Expression of thyroglobulin by regulatory T cells in thyroid tissue

in Journal of Molecular Endocrinology
Correspondence should be addressed to X-M Mao: maoxming@163.com

(*Y-Q Zhu and Y Hu contributed equally to this work)

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The follicles are the minimal functional unit of the thyroid; the morphology and the function of each follicle can vary significantly. However, the reasons for the apparent follicular heterogeneity are poorly understood. Some tissue-resident regulatory T cells (Tregs) have a special phenotype that expresses unique molecules related to local tissue and regulates the tissue functions. The aim of this study was to identify the phenotype of thyroid Tregs and the roles of thyroid Tregs in thyroid physiological regulation. Thyroid tissue and peripheral blood samples were obtained from patients with benign thyroid nodules. Microarray-based gene expression, flow cytometry, immunofluorescence microscopy, and functional analysis of thyroid Tregs were performed. Here, we demonstrated that human thyroid Tregs expressed high level of thyroglobulin (Tg), both gene and protein. The immunofluorescence microscopy of thyroid section showed that the FOXP3+Tg+ cells concentrated in some of the thyroid follicles, at the side of the thyroid follicle. The peripheral blood Tregs expressed minimal levels of Tg, and low levels of Tg could effectively induce peripheral blood Tregs to express Tg, which was independent of thyrotropin simulation. Furthermore, the Tg secreted freely from thyroid Tregs that negatively regulated some thyroid-related genes expression. Our results revealed that the thyroid Tregs was a distinct population of Tregs, which expressed high level of Tg. The thyroid Tregs regulate thyroid function by Tg that is paracrine from the cells.

 

      Society for Endocrinology

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    Proportion of thyroid tissue Tregs. The thyroid tissue and peripheral blood were isolated and collected from the patients who underwent thyroid surgery; the mononuclear cells were stained for CD4, CD25, FOXP3. (A) Upper panel: T cell distribution in mononuclear cells from the thyroid tissue. Lower panel: T cell distribution in mononuclear cells from the peripheral blood. Percentage of FOXP3+CD25+ T cells in the thyroid gated on CD4+ T cells and representative dot plots are shown. (B) Frequency of CD25+FOXP3+/CD4+ T cells in the thyroid tissue and the peripheral blood. Mean ± s.d. from 12 independent experiments are shown. ***P < 0.001; Student’s t-test.

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    Functional comparison of Tregs from thyroid tissue and peripheral blood. CD4+CD25+ Tregs and conventional T (Tconv) cells were isolated from the human thyroid tissue and peripheral blood; a standard in vitro suppression assay was performed. Blood-derived CD4+ effector T cells (responder cells) were incubated with various ratios of CD4+CD25+ Tregs from the thyroid tissue and peripheral blood. (A and B) The different ratios of Tregs inhibited Tconv cell proliferation. (C) Sum of the three ratios of Tregs inhibiting Tconv cells proliferation. (D) The FOXP3 mRNA expression in the thyroid and peripheral blood Tregs. Mean ± s.d. from at least six independent experiments are shown. ***P < 0.001; Student’s t-test.

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    A particular gene-expression profile of Tregs in the thyroid. (A–F) Analysis with Agilent Human 4x44K Gene Expression Microarrays and (G–J) real-time PCR. (A) Normalized expression values for profiles directly comparing Tregs between the thyroid and the peripheral blood and (B) for profiles directly comparing Tconv between the thyroid and the peripheral blood. The numbers are calculated on the basis of a cut-off of two-fold from individual comparisons, and volcano plot comparing P values between Tregs and Tconvs signature in the peripheral blood (C) and the thyroid (D). (E) Volcano plot comparing gene expression of the peripheral blood and thyroid Tregs. (F) Fold-change to fold-change plots comparing Tregs/Tconvs expression profiles between the peripheral blood and the thyroid. Real-time PCR comparing (G) thyroglobulin (Tg), (H) IL-4, (I) IFN-γ, and (J) IL-10 mRNA expressions between the peripheral blood and thyroid Tregs. Mean ± s.d. from six independent experiments are shown. *P < 0.05; ***P < 0.001; Student’s t-test.

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    Protein expression of Tg in thyroid Tregs. (A) Immunofluorescence microscopy of thyroid sections. DAPI was used to label nuclear DNA. Original magnification ×400. (B) Upper panel: The percentage of Tg expression in CD25+ cells from the peripheral blood gated on CD4+ T cells. Lower panel: The percentage of Tg expression in CD25+ T cells from the thyroid tissue gated on CD4+ T cells. The representative dot plots are shown. (C) The proportion of Tg+ in CD4+CD25+ cells. **P<0.01; Student’s t-test. (D) Thyroid Tregs were isolated and various concentrations of Tregs were cultured; after 6 h, the supernatants were collected and Tg concentration was tested. Mean ± s.d. from at least five independent experiments are shown. *P < 0.05; **P < 0.01; Student’s t-test.

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    Tg stimulates peripheral blood Tregs to express Tg. T-Treg represents thyroid Tregs, B-Treg represents peripheral blood Tregs. The B-Treg and T-Treg were isolated from normal human peripheral blood and thyroid, respectively. (A) TSH mRNA expression in peripheral blood and thyroid Tregs. (B) 1.0 mU/mL TSH or 1.0 mg/mL Tg was used to stimulate B-Treg. (C) The various concentrations of Tg were used to stimulate B-Treg. The mean ± s.d. from at least five independent experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t-test. (D) Tg protein expression in peripheral blood CD25+ cells gated on CD4+ cells after 1.0 mg/mL Tg stimulation.

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    Thyroid tissue Tregs affect thyroid-related gene expression. The thyroid Tregs and follicular epithelial cells were isolated from the human thyroid tissue. (A–C) The thyroid follicular epithelial cells and various concentrations of Tg or thyroid Tregs were co-cultured; the thyroid follicular epithelial cells were isolated; and real-time PCR was conducted. Mean ± s.d. from at least five independent experiments are shown, compared with the control. *P < 0.05;**P < 0.01; ***P < 0.001; Student’s t-test.

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