Study On The Variation Of Th17Cells And Related Regulatory Factors In Patients With Hashimoto’s Thyroiditis

Objective:To investigate the variations of CD4+T-helper17(Th17) cells and the level of Thl7cells related cytokines in patients with Hashimoto’s Thyroiditis (HT), analyze the relations between Th17cells and autoantibodies, evaluate the role of Th17cells and related cytokines in the pathogenesis of patients with HT. To investigate the proportion of Th17and regulatory T cells (Treg) and the expression of specific transcription factors, analyze the relations between the ratio of Th17/Treg and autoantibodies, explore the role of immune deviation of Th17/Treg in HT patients.To investigate the level of Glucocorticoid-induced TNF receptor-related protein ligand (GITRL) in peripheral blood of patients with HT and its correlation with Th17cells or Treg cells, explore the role of GITR/GITRL in the balance of Th17/Treg. To study miRNAs of regulating interleukin-23receptor (IL-23R) expression, explore the regulation role of miR-125a-3p on IL-23R in peripheral blood mononuclear cells (PBMC) of patients with HT.Methods:1. The frequency of CD3+CD8-IL-17+T (Thl7) cells in PBMC of patients with HT and healthy controls was analyzed by flow cytometry (FCM), the concentration of plasma IL-6、IL-23were measured by enzyme-linked immunosorbent assay (ELISA). QRT-PCR was used to measure the expression of RORyt mRNA in PBMC, the expression of RORyt and IL-17mRNA in thyroid tissue was measured by RT-PCR.2. The levels of anti-thyroglobulin antibody (TG-Ab) and anti-thyroperoxidase antibody (TPO-Ab) in patients with HT were detected by chemiluminescence immunoassay(CIA), correlation between Th17and the levels of autoantibodies was analyzed.3. The frequeney of CD4+CD25+CD127lowT (Treg) cells in PBMC of patients with HT and healthy controls was analyzed by FCM, qRT-PCR was used to measure the expression of Foxp3mRNA in PBMC.4. The ratio of Th17/Treg and correlation with the levels of TG-Ab was analyzed.5. The concentration of plasma GITRL in HT patients and healthy controls were measured by ELISA. QRT-PCR was used to measure the expression of GITRL mRNA in thyroid tissue of patients with HT and healthy controls. Correlation between the levels of plasma GITRL and ratio of T17/Treg was analyzed.6. Naive CD4+T cells were purified by immune magnetic beads from normal murine, and incubated under the Th17-differentiation condition. The role of GITRL on the differentiation of Th17cells was observed.7. QRT-PCR was used to measure the expression of IL-23R mRNA in PBMC of patients with HT and healthy controls. Targing IL-23R gene, through miRanda and PicTar software analysis, we listed the miRNAs probably target to IL-23R.8. The expression of the predicted miRNAs in HT patients and healthy controls were detected by qRT-PCR. The miRNA in line with expectations was chosen for further research.9. The targeting role of miR-125a-3p on IL-23R was validated by luciferase reporter assay, to clear the binding site strength of miR-125a-3p and IL-23R3’UTR. MiR-125a-3p mimics and control transfection was done in PBMC expressing IL-23R from the people conducted health examination as target cells. The expression of IL-23R in PBMC was measured by FCM.10. QRT-PCR was used to measure the expression of miR-125a-3p in PBMCs of HT patients and healthy controls. Correlation between the levels of miR-125a-3p in PBMCs and the levels of autoantibodies was analyzed.Results:1. The proportion of Th17cells were significantly increased in HT patients compared with healthy controls[(0.75±0.79)%versus (0.28±0.23)%, P<0.05]. The level of RORγt mRNA in PBMC was significantly higher in HT patients compared with that from healthy controls (0.30±0.38versus0.04±0.02, P<0.05). The concentrations of plasma IL-6、IL-23were increased in HT patients compared with healthy controls (3.66±4.70versus0.47±1.11pg/ml, P<0.05)(154.7±75.81versus80.65±61.41pg/ml, P<0.05). Compared with the simple goiter group, the expression of RORyt and IL-17mRNA in thyroid tissue was measured in HT patients.2. A strong positive correlation between the proportion of Th17cells in PBMC and levels of TG-Ab (r=0.8484, P=0.0077) and a borderline correlation that with the levels of TPO-Ab (r=0.6224, P=0.0994) were found in HT patients.3. The proportion of Treg cells was significantly decreased in HT patients(P<0.01). The expression of Foxp3mRNA was significantly decreased in HT patients compared with healthy controls (P<0.05).4. The ratio of Thl7/Treg was significantly increased in HT patients (P<0.01) and had significant correlation with the levels of TG-Ab (r=0.5243, P=0.0176)5. The concentration of plasma GITRL was significantly increased in HT patients (P<0.05). The levels of GITRL had significantly positive correlation with proportion of Th17cells (r=0.5282, P=0.0167). mGITRL can significantly promote the differentiation of Thl7cells under the Th17-differentiation condition.6. The expression of RORγt、IL-17mRNA and GITRL mRNA in thyroid tissues was significantly increased in HT patients compared with the simple goiter group (P<0.05).7. The level of IL-23R mRNA in PBMCs was significantly increased in HT patients (P<0.05). Through software analysis, we listed two miRNAs probably target to IL-23R:miR-155and miR-125a-3p. The expression level of miR-125a-3p was significantly decreased in HT patient (P<0.05), in contrast with the upregulation of IL-23R mRNA level.8. The luciferase reporter assay confirmed that miR-125a-3p can target to the seed region of IL-23R and can inhibit the expression of downstream IL-23R gene, fluorescence intensity was decreased. The results show that miR-125a-3p had direct targeted relationship with IL-23R gene.The percentage of IL-23R+cells was decreased after miR-125a-3p transfection in PBMC, indicated miR-125a-3p can inhibit the expression of IL-23R.9. The expression levels of miR-125a-3p measured by qRT-PCR were significantly decreased in PBMC of HT (P<0.05). A negative correlation was found between the levels of miR-125a-3p and the levels of IL-23R mRNA in HT (r=-0.5195, P=0.0226). The expression levels of miR-125a-3p had a significantly negative correlation with the levels of TG-Ab(r=-0.4811, P=0.0371).Conclusions:1. The increased proportion of Th17cells and related cytokine were observed in patients with HT. The proportion of Th17cells was positively correlated with the levels of autoantibodies. The results show that Th17cells and related cytokines may play an important role in development of HT.2. The elevated of Th17cells were accompanied with the decreased Treg cells as same as changes of transcription factors RORγt and Foxp3with the significant correlation between the ratio of Th17/Treg and TG-Ab, suggesting that Th17/Treg imbalance may be involved in HT patients. It provided a new insight in elucidating the pathogenesis of HT.3. GITRL could promote the development of Th17cells in vitro. The increased expression levels of GITRL in HT patients were positively correlated with Th17cells. HT patients had significantly increased levels of GITRL、RORγt、 IL-17mRNA in thyroid tissues. The results show that GITRL may be involved in the regulation the balance of Thl7/Treg cells.4. MiR-125a-3p can derectly regulate the expression of IL-23R. The decreased expression levels of miR-125a-3p in PBMCs of HT patients were negatively correlated with increased expression levels of IL-23R mRNA. Moreover, the expression levels of miR-125a-3p had negative correlation with the levels of autoantibodies. It is indicated the possible involvement of miR-125a-3p in the pathogenesis of HT, and also in the severity of desease.