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Suppression of Iodide Uptake and Thyroid Hormone Synthesis with Stimulation of the Type I Interferon System by Double-Stranded Ribonucleic Acid in Cultured Human Thyroid Follicles

Thyroid Disease Institute (K.Y., E.Y., T.Y.), Kanaji Hospital, Tokyo 114-0015, Japan; Department of Bioregulation (Ko.S.), National Institute of Infectious Diseases, Tokyo 189-0002, Japan; Department of Molecular Biodefense Research (F.T.), Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; Department of Microbiology and Immunology (M.M.), Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan; Saitama Medical School (T.M.), Saitama 350-3469, Japan; and Department of Surgery (K.Y., T.O.), and Department of Medicine (K.T., Ka.S.), Institute of Clinical Endocrinology, Tokyo Women’s Medical University, Tokyo 162-8666, Japan

Address all correspondence and requests for reprints to: Kanji Sato, M.D., Ph.D., Institute of Clinical Endocrinology, Tokyo Women’s Medical University, Kawada-cho 8-1, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: satokan{at}attglobal.net.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 human TSH, 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 retinoic-acid-inducible protein-1) comparable with the TSH receptor. DNA microarray and real-time PCR analyses revealed that dsRNA increased the expression of mRNA for TLR3, IFN-ß, IFN-regulating factors, proinflammatory cytokines, and class I major histocompatibility complex (MHC), whereas genes associated with thyroid hormonogenesis (sodium/iodide symporter, 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 /ß 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 (1, 2). It was suggested that viral infection and/or tissue damage result in release of double-stranded DNA (dsDNA), which in turn enhances antigen presentation in the thyroid (3). Recently, it was shown that pathogen-associated molecular patterns (PAMPs) stimulate Toll-like receptors (TLRs) and activate innate immune responses by producing type I interferons (IFN) (4, 5, 6). Type I IFNs (both IFN-ß and multiple IFN- molecules) are the key cytokines produced after viral infection that mediate induction of both the innate immune response and subsequent development of adaptive immunity to viruses (4, 5, 6). Type I IFNs reversibly inhibit iodine incorporation and thyroid hormone release in human thyroid follicles (7) and confluent monolayers by inhibiting the thyroid peroxidase (TPO), sodium/iodide symporter (NIS), and thyroglobulin genes (8). TLR activation by PAMPs is also associated with production of proinflammatory cytokines, such as IL-1, TNF-, and IL-6, which modulate thyroid hormonogenesis in vitro (9, 10, 11). A recent prospective study of hepatitis C virus-positive patients treated with IFN- has suggested that both the innate and acquired immune system are involved in autoimmune thyroiditis (12).

Among more than 10 members of the TLR family identified in mammals (5, 6), TLR1, TLR2, TLR6, and TLR4 are involved in the recognition of bacteria, whereas TLR3, TLR7/TLR8, and TLR9 are involved in the recognition of double-stranded RNA(dsRNA), single-stranded RNA (ssRNA), and unmethylated CpG DNA, respectively (6, 13, 14). dsRNA can be generated during viral infection as a replication intermediate for ssRNA viruses or as a by-product of symmetrical transcription in DNA viruses (15). A synthetic analog of viral dsRNA, polyinosinic-polycytidylic acid [Poly(I:C)], is also recognized by TLR3, and has been used extensively to mimic viral infection (6). TLR3 is expressed not only in immunocompetent cells, but also in various epithelial cells, such as human respiratory cells, intestinal cells, and rat thyroid-like cells (16, 17, 18). Very recently, it was demonstrated that dsRNA-mediated TLR3 activation is enhanced by CD14, a ligand for endotoxin and TLR3 (19). Furthermore, dsRNA synthesized in the cytoplasm is recognized intracellularly by cytoplasmic helicase proteins, such as retinoic-acid-inducible protein-1 (RIG-1) and melanoma differentiation-associated gene 5 (MDA5) (5, 6, 20).

Using suspension-cultured human thyroid follicles, which release de novo synthesized thyroid hormones into the culture medium in the presence of physiological concentrations of human TSH (21), we studied the effects of Poly(I:C) on TSH-induced thyroid function. Because stimulation of the type I IFN system activates a number of genes (5, 6), we studied the effects of Poly(I:C) on human thyrocytes using an oligo-DNA microarray in which the whole human genome can be analyzed in a single run (22).

	Materials and Methods

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Reagents
Lipopolysaccharide (LPS) from Salmonella Minnesota Re-595 was purchased from Sigma (St. Louis, MO). Poly(I:C) and CpG oligodeoxynucleotide were purchased from Hokkaido System Science (Hokkaido, Japan). Anti-IFN/ß receptor antibody was obtained from PBL Biological Laboratories (Piscataway, NJ). Antihuman TLR3 monoclonal antibody (Anti-TLR3-Ab) was prepared as described previously (23), and a lot containing the same neutralizing activity was used (23).

Thyroid follicles in suspension culture
This study was approved by the Ethics Committee of Tokyo Women’s Medical University. Informed consent was obtained from all patients with Graves’ disease before subtotal thyroidectomy. Human thyroid follicles were cultured as reported previously (24). In brief, thyroid tissue (15–30 g) obtained by subtotal thyroidectomy from patients with Graves’ disease was minced with scissors into small pieces (3 x 3 x 3 mm). The dissected thyroid tissue was digested with 0.3 mg/ml collagenase (type IV; Worthington Biochemical Corp., Lakewood, NJ) and 5 mg/ml dispase (Godo Shusei Co., Tokyo, Japan) in Hanks’ balanced salt solution at 32 C for 30 min. The digested material was filtered through nylon mesh (80 mesh), and the undigested tissue fragments were processed in the same manner once more. The second filtrate containing thyroid follicles was centrifuged at 70 x g for 5 min, and the pellet was washed three times with F-12/RPMI-1640 medium until the color of the pellet became white.

To investigate the effect of Poly(I:C) on gene expression, thyroid follicles were resuspended in F-12/RPMI-1640 medium containing NaI (10^–8 M) and 0.5% fetal calf serum (about 2000–3000 thyroid follicles/ml), and about 15 ml were added to 10-cm culture dishes, the bottom of which had been coated with agarose (7, 8, 10). After culturing for 4–5 d, bovine TSH (bTSH) was added to a final concentration of 30 µU/ml, and cultured for additional 1 d, then Poly(I:C) was added to a final concentration of 25 µg/ml. After an additional 6 or 24 h culture, total RNA was prepared from thyroid follicles treated with or without Poly(I:C).

RNA extraction
Total RNA was extracted from thyroid follicles using the RNeasy Minikit (QIAGEN, Tokyo, Japan) according to the manufacturer’s suggested protocol (22, 25). Concentration and purity were determined using a GeneQuant spectrophotometer (Amersham Pharmacia Biotech, Cambridge, UK). The quality of the RNA was checked by electrophoresis of 1-µg samples in 1.0% formaldehyde agarose gel, followed by staining with ethidium bromide. The 28S and 18S rRNA bands were examined on a UV transilluminator. No significant degradation was observed in any of the RNA samples.

Oligo-DNA microarray
After the RNA had been extracted, oligo-DNA microarray was performed as described previously (22). In brief, RNA was converted to cDNA using a Human cDNA System I Direct Kit (NEN Life Science Products, Boston, MA). cDNA obtained from thyroid follicles cultured in the control and treated media was labeled with Cy3 (green, control) and Cy5 [red, Poly(I:C)], respectively, and the expression levels of 41000 gene spots were analyzed using cDNA microarray (Whole Human Genome Oligo Microarray Kit, Product No. G4112A; Agilent Technologies, Palo Alto, CA).

Laser detection of the Cy3 and Cy5 signals on the microarray was performed with a nonconfocal laser leader, Gene PX 4000A (Axon Instruments, Union, CA). Fluorescence signal intensities and the Cy5/Cy3 ratios for each of the 41000 oligo-DNAs were analyzed using the Gene PixPro 30 software package (Axon Instruments).

Real-time RT-PCR
Total RNA (250 ng) was reverse-transcribed in a total reaction volume of 100 µl using a high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) in accordance with the manufacturer’s instructions. cDNA was synthesized using a thermal cycler (program temperature control system PC-7000; ASTEK, Fukuoka, Japan) by incubation at 25 C for 10 min, and at 37 C for 120 min. Single-stranded cDNA products were then analyzed by real-time PCR using the TaqMan Gene Expression Assay in accordance with the manufacturer’s instructions (Applied Biosystems). Single-stranded cDNA products were analyzed using an ABI PRISM 7700 Sequence Detector (Applied Biosystems). Twelve assays were performed: TLR-2 (Hs00152932-m1), TLR-3 (Hs00152933-m1), TLR-4 (Hs00152937-m1), TLR-9 (Hs00152973-m1), IFN- (Hs00256882-s1), IFN-ß (Hs00277188-s1), RIG-1 (Hs00204833-m1), MDA5 (Hs00223420-m1), thyroglobulin (Hs 00174974-m1), peroxidase (Hs00174927-m1), type I deiodinase (Hs00174944-m1), and type II deiodinase (Hs00255341-m1), using 18S rRNA (Hs99999901-s1) as a control. The PCR thermal cycle conditions were set at 50 C for 2 min and 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. In each case, triplicate threshold cycle (Ct) values were obtained and averaged, and the expression levels were then evaluated using a relative quantification method. The fold change in target genes was normalized to the 18S rRNA (reference gene) and compared with the control (calibrator) sample, using the formula: Fold change = 2^–Ct, where Ct = (Ct-target – Ct-reference)sample-n – (Ct-target – Ct-reference)calibrator-sample. Sample-n corresponds to any sample for the target gene normalized to the reference gene, and calibrator-sample represents the expression level (1x) of the target gene normalized to the reference gene.

Effects of PAMPs on TSH-induced thyroid function
To investigate the effects of PAMPs on TSH-induced thyroid function, follicles were resuspended in the supplemented F-12/RPMI-1640 medium, which was a 1:1 mixture of F-12 and RPMI-1640 medium supplemented with 2 mg/ml BSA, bovine insulin (5 µg/ml), hydrocortisone (10^–8 M), bovine transferrin (5 µg/ml) and NaI (10^–8 M). One milliliter of follicular suspension (containing 200–300 follicles) was cultured in the presence of 30 µU/ml TSH in a humidified atmosphere of 5% CO₂ and 95% air at 37 C in 24 multiwell dishes the bottom of which had been coated with agarose (7, 8, 10).

After 5 d of culture, the culture medium was changed to supplemented F-12/RPMI-1640 medium containing 30 µU/ml bTSH and various concentrations of LPS, peptidoglycans, Poly(I:C), PolyC, or Poly(dI:dC), and cultured for 1 h. Then, 1.5–2.0 x 10⁴ cpm ¹²⁵I was added. After an additional 3 d of culture, the medium and the thyroid follicles were transferred to glass tubes each with a sharp-pointed bottom (12 x 75 mm, made to order by Sugiyama-gen, Tokyo, Japan) and centrifuged at 2000 x g for 10 min. The conditioned medium was transferred to another standard-type glass tube, and then 100 µl of fetal bovine serum and 200 µl of 50% trichloroacetic acid (TCA) were added. After a through mixing, the tubes were centrifuged, and the TCA-precipitates were washed once with 5% TCA. To measure iodide incorporation into follicles, the tubes containing thyroid follicles were gently washed with 1 ml Hanks’ solution. The radioactivity in the pellets (organic ¹²⁵I released) and the follicles (¹²⁵I incorporated) was counted in a -spectrometer (Aloka, Tokyo, Japan). All assays were performed in triplicate or quadruplicate.

To study whether it is possible to prevent dsRNA-induced thyroid dysfunction using Anti-TLR3-Ab, thyroid follicles were preincubated with the antibody at 5 and 20 µg/ml as reported elsewhere (23). After a 1-h incubation, dsRNA was added at a final concentration of 1 µg/ml, and then ¹²⁵I was added. After an additional 3 d of culture, ¹²⁵I incorporated into the thyroid follicles and organic ¹²⁵I secreted into the culture medium were counted.

Analysis of ¹²⁵I-labeled metabolites by thin-layer chromatography
After thyroid follicles had been cultured in the medium containing various concentrations of Poly(I:C) and bTSH (50 µU/ml) for 3 d, they and the conditioned medium were stored at –20 C. The thyroid follicles were then treated with proteinase A at room temperature overnight. After lyophilization of the samples, ¹²⁵I-metabolites in the thyroid follicles and conditioned medium were extracted with butanol, and analyzed by thin-layer chromatography as described previously (26).

Determination of IFN- and -ß concentrations in the conditioned medium
Culture media were centrifuged to remove cell debris, and the supernatants were stored at –80 C until assay. The levels of IFNs secreted into the culture medium were determined with ELISA kits for human IFN- (Funakoshi TBL, Tokyo, Japan) and human IFN-ß (Torei, Tokyo, Japan) in accordance to the manufacturers’ protocols. The sensitivities for IFN- and -ß were less than 12.5 pg/ml and 2.5 pg/ml, respectively.

Immunohistochemistry
Graves’ thyroid tissues were fixed in 10% neutral buffered formalin for 24 h, then embedded in paraffin as a routine procedure. Modified antigen retrieval method (27) was used for the immunohistochemical visualization of TLR3. Briefly, sections were placed in a plastic Coplin jar filled with 0.001 N NaOH and heated for 5 min at 120 C in an autoclave. Sections were then exposed to a 3% H₂O₂ to inactivate endogenous peroxidase and incubated at 4 C overnight with 1:100 dilution of purified monoclonal antihuman TLR3 antibody (Anti-TLR3-Ab), as described previously (23). Labeled Streptavidin-Biotin method (LSAB+ System HRP kit; Dako, Glostrup, Denmark) was used according to the manufacture’s protocol. 3,3'-Diaminobenzidine tetrahydrochloride was used to detect peroxidase activity. Sections were lightly counterstained with hematoxylin for 10 sec to visualize nuclei.

Statistical analysis
Differences between groups were analyzed by ANOVA with pairwise comparison by Sheffe’s method, using Statview (Abacus Concepts, Berkley, CA). Statistical significance was accepted at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of mRNA for pattern recognition receptors in human thyroid follicles cultured in the presence or absence of TSH
In accordance with a previous report (28), human thyroid follicles expressed significant levels of TLR mRNAs. Because human thyrocytes expressed TSH receptor (TSHR) [NM_000369] constitutively and its expression was not modulated by TSH (22), and because all TLRs were expressed to the same extent as TSHR, the levels of TLR expression were compared with those of TSHR. As shown in Table 1, TLR-4, -8, and -10 were hardly expressed, whereas TLR-1, -2, -3, and -6 were expressed almost equivalently in the thyroid follicles. RIG-I, MDA5, and CD14, other molecules responsible for the recognition of dsRNA, were expressed at levels nearly double that of TSHR (Table 1). The expression levels of these molecules were not significantly modulated by TSH (data not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 1. Oligo-DNA microarray analysis of Poly(I:C) effect on mRNA expression in cultured human thyroid follicles. Human thyroid follicles were precultured in the absence of bTSH for 4 d and in the presence of bTSH (30 µU/ml) for 1 d, and then Poly(I:C) was added to a final concentration of 25 µg/ml. After 6-h incubation, total RNA was extracted and oligo-DNA microarray was performed as described in Materials and Methods. Representative data from three experiments are shown. CXCL, Chemokine (C-X-C) ligand; DIO, deiodinase; HLA, human leukocyte antigen; IFIT, IFN-induced protein with tetratricopeptide repeat; IRF, IFN regulatory factor; SLC26A4, pendrin; VEGF, vascular endothelial growth factor.

View larger version (9K): [in this window] [in a new window]   FIG. 2. Real-time PCR analysis of Poly(I:C) effect on expression levels of thyroglobulin, TPO, and type I and type II deiodinases mRNA. Human thyroid follicles were cultured in the presence of bTSH (30 µU/ml) for 5 d, when Poly(I:C) was added to a final concentration of 25 µg/ml. After 3, 6, 24, and 48 h of incubation, total RNA was extracted and real-time PCR was performed as described in Materials and Methods. –, Thyroglobulin; –, TPO (A); –, type I deiodinase; –, type II deiodinase. Data are mean ± SD of triplicate determinations. Where error bars are not seen, SD is less than the squares. Representative data from two experiments are shown.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Effects of peptidoglycans, Poly(I:C), and LPS on TSH-induced thyroid function. Human thyroid follicles were cultured as described in Materials and Methods in the presence or absence of bTSH (30 µU/ml) for 5 d, then peptidoglycans, Poly(I:C), or LPS were added at various concentrations as indicated in the figure. One hour later, 125I was added (about 20,000 cpm/well). After an additional 3 d of culture, 125I incorporated into the thyroid follicles (columns) and organic 125I released into the culture medium () were determined. Results are expressed as mean ± SD of triplicate cultures. Representative data from six experiments are shown. *, P < 0.05; **, P < 0.001, Poly(I:C) (–) vs. (+).

View larger version (27K): [in this window] [in a new window]   FIG. 4. 125I-metabolites in the thyroid follicles and conditioned medium determined by thin-layer chromatography. Human thyroid follicles were cultured in the presence or absence of bTSH (30 µU/ml) for 5 d, and then Poly(I:C) was added at various concentrations (1–25 µg/ml). After an additional 3 d of culture, 125I metabolites released into the medium (upper panel), and 125I metabolites in the thyroid follicles (lower panel) were analyzed by thin-layer chromatography (26 ). The ratios of 125I-T3/125I-T4 released in the medium at Poly(I:C) concentrations of 0, 1, 5, and 25 µg/ml, were 0.81, 0.79, 0.70, and 0.62, respectively. MIT, Monoiodotyrosine; DIT, diiodotyrosine; O, origin.

To elucidate whether the observed effect was specific for Poly(I:C), we tested the effect of other nucleic acids, i.e. ssRNA and dsDNA, on follicular function. Addition of ssRNA [Poly(C)] and dsDNA [Poly(dI:dC)] to the culture medium of human thyroid follicles had no significant effect on ¹²⁵I uptake or thyroid hormone secretion (Fig. 5).

View larger version (33K): [in this window] [in a new window]   FIG. 5. Effects of Poly(I:C) (A), PolyC (B), and Poly(dI:dC) (C) on TSH-induced thyroid function. Human thyroid follicles were cultured as described in Materials and Methods in the presence or absence of bTSH (30 µU/ml) for 5 d, and then Poly(I:C), PolyC, or Poly(dI:dC) was added at various concentrations as indicated in the figure. One hour later, 125I was added (15,000 cpm/well). After an additional 3 d of culture, 125I incorporated into the thyroid follicles (columns) and organic 125I released into the culture medium () were determined. Results are expressed as mean ± SD of triplicate cultures. Representative data from three experiments are shown. *, P < 0.05; **, P < 0.01, Poly(I:C) (–) vs. (+).

View larger version (120K): [in this window] [in a new window]   FIG. 6. Immunohistochemistry of thyroid gland with anti-TLR3-antibody. Thyroid tissue, obtained from patients with Graves’ disease at subtotal thyroidectomy, was stained with anti-TLR3 monoclonal antibody using antigen retrieval method as described in Materials and Methods. Five representative Graves’ thyroids were shown in A–E. Immunostaining without antigen retrieval method gave no staining (F). G and H are high magnification of follicular epithelium stained with anti-TLR3. Original magnification: A–F, x100; G and H, x1000.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Real-time PCR analysis of Poly(I:C) effect on IFN- and IFN-ß mRNA expression levels and IFN-ß concentration in the conditioned medium. Human thyroid follicles were precultured in the absence of bTSH for 4 d and in the presence of bTSH (30 µU/ml) for 1 d, and then Poly(I:C) was added to a final concentration of 25 µg/ml. After 1-, 2-, 6-, 24-, 48-, and 72-h incubation, total RNA was extracted and real-time PCR was performed as described in Materials and Methods (A). –, IFN-ß; –, IFN-. Representative data from two experiments are shown. IFN-ß concentration in the conditioned medium was measured using ELISA with an assay sensitivity of less than 2.5 pg/ml. Data are mean ± SD of triplicate cultures (A and B). Where error bars are not seen, SD is less than the circles. IFN- concentration was below the detection limit of the assay (<12.5 pg/ml) (data not shown).

View this table: [in this window] [in a new window]   TABLE 6. Effect of anti-IFN/ß-R monoclonal antibody on TSH-stimulated thyroid function in human thyroid follicles treated with IFN-ß (I) or Poly(I:C) (II)

View larger version (11K): [in this window] [in a new window]   FIG. 8. Real-time PCR analysis of Poly(I:C) effect on expression levels of TLR-2, -3, -4, and -9 mRNA. Human thyroid follicles were precultured in the absence of bTSH for 4 d and in the presence of bTSH (30 µU/ml) for 1 d, and then Poly(I:C) was added to a final concentration of 25 µg/ml. After 0.5-, 2-, 6-, and 24-h incubation, total RNA was extracted and real-time PCR was performed as described in Materials and Methods. Data are mean ± SD of triplicate determinations. Where error bars are not seen, SD is less than the triangles. –, TLR2; –, TLR3; –, TLR4; –, TLR9. Representative data from two experiments are shown.

View larger version (10K): [in this window] [in a new window]   FIG. 9. Real-time PCR analysis of Poly(I:C) effect on expression levels of RIG-1 and MDA5 mRNA. Human thyroid follicles were precultured in the absence of bTSH for 4 d and in the presence of bTSH (30 µU/ml) for 1 d, and then Poly(I:C) was added to a final concentration of 25 µg/ml. After 3, 6, 24, and 48 h of incubation, total RNA was extracted and real-time PCR was performed as described in Materials and Methods. –, RIG-1; –, MDA5. Data are mean ± SD of triplicate determinations. Representative data from two experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cellular production of type I IFNs occurs after viral infection. It is now at least partly clear that dsRNA, which is produced by most viruses at some point during their replication cycle, triggers such a response (6). To study the effect of dsRNA on thyroid function, we have used suspension culture of human thyroid follicles in which thyroid hormones are synthesized de novo and secreted into the culture medium in the presence of a physiological concentration of human TSH (21). We have demonstrated that addition of a dsRNA analog to the culture medium without transfection by lipofectamine dose dependently and almost completely suppressed TSH-induced iodide uptake and thyroid hormone secretion, accompanied with decreased mRNA expression of TSHR, NIS, thyroglobulin, peroxidase, and type I and II deiodinases. The effect was specific for RNA, but not DNA, and also specific for the double-stranded structure.

The present in vitro findings support previous reports on the modulation of thyroid function after viral infection in the thyroid. Thus, lymphocytic choriomeningitis virus can persistently infect thyroid epithelial cells and perturb thyroid hormone production (32). Similar findings have been reported in mice neonatally infected with lymphocytic choriomeningitis virus, which persists in the thyroid gland without necrosis or inflammation, and reduces the expression level of thyroglobulin mRNA and circulating thyroid hormones (33). When 10-d-old chicken embryos were infected with an avian leukosis virus, RAV-7, hypothyroidism developed within 3 wk after hatching, accompanied by extensive infiltration of lymphoblastoid cells by 7 d after hatching (34).

We have also shown that dsRNA increases the expression level of MHC class I. This is consistent with previous in vitro findings that reovirus enhances the expression of MHC class I molecules in cultured human thyroid follicular cells through induction of type 1 IFN (35). Furthermore, viruses are also reported to induce class II MHC expression in thyroid cells (36, 37). Although the precise mechanism of these effects is unknown, it would allow nonprofessional antigen-presenting cells such as thyrocytes to present self or foreign antigens to immune cells. As a result, autoimmune thyroiditis may occur after viral infection, as has been reported in mice infected with reovirus (38). In our in vitro study, however, the effect of dsRNA on MHC gene expression was restricted to class I antigens, because few immunocompetent cells were present in our experimental model (39), suggesting that addition of dsRNA to the culture medium was not enough to induce MHC class II expression.

The underlying molecular mechanism by which dsRNA inhibits ¹²⁵I incorporation and thyroid hormonogenesis remains to be elucidated. One of the likely explanations is that IFN-ß produced by dsRNA stimulation is secreted to the outside of the cell, where it exerted inhibitory effects in an autocrine or paracrine manner through type I IFN receptors expressed on the plasma membrane. However, this was unlikely under the present experimental conditions, because anti-IFN-/ß receptor neutralizing antibody did not block dsRNA-induced thyroid dysfunction (Table 6). Recently, Lee et al. (19) demonstrated that dsRNA binds to CD14 expressed on the plasma membrane and that dsRNA/CD14 complex activates TLR3. Furthermore, dsRNA activates nuclear factor-B or MAPK pathways independent of the signaling pathway through IFN-/ß receptor (5, 6). Because thyrocytes treated with dsRNA show increased levels of various cytokines, and chemokines in addition to IFNs, which inhibit thyroid function (9, 10, 11), these might be responsible for the dsRNA-initiated inhibitory action in the thyroid.

Very recently, Harii et al. (18), using monolayer cultures of rat thyroid FRTL-5 cells, observed that dsRNA activates various signaling molecules such as nuclear factor-B, MAPK, IFN regulatory factor-3, and IFN-ß. They also demonstrated that transfection of dsRNA, infection with influenza A virus, or incubation with IFN-ß, but not incubation with dsRNA or IFN, enhanced the expression of TLR3, and suggested that thyrocytes express functional TLR3. In contrast to human fibroblasts, in which TLR3 is expressed on the cell surface and an anti-TLR3 monoclonal antibody inhibits dsRNA-induced IFN-ß production (23), pretreatment of thyroid follicles with the same antibody did not block dsRNA-induced suppression of thyroid function, suggesting that TLR3 was predominantly located intracellularly in the thyroid follicles, probably in endosome-like vesicles, rather than in the plasma membrane, as reported in immature human dendritic cells and human alveolar and bronchial epithelial cells (16, 30). Our immunohistochemical studies also provided support for the intracellular expression of TLR3 in human thyroid follicles from Graves’ disease patients (Fig. 6). Although Harii et al. (18) demonstrated TLR3 expression in thyrocytes obtained from patients with Hashimoto’s thyroiditis, but not in those with Graves’ disease, our present findings suggest that TLR3 is present even in the Graves’ thyroid. The present data seem to be quite reasonable because we have detected TLR3 mRNA from Graves’ thyroid using DNA microarray and real time PCR. Also, Harii et al. (18) actually demonstrated TLR3 mRNA in normal mouse thyroid, rat thyroid FRTL-5 cells, and NPA human papillary thyroid cells. Our results suggest that the efficiency of antigen-retrieval methods and the sensitivity of immunohistochemistry significantly affect the staining.

It is well known that antiviral antibodies against single-stranded RNA viruses (influenza, coxsackie, mumps, and echo viruses) as well as dsDNA virus (adenovirus) transiently increase in patients with de Quervain subactue thyroiditis (40). There is a general consensus that once these viruses enter thyrocytes through various pathways (41), dsRNA will be produced. Thus, without interacting with the TLR3-mediated signaling pathway (5, 6), the dsRNA can interact with RIG-1 and MDA5 in the cytoplasm, and these complexes also stimulate the type I IFN system (5, 6). Our in vitro data are compatible with clinical observations that no particular, pathognomonic viruses have been isolated from patients with subacute thyroiditis (42). Furthermore, our data suggest that in the prodromal period of subacute thyroiditis, serum levels of T₄ and, to a greater extent, T₃ would be transiently decreased.

The present findings are compatible with some clinical reports describing that a euthyroid patient with mumps, who developed thyroid swelling and a diffusely reduced uptake in a thyroid scan (43). However, in general, serum levels of thyroid hormones are not measured in the prodromal period in patients with subacute thyroiditis, but are routinely measured during the thyrotoxic period when thyroid follicles have been extensively destroyed. Therefore, any transient decrease in serum thyroid hormone levels during the prodromal period, or subclinical viral infections, would be obscured.

In summary, using human thyroid follicles de novo synthesizing and releasing thyroid hormones in the presence of physiological TSH concentration, we have shown that TLR signaling via dsRNA [Poly(I:C)] results in the production of type I IFN and inflammatory cytokines, leading to establishment of antiviral immunity, accompanied by thyroid dysfunction. These in vitro findings may be associated, at least in part, with development of thyroid dysfunction and would be related to the development of autoimmune thyroid disorders.

	Footnotes

 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (17590967 and 15390296).

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 29, 2007

Abbreviations: bTSH, Bovine TSH; dsRNA, double-stranded RNA; Ct, threshold cycle; IFN, interferon; LPS, lipopolysaccharide; MDA5, melanoma differentiation-associated gene 5; MHC, major histocompatibility complex; NIS, sodium/iodide symporter; PAMPs, pathogen-associated molecular patterns; Poly(I:C), polyinosinic-polycytidylic acid; RIG-1, retinoic-acid-inducible protein-1; ssRNA, single-stranded RNA; TCA, trichloroacetic acid; TLR, Toll-like receptor; TPO, thyroid peroxidase; TSHR, TSH receptor.

Received December 7, 2006.

Accepted for publication March 16, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Tomer Y, Davies TF 1993 Infection, thyroid disease, and autoimmunity. Endocr Rev 14:107–120[Abstract/Free Full Text]
2. Prummel MF, Strieder T, Wiersinga WM 2004 The environment and autoimmune thyroid diseases. Eur J Endocrinol 150:605–618[Abstract]
3. Suzuki K, Mori A, Ishii KJ, Saito J, Singer DS, Klinman DM, Krause PR, Kohn LD 1999 Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides. Proc Natl Acad Sci USA 96:2285–2290[Abstract/Free Full Text]
4. Theofilopoulos AN, Baccala R, Beutler B, Kono DH 2005 Type I interferons (/ß) in immunity and autoimmunity. Annu Rev Immunol 23:307–336[CrossRef][Medline]
5. Akira S, Uematsu S, Takeuchi O 2006 Pathogen recognition and innate immunity. Cell 124:783–801[CrossRef][Medline]
6. Kawai T, Akira S 2006 Innate immune recognition of viral infection. Nature Immunol 7:131–137[CrossRef][Medline]
7. Yamazaki K, Kanaji Y, Shizume K, Yamakawa Y, Kanaji Y, Obara T, Sato K 1993 Reversible inhibition by interferons and ß of ¹²⁵I incorporation and thyroid hormone release by human thyroid follicles in vitro. J Clin Endocrinol Metab 77:1439–1441[Abstract]
8. Caraccio N, Giannini R, Cuccato S, Faviana P, Berti P, Galleri D, Dardano A, Basolo F, Ferrannini E, Monzani F 2005 Type I interferons modulate the expression of thyroid peroxidase, sodium/iodide symporter, and thyroglobulin genes in primary human thyrocyte cultures. J Clin Endocrinol Metab 90:1156–1162[Abstract/Free Full Text]
9. Sato K, Satoh T, Shizume K, Ozawa M, Han DC, Imamura H, Tsushima T, Kanaji Y, Ito Y, Obara T, Fujimoto Y, Kanaji Y 1990 Inhibition of ¹²⁵I organification and thyroid hormone release by interleukin-1, tumor necrosis factor-, and interferon- in human thyrocytes in suspension culture. J Clin Endocrinol Metab 70:1735–1743[Abstract/Free Full Text]
10. Sato K, Yamazaki K, Shizume K, Yamakawa Y, Satoh T, Kanaji Jr Y, Obara T, Fujimoto Y, Aiba M, Kakiuchi T, Kanaji Y 1993 Pathogenesis of autoimmune hypothyroidism induced by lymphokine-activated killer (LAK) cell therapy: in vitro inhibition of human thyroid function by interleukin-2 in the presence of autologous intrathyroidal lymphocytes. Thyroid 3:179–188[Medline]
11. Yamazaki K, Yamada E, Kanaji Y, Shizume K, Wang DS, Maruo N, Obara T, Sato K 1996 Interleukin-6 (IL-6) inhibits thyroid function in the presence of soluble IL-6 receptor in cultured human thyroid follicles. Endocrinology 137:4857–4863[Abstract]
12. Mazziotti G, Sorvillo F, Piscopo M, Morisco F, Cioffi M, Stornaiuolo G, Gaeta GB, Molinari AM, Lazarus JH, Amato G, Carella C 2005 Innate and acquired immune system in patients developing interferon--related autoimmune thyroiditis: a prospective study. J Clin Endocrinol Metab 90:4138–4144[Abstract/Free Full Text]
13. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA 2001 Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 413:732–738[CrossRef][Medline]
14. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S,Lipford G, Wagner H, Bauer S 2004 Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526–1529[Abstract/Free Full Text]
15. Haller O, Kochs G, Weber F 2006 The interferon response circuit: induction and suppression by pathogenic viruses. Virology 344:119–130[CrossRef][Medline]
16. Guillot L, Le Goffic R, Bloch S, Escriou N, Akira S, Chignard M, Si-Tahar M 2005 Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus. J Biol Chem 280:5571–5580[Abstract/Free Full Text]
17. Cario E, Podolsky DK 2000 Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 68:7010–7017[Abstract/Free Full Text]
18. Harii N, Lewis CJ, Vasko V, McCall K, Benavides-Peralta U, Sun X, Ringel MD, Saji M, Giuliani C, Napolitano G, Goetz DJ, Kohn LD 2005 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. Mol Endocrinol 19:1231–1250[Abstract/Free Full Text]
19. Lee HK, Dunzendorfer S, Soldau K, Tobias PS 2006 Double-stranded RNA-mediated TLR3 activation is enhanced by CD14. Immunity 24:153–163[CrossRef][Medline]
20. Yoneyama M, Kikuchi M, Matsumoto K, Imaizumi T, Miyagishi M, Taira K, Foy E, Loo YM, Gale Jr M, Akira S, Yonehara S, Kato A, Fujita T 2005 Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J Immunol 175:2851–2958[Abstract/Free Full Text]
21. Yamazaki K, Sato K, Shizume K, Kanaji Y, Ito Y, Obara T, Nakagawa T, Koizumi T, Nishimura R 1995 Potent thyrotropic activity of human chorionic gonadotropin variants in terms of ¹²⁵I incorporation and de novo synthesized thyroid hormone release in human thyroid follicles. J Clin Endocrinol Metab 80:473–479[Abstract]
22. Yamada E, Yamazaki K, Takano K, Obara T, Sato K 2006 Iodide inhibits vascular endothelial growth factor (VEGF)–a expression in cultured human thyroid follicles: a microarray search for effects of TSH and iodide on angiogenesis factors. Thyroid 16:545–554[CrossRef][Medline]
23. Matsumoto M, Kikkawa S, Kohase M, Miyake K, Seya T 2002 Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem Biophys Res Commun 293:1364–1369[CrossRef][Medline]
24. Sato K, Yamazaki K, Shizume K, Kanaji Y, Obara T, Ohsumi K, Yamaguchi S, Shibuya M 1995 Stimulation by thyroid-stimulating hormone and Grave’s immunoglobulin G of vascular endothelial growth factor mRNA expression in human thyroid follicles in vitro and flt mRNA expression in the rat thyroid in vivo. J Clin Invest 96:1295–302[Medline]
25. Suzuki K, Kohn LD 2006 Differential regulation of apical and basal iodide transporters in the thyroid by thyroglobulin. J Endocrinol 189:247–255[Abstract/Free Full Text]
26. Sato K, Robbins J 1981 Thyroid hormone metabolism in primary cultured rat hepatocytes. Effects of glucose, glucagon, and insulin. J Clin Invest 68:475–483[Medline]
27. Katoh R, Bray CE, Suzuki K, Komiyama A, Hemmi A, Kawaoi A, Oyama T, Sugai T, Sasou S 1995 Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure using monoclonal antibody MIB-1. Hum Pathol 26:139–146[CrossRef][Medline]
28. Nishimura M, Naito S 2005 Tissue-specific mRNA expression profiles of human toll-like receptors and related genes. Biol Pharm Bull 28:886–892[CrossRef][Medline]
29. Velez ML, Costamagna E, Kimura ET, Fozzatti L, Pellizas CG, Montesinos MM, Lucero AM, Coleoni AH, Santisteban P, Masini-Repiso AM 2006 Bacterial lipopolysaccharide stimulates the TSH-dependent thyroglobulin gene expression at transcriptional level by involving the transcription factors TTF-1 and Pax8. Endocrinology 147:3260–3275[Abstract/Free Full Text]
30. Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, Yamamoto A, Seya T 2003 Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 171:3154–3162[Abstract/Free Full Text]
31. Samuel CE 2001 Antiviral actions of interferons. Clin Microbiol Rev 14:778–809[Abstract/Free Full Text]
32. Klavinskis LS, Notkins AL, Oldstone MB 1988 Persistent viral infection of the thyroid gland: alteration of thyroid function in the absence of tissue injury. Endocrinology 122:567–575[Abstract/Free Full Text]
33. Klavinskis LS, Oldstone MB 1987 Lymphocytic choriomeningitis virus can persistently infect thyroid epithelial cells and perturb thyroid hormone production. J Gen Virol 68(Pt 7):1867–1873
34. Carter JK, Smith RE 1983 Rapid induction of hypothyroidism by an avian leukosis virus. Infect Immun 40:795–805[Abstract/Free Full Text]
35. Atta MS, Irving WL, Powell RJ, Todd I 1995 Enhanced expression of MHC class I molecules on cultured human thyroid follicular cells infected with reovirus through induction of type 1 interferons. Clin Exp Immunol 101:121–126[Medline]
36. Neufeld DS, Platzer M, Davies TF 1989 Reovirus induction of MHC class II antigen in rat thyroid cells. Endocrinology 124:543–545[Abstract/Free Full Text]
37. Khoury EL, Pereira L, Greenspan FS 1991 Induction of HLA-DR expression on thyroid follicular cells by cytomegalovirus infection in vitro. Evidence for a dual mechanism of induction. Am J Pathol 138:1209–1223[Abstract]
38. Srinivasappa J, Garzelli C, Onodera T, Ray U, Notkins AL 1988 Virus-induced thyroiditis. Endocrinology 122:563–566[Abstract/Free Full Text]
39. Nakajima K, Yamazaki K, Yamada E, Kanaji Y, Kosaka S, Sato K, Takano K 2001 Amiodarone stimulates interleukin-6 production in cultured human thyrocytes, exerting cytotoxic effects on thyroid follicles in suspension culture. Thyroid 11:101–109[CrossRef][Medline]
40. Volpe R, Row VV, Ezrin C 1967 Circulating viral and thyroid antibodies in subacute thyroiditis. J Clin Endocrinol Metab 27:1275–284[Abstract/Free Full Text]
41. Smith AE, Helenius A 2004 How viruses enter animal cells. Science 304:237–242[Abstract/Free Full Text]
42. Lazarus JH 1996 Silent thyroiditis and subacute thyroiditis. In: Braverman LE, Utiger RD, eds. Werner and Ingbar’s the thyroid: a fundamental and clinical text. 7th ed. Lippincott-Raven, Philadelphia; 577–591
43. Parmar RC, Bavdekar SB, Sahu DR, Warke S, Kamat JR 2001 Thyroiditis as a presenting feature of mumps. Pediatr Infect Dis J 20:637–638[CrossRef][Medline]

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