Cd4 ~ + ~ Of Cd25 + Regulatory T Cells And Th17 Cells And Hashimoto's Thyroiditis-related Research

Objective: Numerous epidemiologic studies indicated that Hashimoto's thyroiditis (HT) is one of the most common organ specific autoimmune diseases, and is common in women. There is no effective therapy for HT. Many factors involved in its pathogenesis,immunologic injury was considered to be one of them. For many years, it is believed that after the self immune tolerance to thyroid tissue was broken, the immunocyte were activated by organ specificity antigen, the autoantibody and relative cytokines were produced, and finally result in lymphocyte infiltration and hypothyroidism. The mechanism which self immune tolerance was broken is still unclear. CD4~+CD25~+T regulator cells ( Tregs ) is a subset of T helper cells which has suppressive function on autoreactive T cells. It plays important role in immune homeostasis and preventing autoimmune disease. HumanCD4~+CD25~+Tregs constitutively express CD25 ( IL-2 receptorαchain ) as well as forkhead transcription factor protein (Foxp3), and suppress autoreactive T cells through the secretion of suppressive cytokine transforming growth factor-beta 1(TGF-β1). The decreased proportion and/or function of CD4~+CD25~+Tregs have been observed in many autoimmune diseases, including rheumatoid arthritis, psoriasis and systemic lupus erythematosus. In order to elucidate whether the changes of amount and/or function of CD4~+CD25~+Tregs have relationship with the development of HT, in the present study we detected the number of circuling CD4~+CD25~+Tregs by flow cytometry. CD4~+CD25~+Tregs were isolated using magnetic affinity cell sorting and suppressive function were assessed by MTT method. The expression levels of TGF-β1 and Foxp3 mRNA were measured by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The object of this study is to provide new strategies for the treatment of HT. Methods :1 Objects of study 20 new-onset and untreated HT patients were enrolled in this study. Diagnostic criteria refer to〈Guide line for the diagnosis and treatment of thyriod disease in China〉(Chinese Society of Endocrinology, 2008); 20 age- and sex-matched healthy subjects were as controls.2 Methods2.1 The serum levels of thyroid stimulating hormono ( TSH ), total triiodothyronine ( TT3 ), total thyroxine ( TT4 ), thyroglobulin antibody(Tg-Ab), and thyroid peroxidase antibody ( TPO-Ab ) were measured with a commercial electrochemiluminescence ( ECL ) Kit.2.2 Peripheral blood mononuclear cells ( PBMC) were freshly isolated by density-gradient centrifugation from new-onset HT patients and healthy control subjects. The proportion of circulating CD4~+T cells and CD4~+CD25~+Tregs were assessed by flow cytometry.2.3 CD4~+CD25~+Tregs and CD4~+CD25-T cells were isolated using magnetic affinity Cells Sorting from new-onset HT patients and healthy control subjects. CD4~+CD25~+Tregs suppressive function on post stimulused autologus CD4~+CD25-T cells was assessed by MTT method .2.4 Total RNA were isolated from in PBMC. Reverse transcription was performed immediately. The expression levels of TGF-β1 and Foxp3 mRNA were detected by qRT-PCR. Human GAPDH gene as internal control.2.5 The correlations between the serum levels of Tg-Ab, TPO-Ab and the proportion of CD4~+ T cells, CD4~+CD25~+Tregs and the expression levels of TGF-β1, Foxp3 mRNA was analyzed with spearman's.Results:1 The serum concentrations of TSH,TT3,TT4, Tg-Ab and TPO-Ab The serum concentrations of TSH, TT3, TT4 has no significant difference between new-onset HT patients and healthy control group ( p>0.05);The serum concentrations of Tg-Ab and TPO-Ab was much more enhanced than that of healthy control subjects enrolled in the study. ( Table 1 )2 The frequency of circulating CD4~+T cells and CD4~+CD25~+ Tregs A significant decrease proportion of the circulating CD4~+CD25~+ Tregs were observed in HT patients ( 1.55±0.49% vs 2.86±1.04% ) ( p<0.05 ). There is no significant difference in proportion of the circulating CD4~+T cells between HT patients and healthy control subjects ( p>0.05 ). (Table.2)3 The suppressive function of CD4~+CD25~+Tregs on autologous CD4~+CD25-T cellsIn order to evaluate the immunosuppressive properties of Tregs, CD4~+CD25~+T cells and CD4~+CD25-T cells were isolated from 5 new-onst HT patients and healthy controls using magnetic affinity cells sorting. proliferation and coincubation ( at ration of 1:1 ) studies were performed.Upon induction by solution anti-CD3 Ab and inactived antigen present cell (APC), sorted CD4~+CD25-T cells sorted from both HT patients and contols showed a high level of proliferation. In contrast, CD4~+CD25~+Tregs sorted from neither the patients nor the controls proliferated lightly. The suppressive function of CD4~+CD25~+Tregs from HT patients showed diminished suppression on autologous responder T cell proliferation compared with controls (15.7±5.36% vs 41.7±9.87%) ( p<0.05 ).(Fig. 7)4 The expression levels of TGF-β1 and Foxp3 mRNALower expression levels of TGF-β1 ( 0.43±0.10 vs 1.02±0.04 )and Foxp3 mRNA were observed in HT patients compared to healthy control subjects ( 0.42±0.29 vs 0.99±0.17 ) ( p<0.05 ).( Fig. 11, Fig. 12 )5 The correlations between the levels of Tg-Ab, TPO-Ab and the proportion of CD4~+T cells, CD4~+CD25~+Tregs and the expression levels of TGF-β1, Foxp3 mRNA from HT patientsThe serum concentrations of Tg-Ab and TPO-Ab has no significant correlation with the proportion of CD4~+T cells in PBMC (P>0.05); (Fig. 13A, Fig. 13B) The serum concentrations of Tg-Ab correlated negatively with the CD4~+CD25~+T cells proportion in CD4~+T cells ( r=-0.548,P<0.05 ); (Fig. 14A) The serum concentrations of TPO-Ab correlated negatively with the CD4~+CD25~+T cells proportion in CD4~+T cells ( r=-0.585,P<0.05 ) ; (Fig. 14B) The serum concentrations of TPO-Ab correlated negatively with the levels of TGF-β1 ( r=-0.695,P<0.05 ) and Foxp3 mRNA ( r=-0.629,P<0.05 ) in PBMC; (Fig. 15A, Fig. 15B) The serum concentrations of Tg-Ab has no significant correlation with the levels of TGF-β1 and Foxp3 mRNA in PBMC(P>0.05); (Fig. 16A, Fig. 16B ) The CD4~+CD25~+T cells proportion in CD4~+T cells correlated positively with levels of TGF-β1 mRNA ( r=0.709,P<0.05); (Fig 17A) The CD4~+CD25~+T cells proportion in CD4~+T cells correlated positively with levels of Foxp3 mRNA ( r=0.591,P<0.05).( Fig 17B )Conclusions:1 A significant decrease proportion of the circulating CD4~+CD25~+Tregs was observed in new-onset HT patients.2 The suppressive function of CD4~+CD25~+Tregs from new-onset HT patients showed diminished suppression on autologous CD4~+CD25-T cell proliferation.3 Lower expression levels of TGF-β1 and Foxp3 mRNA were observed in new-onset HT patients compared with healthy control subjects.4 The serum concentrations of Tg-Ab, TPO-Ab correlated negatively with the CD4~+CD25~+Tregs proportion in CD4~+T cells; The serum concentrations of TPO-Ab correlated negatively with the levels of TGF-β1 and Foxp3 mRNA in PBMC. The CD4~+CD25~+Tregs proportion in CD4~+T cells correlated positively with levels of TGF-β1 and Foxp3 mRNA in PBMC.5 The decreasing amount and function of circuling CD4~+CD25~+Tregs lead to the increase of TPO-Ab production may involved in the development of HT. Objective: T help 17( Th17 ) cell is a new subset of CD4~+ T cells that is apparently different from Th1, Th2 cells. It has independence of cell differentiation, development and biological function. Th17 cells specificly express transcription factor of retinoic acid receptor-related orphan receptorγt (RORγt) and mainly secrete cytokine interleukin-17 ( IL-17A ). IL-17 is a proinflammatory cytokine that can recruit and activate neutrophile, enhance T cells immune response and promote the release of inflammation mediator. It is much more important for host clearing pathogen and promoting autoimmune disease as well as inducing tissue inflammatory.The presence of IL-17 in plasma and biopsies of many human autoimmune disease patients ( such as Crohn's disease, psoriasis and atopic dermatitis and so on) was reported long before. Latest studies confirmed that Th17 cells were characterized as a distinct T helper cell lineage certainly reside in these disease. But there are few studies focus on Th17 cells in human Hashimoto's thyroiditis. Thus, in this study we assessed the plasma concentrations of IL-17 by ELISA method. The expression levels of RORγt, IFN-γ, IL-4 mRNA were measured by qRT-PCR. The distribution and the amount of IL-17~+ cells in thyroid gland were observed by Immunohistochemistry. The purpose of this study is to explore the role of Th17 cells in the development of HT.Methods :1 Objects of study and materials 20 new on-set HT patients and controls are from the same groups with part one. Thyroid glands surgical specimens were obtained from 12 HT patients and 4 healthy controls dead from trauma.2 Methods:2.1 The plasma levels of IL-17 from HT patients and healthy control subjects were determined by ELISA kit.2.2 PBMC were isolated from new on-set HT patients as well as healthy control subjects. Total RNA were extracted from PBMC.The expression levels of RORγt, IFN-γand IL-4 mRNA were detected by qRT-PCR. Human GAPDH gene as internal control.2.3 IL-17~+ cells in thyroid gland were observed by immunohistochemistry.Result:1 The plasma levels of IL-17 The plasma levels of IL-17 from HT patients and healthy control subjects was too low to detected by kit.2 The expression levels of RORγt, IFN-γand IL-4 mRNA in PBMC Higher expression levels of RORγt (1.95±0.65) was observed in PBMC from HT patients compared to healthy control subjects (1.3±0.71)(P<0.05). However, there was no significant difference with the expression levels of IFN-γ, IL-4 mRNA between two groups ( P>0.05 ). ( Fig.1 )3 Distribution of IL-17~+ cells in thyroid gland Immunohistochemical analysis of thyroid sections revealed that IL-17~+ cells appeared in thyroid gland from both HT patients and healthy control subjects, and mainly within lymphocyte. There was a significant increase of IL-17~+ cells in thyroid gland from HT patients compared with healthy control subjects. ( Fig.2, Fig.3, Fig.4 )Conclusion:1 The concentration of IL-17 in plasma from HT patients was not detected.2 The expression levels of RORγt mRNA in new on-set HT patients were higher than healthy control subjects. There was bulk of IL-17~+ lymphocyte in thyroid gland from HT patients. It shows that Th17 cells may play a role in the development of HT.