Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disorder characterized by progressive thyroid failure. Th1 and Treg subset of CD4(+) cells have been implicated in the pathogenesis; however, less is known about their respective roles across the spectrum of HT clinical presentations. To shed more light on CD4(+) subsets role in HT, we investigated the mRNA expression levels of several Th1/Treg-associated transcription factors (T-bet/ETS1, HIF1α/BLIMP1/FOXP3) in peripheral blood T cells of 10 hypothyroid, untreated HT patients, 10 hypothyroid patients undergoing hormone replacement therapy, 12 euthyroid HT subjects, and 11 healthy controls by the qRT-PCR. Compared to euthyroid HT patients and controls, both hypothyroid (2.34-fold difference versus controls, P < 0.01) and thyroxine-supplemented patients (2.5-fold, P < 0.001) showed an increased FOXP3 mRNA expression in T cells. Similarly, mRNA expression levels of T-bet were upregulated in severely affected but not in euthyroid HT subjects (2.37-fold and 3.2-fold, hypothyroid and thyroxine-supplemented HT patients versus controls, resp., P < 0.01). By contrast, no differences in mRNA expression levels of ETS1, BLIMP1, and HIF1α were observed across the study groups. In summary, severe but not euthyroid HT was associated with robust upregulation of T-bet and FOXP3 mRNA in peripheral T cells, independent of the thyroid hormone status but proportional to disease activity.

Department of Molecular Diagnostics and Tissue Typing Osijek University Hospital Josipa Huttlera 4 31000 Osijek Croatia

Department of Molecular Diagnostics and Tissue Typing Osijek University Hospital Josipa Huttlera 4 31000 Osijek Croatia; Faculty of Medicine University of Osijek Cara Hadrijana 10E 31000 Osijek Croatia; Laboratory of Immunogenomics Department of Pathological Physiology Faculty of Medicine and Dentistry Palacký University 775 20 Olomouc Czech Republic

Faculty of Medicine University of Osijek Cara Hadrijana 10E 31000 Osijek Croatia

Faculty of Medicine University of Osijek Cara Hadrijana 10E 31000 Osijek Croatia; Clinical Institute of Nuclear Medicine and Radiation Protection Osijek University Hospital Josipa Huttlera 4 31000 Osijek Croatia

Laboratory of Immunogenomics Department of Pathological Physiology Faculty of Medicine and Dentistry Palacký University 775 20 Olomouc Czech Republic

Zobrazit více v PubMed

Stassi G., De Maria R. Autoimmune thyroid disease: new models of cell death in autoimmunity. Nature Reviews Immunology. 2002;2(3):195–204. doi: 10.1038/nri750. PubMed DOI

Horie I., Abiru N., Nagayama Y., et al. T helper type 17 immune response plays an indispensable role for development of iodine-induced autoimmune thyroiditis in nonobese diabetic-H2h4 mice. Endocrinology. 2009;150(11):5135–5142. doi: 10.1210/en.2009-0434. PubMed DOI

Figueroa-Vega N., Alfonso-Pérez M., Benedicto I., Sánchez-Madrid F., González-Amaro R., Marazuela M. Increased circulating pro-inflammatory cytokines and Th17 lymphocytes in Hashimoto's thyroiditis. Journal of Clinical Endocrinology and Metabolism. 2010;95(2):953–962. doi: 10.1210/jc.2009-1719. PubMed DOI

Shi Y., Wang H., Su Z., et al. Differentiation imbalance of Th1/Th17 in peripheral blood mononuclear cells might contribute to pathogenesis of Hashimoto's thyroiditis. Scandinavian Journal of Immunology. 2010;72(3):250–255. doi: 10.1111/j.1365-3083.2010.02425.x. PubMed DOI

Li D., Cai W., Gu R., et al. Th17 cell plays a role in the pathogenesis of Hashimoto's thyroiditis in patients. Clinical Immunology. 2013;149:411–420. doi: 10.1016/j.clim.2013.10.001. PubMed DOI

Nanba T., Watanabe M., Inoue N., Iwatani Y. Increases of the Th1/Th2 cell ratio in severe Hashimoto's disease and in the proportion of Th17 cells in intractable Graves' disease. Thyroid. 2009;19(5):495–501. doi: 10.1089/thy.2008.0423. PubMed DOI

Liu Y., Tang X., Tian J., et al. Th17/Treg cells imbalance and GITRL profile in patients with Hashimoto’s thyroiditis. International Journal of Molecular Sciences. 2014;15(12):21674–21686. doi: 10.3390/ijms151221674. PubMed DOI PMC

Xue H., Yu X., Ma L., et al. The possible role of CD4+CD25highFoxp3+/CD4+IL-17A+ cell imbalance in the autoimmunity of patients with Hashimoto thyroiditis. Endocrine. 2015;50(3):665–673. doi: 10.1007/s12020-015-0569-y. PubMed DOI

Grenningloh R., Bok Y. K., Ho I.-C. Ets-1, a functional cofactor of T-bet, is essential for Th1 inflammatory responses. Journal of Experimental Medicine. 2005;201(4):615–626. doi: 10.1084/jem.20041330. PubMed DOI PMC

Tsao H.-W., Tai T.-S., Tseng W., et al. Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(39):15776–15781. doi: 10.1073/pnas.1304343110. PubMed DOI PMC

Mouly E., Chemin K., Nguyen H. V., et al. The Ets-1 transcription factor controls the development and function of natural regulatory T cells. Journal of Experimental Medicine. 2010;207(10):2113–2125. doi: 10.1084/jem.20092153. PubMed DOI PMC

Cimmino L., Martins G. A., Liao J., et al. Blimp-1 attenuates Th1 differentiation by repression of ifng, tbx21, and bcl6 gene expression. Journal of Immunology. 2008;181(4):2338–2347. doi: 10.4049/jimmunol.181.4.2338. PubMed DOI

Martins G. A., Cimmino L., Liao J., Magnusdottir E., Calame K. Blimp-1 directly represses Il2 and the Il2 activator Fos, attenuating T cell proliferation and survival. Journal of Experimental Medicine. 2008;205(9):1959–1965. doi: 10.1084/jem.20080526. PubMed DOI PMC

Cretney E., Xin A., Shi W., et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nature Immunology. 2011;12(4):304–311. doi: 10.1038/ni.2006. PubMed DOI

Dang E. V., Barbi J., Yang H.-Y., et al. Control of TH17/Treg balance by hypoxia-inducible factor 1. Cell. 2011;146(5):772–784. doi: 10.1016/j.cell.2011.07.033. PubMed DOI PMC

Štefanić M., Papić S., Suver M., Glavaš-Obrovac L., Karner I. Association of vitamin D receptor gene 3'-variants with Hashimoto's thyroiditis in the Croatian population. International Journal of Immunogenetics. 2008;35(2):125–131. doi: 10.1111/j.1744-313x.2008.00748.x. PubMed DOI

Böyum A. Separation of leukocytes from blood and bone marrow. Introduction. Scandinavian Journal of Clinical And Laboratory Investigation, Supplementum. 1968;97, article 7 PubMed

Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 1987;162(1):156–159. doi: 10.1016/0003-2697(87)90021-2. PubMed DOI

Pfaffl M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. 2001;29(9, article e45) doi: 10.1093/nar/29.9.e45. PubMed DOI PMC

Horie I., Abiru N., Sakamoto H., Iwakura Y., Nagayama Y. Induction of autoimmune thyroiditis by depletion of CD4+CD25+ regulatory T cells in thyroiditis-resistant IL-17, but not interferon-γ receptor, knockout nonobese diabetic-H2h4 mice. Endocrinology. 2011;152(11):4448–4454. doi: 10.1210/en.2011-1356. PubMed DOI

Oh S., Hwang E. S. The role of protein modifications of T-bet in cytokine production and differentiation of t helper cells. Journal of Immunology Research. 2014;2014:7. doi: 10.1155/2014/589672.589672 PubMed DOI PMC

Qin Q., Liu P., Liu L., et al. The increased but non-predominant expression of Th17- and Th1-specific cytokines in hashimoto's thyroiditis but not in graves' disease. Brazilian Journal of Medical and Biological Research. 2012;45(12):1202–1208. doi: 10.1590/s0100-879x2012007500168. PubMed DOI PMC

Karanikas G., Schuetz M., Wahl K., et al. Relation of anti-TPO autoantibody titre and T-lymphocyte cytokine production patterns in Hashimoto's thyroiditis. Clinical Endocrinology. 2005;63(2):191–196. doi: 10.1111/j.1365-2265.2005.02324.x. PubMed DOI

Gérard A.-C., Boucquey M., Van Den Hove M.-F., Colin I. M. Expression of TPO and ThOXs in human thyrocytes is downregulated by IL-1α/IFN-γ, an effect partially mediated by nitric oxide. American Journal of Physiology—Endocrinology and Metabolism. 2006;291(2):E242–E253. doi: 10.1152/ajpendo.00439.2005. PubMed DOI

Marique L., Van Regemorter V., Gérard A.-C., et al. The expression of dual oxidase, thyroid peroxidase, and caveolin-1 differs according to the type of immune response (TH1/TH2) involved in thyroid autoimmune disorders. Journal of Clinical Endocrinology and Metabolism. 2014;99(5):1722–1732. doi: 10.1210/jc.2013-3469. PubMed DOI

Su H. W., Bretz J. D., Phelps E., et al. A unique combination of inflammatory cytokines enhances apoptosis of thyroid follicular cells and transforms nondestructive to destructive thyroiditis in experimental autoimmune thyroiditis. Journal of Immunology. 2002;168(5):2470–2474. doi: 10.4049/jimmunol.168.5.2470. PubMed DOI

Marazuela M., García-López M. A., Figueroa-Vega N., et al. Regulatory T cells in human autoimmune thyroid disease. Journal of Clinical Endocrinology and Metabolism. 2006;91(9):3639–3646. doi: 10.1210/jc.2005-2337. PubMed DOI

De Rosa V., Galgani M., Porcellini A., et al. Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants. Nature Immunology. 2015;16(11):1174–1184. doi: 10.1038/ni.3269. PubMed DOI PMC

Kristensen B., Hegedüs L., Madsen H. O., Smith T. J., Nielsen C. H. Altered balance between self-reactive T helper (Th)17 cells and Th10 cells and between full-length forkhead box protein 3 (FoxP3) and FoxP3 splice variants in Hashimoto's thyroiditis. Clinical and Experimental Immunology. 2015;180(1):58–69. doi: 10.1111/cei.12557. PubMed DOI PMC

Ryder L. R., Woetmann A., Madsen H. O., et al. Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis. Scandinavian Journal of Rheumatology. 2010;39(4):279–286. doi: 10.3109/03009740903555374. PubMed DOI

Ban H., Andoh A., Shioya M., Nishida A., Tsujikawa T., Fujiyama Y. Increased number of FoxP3+CD4+ regulatory T cells in inflammatory bowel disease. Molecular Medicine Reports. 2008;1(5):647–650. PubMed

Nakano A., Watanabe M., Iida T., et al. Apoptosis-induced decrease of intrathyroidal CD4+CD25+ regulatory T cells in autoimmune thyroid diseases. Thyroid. 2007;17(1):25–31. doi: 10.1089/thy.2006.0231. PubMed DOI

Mao C., Wang S., Xiao Y., et al. Impairment of regulatory capacity of CD4+CD25+ regulatory T cells mediated by dendritic cell polarization and hyperthyroidism in Graves' disease. Journal of Immunology. 2011;186(8):4734–4743. doi: 10.4049/jimmunol.0904135. PubMed DOI

van Amelsfort J. M. R., Jacobs K. M. G., Bijlsma J. W. J., Lafeber F. P. J. G., Taams L. S. CD4+CD25+ regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis and Rheumatism. 2004;50(9):2775–2785. doi: 10.1002/art.20499. PubMed DOI

González-Amaro R., Marazuela M. T regulatory (Treg) and T helper 17 (Th17) lymphocytes in thyroid autoimmunity. Endocrine. 2016;52(1):30–38. doi: 10.1007/s12020-015-0759-7. PubMed DOI

Veldhoen M., Hocking R. J., Atkins C. J., Locksley R. M., Stockinger B. TGFβ in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity. 2006;24(2):179–189. doi: 10.1016/j.immuni.2006.01.001. PubMed DOI

Li L., Boussiotis V. A. Molecular and functional heterogeneity of T regulatory cells. Clinical Immunology. 2011;141(3):244–252. doi: 10.1016/j.clim.2011.08.011. PubMed DOI PMC

Koch M. A., Tucker-Heard G., Perdue N. R., Killebrew J. R., Urdahl K. B., Campbell D. J. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nature Immunology. 2009;10(6):595–602. doi: 10.1038/ni.1731. PubMed DOI PMC

Yu F., Sharma S., Edwards J., Feigenbaum L., Zhu J. Dynamic expression of transcription factors T-bet and GATA-3 by regulatory T cells maintains immunotolerance. Nature Immunology. 2015;16(2):197–206. doi: 10.1038/ni.3053. PubMed DOI PMC

Tian L., Altin J. A., Makaroff L. E., et al. Foxp3+ regulatory T cells exert asymmetric control over murine helper responses by inducing Th2 cell apoptosis. Blood. 2011;118(7):1845–1853. doi: 10.1182/blood-2011-04-346056. PubMed DOI PMC

Hua J., Davis S. P., Hill J. A., Yamagata T. Diverse gene expression in human Regulatory T cell subsets uncovers connection between regulatory T cell genes and suppressive function. Journal of Immunology. 2015;195(8):3642–3653. doi: 10.4049/jimmunol.1500349. PubMed DOI

Li X., Liang Y., Leblanc M., Benner C., Zheng Y. Function of a foxp3 cis-element in protecting regulatory T cell identity. Cell. 2014;158(4):734–748. doi: 10.1016/j.cell.2014.07.030. PubMed DOI PMC

Lin M. H., Yeh L. T., Chen S. J., et al. T cell-specific BLIMP-1 deficiency exacerbates experimental autoimmune encephalomyelitis in nonobese diabetic mice by increasing Th1 and Th17 cells. Clinical Immunology. 2014;151(2):101–113. doi: 10.1016/j.clim.2014.02.006. PubMed DOI

Moisan J., Grenningloh R., Bettelli E., Oukka M., Ho I.-C. Ets-1 is a negative regulator of Th17 differentiation. Journal of Experimental Medicine. 2007;204(12):2825–2835. doi: 10.1084/jem.20070994. PubMed DOI PMC

Polansky J. K., Schreiber L., Thelemann C., et al. Methylation matters: Binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells. Journal of Molecular Medicine. 2010;88(10):1029–1040. doi: 10.1007/s00109-010-0642-1. PubMed DOI PMC

Cretney E., Xin A., Shi W., et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nature Immunology. 2011;12(4):304–312. doi: 10.1038/ni.2006. PubMed DOI

miR-29a-3p/T-bet Regulatory Circuit Is Altered in T Cells of Patients With Hashimoto's Thyroiditis