EZH2 Expression in B Cell Lymphocyte Subsets of Hashimoto's Thyroiditis and the Therapeutic Mechanism and Effect of Its Inhibitors

Abstract

Background Thyroid autoantibody is a marker for the diagnosis of Hashimoto's thyroiditis (HT), and B cells are essential in the pathogenesis of HT. Enhancer of Zeste homolog 2 (EZH2), which is an important epigenetic regulator, plays an important role in the regulation of lymphocytes development and function. Objective To investigate EZH2 expression in plasmoblasts and plasma cells in HT, and further explore the therapeutic effect of EZH2 inhibitors in experimental autoimmune thyroiditis (EAT) model. Methods The thyroid tissues from 6 patients who underwent thyroidectomy (3 HT patients with PTC, 3 patients with PTC alone) in Peking University First Hospital between 2010 and 2020 were obtained from the contralateral lobe with thyroid cancer, and screened for the expression of B-lymphocyte-related genes by RNA-seq; thyroid tissues from 16 HT patients and 8 normal thyroid tissues were collected and verified for the the expression of EZH2 in B cells in HT thyroid tissues by immunohistochemistry or immunofluorescence, respectively. Fine-needle aspiration (FNA) samples from patients with HT (n=25), and peripheral blood from patients with HT (n=19) or healthy donors (n=12) were analyzed by flow cytometry to define altered EZH2 expression in plasmablasts and plasma cells. Fifteen seven-week-old NOD.H-2h4 mice were randomly divided into the control (n=5), EAT without injection group (n=5), and EZH2 inhibitor+ GSK126 injection group (10 mg/kg, intraperitoneal injection 3 times /week, n=5). The degree of thyroid inflammation and changes in TgAb levels were observed after 8 weeks. Results RNA sequencing analysis showed that EZH2 and genes associated with the B-cell phenotype such as CD19, CD27, CD38, CD52 were higher expressed in HT hyroid tissues compared with normal thyroid tissues. Immunohistochemical results showed that immunohistochemical staining for EZH2 in 16 HT thyroid tissue specimens was strongly positive with positive cells observed in the GC region, and no positive cells were observed in the staining of 8 normal thyroid tissues. EZH2 staining in HT thyroid tissue was highly expressed in the GC region, and EZH2 was specifically expressed in CD19+ B cells. The results of flow cytometry assay showed that the proportion of CD19+ B cells, plasmablasts and plasma cells in HT FNA samples was higher than that of HD peripheral blood and HT peripheral blood samples (P<0.01), and the proportion of EZH2 positivity in CD19+ B cells and plasma cells was higher in HT FNA samples than that of HT peripheral blood (P<0.005). In the mouse experiments, lymphocytic infiltration of the thyroid tissues was increased in the EAT group compared to the control group. In the GSK126 treatment groups, the thyroid inflammatory score and serum TgAb titer were significantly higher than the control group and lower than the EAT group. Conclusion EZH2 over-expression in CD19+ B cells of HT hyroid tissues may promote the differentiation of B cells into plasma cells and auto-antibody production, which leads to the destruction of thyroid tissues. EZH2 inhibitors can slow down the degree of thyroid inflammation in the EAT model. Increased EZH2 expression in plasmablasts may be involved in the pathogenesis of HT. EZH2 may serve as a therapeutic target for HT, although further studies are needed.

Keywords

• hashimoto thyroiditides|b-lymphocyte subsets|enhancer of zeste homolog 2 protein|hypothyroidism|targeted therapy