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Background: MicroRNAs (miRNAs) are a class of critical epigenetic regulators involved in several autoimmune diseases. Our previous study reported an miR-326-induced increase in T helper (Th) 17 cells in a mouse model of Hashimoto's thyroiditis (HT), but the pathogenic effect of miR-326 in HT patients has not been verified. The goal of the present study was to explore the pathogenic role of miR-326 and its underlying molecular mechanism in HT patients. Methods: A total of 58 HT patients and 55 normal controls were enrolled in this study. We examined whether Th17 cells and miR-326 were aberrantly altered in the peripheral blood mononuclear cells (PBMCs) of HT patients with flow cytometry and real-time polymerase chain reaction. Levels of membrane interleukin (IL)-23R (mIL-23R) were determined by flow cytometry and Western blot to explore the critical role of mIL-23R in the development of Th17 cells. Isolated CD3+ T cells were used to further investigate the ectodomain shedding of mIL-23R by a disintegrin and metalloprotease (ADAM17). Furthermore, miR-326 inhibitor and mimics were transfected into PBMCs derived from HT patients and healthy controls to verify the regulation of ADAM17 by miR-326. Results: We observed elevated miR-326 levels in the PBMCs of HT patients compared with those in the PBMCs of healthy controls. Consistent with IL-23-induced STAT3 overactivation, substantially more HT patient-derived PBMCs differentiated into Th17 cells under polarization culture conditions, which may, at least in part, have resulted from enhanced mIL-23R levels. Furthermore, ADAM17, an ectodomain sheddase of mIL-23R, was targeted and negatively regulated by miR-326. Inhibiting ADAM17 might attenuate the ectodomain shedding of mIL-23R. Conclusions: Our findings suggest that the effect of miR-326 on the IL-23/IL-23R/Th17 cell axis in HT patients might be partially due to the targeting of ADAM17. [ABSTRACT FROM AUTHOR]

Th17 and regulatory lymphocyte subsets such as Tregs and Bregs have been reported to play important roles in autoimmune diseases. The aim of this work was to perform quantitative studies of circulating Th17, Tregs, and Bregs in patients with new-onset Graves’ disease (GD). Twenty GD patients and 20 healthy controls were involved in this study. Blood samples were taken for flow cytometry detection of CD4+IL-17+ Th17, CD4+Foxp3+ Tregs, and CD19+CD1dhiCD5+ Bregs and meanwhile, for real-time PCR measurement of gene expressions of RORγt, IL-17 and IL-10. The proportions of Tregs and Bregs as well as the Foxp3 gene expression but not IL-10 were significantly decreased in GD group compared with the healthy controls. The frequency of Th17 together with the gene expressions of RORγt and IL-17 were significantly increased in the GD group. Furthermore, the Th17/Treg ratio was also significantly higher in GD group. A significant positive correlation between Th17 and TSAb (r=0.656, p<0.001) but significant negative correlations between Treg/Breg and TSAb (r=-0.339, p=0.032; r=-0.759, p<0.001) were identified among the participants. This study indicated that increased Th17 and impaired Treg responses, along with a decreased number of CD19+CD1dhiCD5+ Breg cells, were involved in GD pathogenesis. [ABSTRACT FROM AUTHOR]

Graves' disease (GD) involves auto-immunity against thyroid cell antigens, but the reasons for the induction of auto-immunity are uncertain. We wished to investigate the role of T helper 17 (Th17) and regulatory T cells (Treg) in a mouse model of Graves' hyperthyroidism. The model was generated by immunizing mice with adenovirus expressing the autoantigen thyroid-stimulating hormone receptor (Ad-TSHR289). The frequencies of splenic Th17 and Treg were determined by flow cytometry. The levels of interleukin-17(IL-17), forkhead box P3 (Foxp3), and orphan retinoic acid nuclear receptor (RORγt) mRNA were determined by real-time PCR. The number of CD4CD25Foxp3 T lymphocyte was significantly reduced in the Ad-TSHR289 group compared with the Ad-control ( P < 0.05). mRNA level for Foxp3 was less abundant in Ad-TSHR289 group compared with Ad-control ( P < 0.05). However, CD4IL-17 T-cell subpopulation and expression of RORγt mRNA did not differ significantly between Ad-TSHR289 and Ad-control groups ( P > 0.05). Nevertheless, in Ad-TSHR289 group, a profound increase in the Th17/Treg ratios was found. The present study demonstrates that Th17 is not involved in promoting Graves' hyperthyroidism, while Treg and the ratio of Th17/Treg might play a role in the pathogenesis of Graves' hyperthyroidism. [ABSTRACT FROM AUTHOR]

Exosomes have been intensively studied in autoimmune diseases, and circulating exosomes and microvesicles have also been explored in autoimmune thyroiditis (AITD). However, the role of thyroid cell-derived exosomes in immune responses is unclear. We showed that IFN-γ-treated Nthy-ori 3-1 cell-derived exosomes (IFN-γ-Exo) harbored TPO, HSP60 and MHC-II and activated dendritic cells (DCs) in vitro. Compared with Exo-targeted DCs (DC Exo), IFN-γ-Exo-targeted DCs (DC IFN-γ-Exo) promoted the expression and release of proinflammatory cytokines, such as IFN-γ, IL-17A and IL-22, from CD4+ T lymphocytes and inhibited the expression and release of anti-inflammatory cytokines, such as IL-4, IL-10 and TGF-β1; however, IFN-γ-Exo did not have this effect compared with Nthy-ori 3-1 cell-derived exosomes (Exo). DC IFN-γ-Exo stimulates the expression and release of cytokines from CD4+ T lymphocytes more efficiently than IFN-γ-Exo. Thus, DC IFN-γ-Exo may effectively induce CD4+ T lymphocyte-mediated immune responses and play a role in the occurrence and development of AITD. • IFN-γ-treated thyroid cell-derived exosomes (IFN-γ-Exo) express TPO, HSP60, MHC-II. • IFN-γ-Exo activate dendritic cells (DCs). • IFN-γ-Exo need to activate DCs before triggering CD4+ T lymphocyte differentiation. [ABSTRACT FROM AUTHOR]