinical pathology examinations and pathology examination were performed and the following were measured periodically: serum T3, T4, and TSH, hepatic UDP-glucuronyltransferase activity, and cell proliferation of the thyroid and liver. Under the conditions of this study, the overall no-observed-adverse-effect level (NOAEL) for the subchronic effects of PTI in male and female rats was 10 mg PTI/kg/day. The NOAEL was based on the effects on the thyroid gland in male and female rats dosed with 25 and 75 mg PTI/kg/day, as well as the hepatic centrilobular fatty change, increased severity of chronic progressive nephropathy, fatty change in the adrenal medulla, and the substantial reduction in body weight and body weight gain. The primary target organs were the thyroid and liver. Alterations in thyroid hormones (T3, T4, and TSH) occurred predominantly at 25 and 75 mg/kg/day. Toxicologically significant alterations in T3, T4, and TSH levels, cell proliferation, and UDP-glucuronyltransferase activity occurred in rats dosed with 25 and 75 mg/kg/day, which correlated with organ weight and histopathological effects. Additionally, the effect of PTI on thyroid peroxidase activity, a key step in thyroid hormone synthesis, was evaluated in vitro using microswine thyroid microsomes. PTI was shown to inhibit thyroid peroxidase, with an IC50 of 0.02 M. These data suggest that PTI enhances the excretion of T4 via induction of glucuronyltransferase and inhibits thyroid hormone synthesis via a direct affect on thyroid peroxidase. Both of these effects contribute to the disruption of the hypothalamic-pituitary-thyroid axis and result in sustained elevation of TSH and the corresponding thyroid hypertrophy and hyperplasia.