EZH2 promotes B-cell autoimmunity in primary Sjogren's syndrome via METTL3-mediated m6A modification

Enhancer of zeste homologue 2 (EZH2) plays an important role in promoting B-cell activation and differentiation. This study aimed to elucidate the role of EZH2 in the B-cell autoimmune response in primary Sjögren's syndrome (pSS) and to explore the therapeutic potential of inhibiting EZH2 in pSS. Single-cell RNA sequencing analysis of B cells in peripheral blood from pSS patients was conducted to identify abnormal expression of EZH2 and METTL3 in B-cell subsets. The levels of EZH2 were further validated across multiple B-cell subsets and the salivary glands (SGs) of pSS patients, as well as three different mouse models of Sjögren's syndrome (SS). Correlation analyses were performed to explore the relationship between the expression of EZH2 and clinical features of pSS patients. Following EZH2 inhibition, SS-like signs and antibody production were assessed in an experimental Sjögren syndrome (ESS) mouse model. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data post-EZH2 inhibition were bioinformatically analyzed to identify the EZH2 targets in pSS. ChIP-qPCR was performed to validate the binding of H3K27me3 to the CDKN1A promoter. Flow cytometric apoptosis analysis and Carboxy Fluorescein Succinimidyl Ester (CFSE) assay were used to assess the impact of an EZH2 inhibitor on B-cell apoptosis and proliferation. Additionally, METTL3 expression and its correlation with disease activity were analyzed in pSS patients. EZH2 expression was examined after METTL3 knockdown. METTL3-RNA immunoprecipitation (RIP) and actinomycin D assays were conducted to confirm the direct binding of METTL3 to EZH2 mRNA and its impact on mRNA stability. M6A-RIP-qPCR was performed to validate the presence of m6A modifications on EZH2 mRNA. EZH2 was found upregulated in multiple B-cell subsets from the peripheral blood and SGs of pSS patients, as well as in three different animal models of SS. The expression of EZH2 in B cells was positively correlated with the ESSDAI score, which is a measure of disease activity. With treatment of EZH2 inhibitor, SS-like signs alleviated and autoantibody production reduced in ESS mice. Similarly, in pSS patients, METTL3 expression was increased in the SGs and peripheral blood CD19 + B cells, also showing a positively correlated with the ESSDAI score. With knockdown of METTL3 , the expression of EZH2 reduced. Mechanistically, EZH2 inhibited B-cell apoptosis and promoted B-cell proliferation by catalyzing H3K27me3 modification at the CDKN1A locus. Furthermore, METTL3 bound to EZH2 mRNA and increased m6A modification on EZH2 mRNA, enhancing its stability and promoting EZH2 expression. The upregulation of EZH2 mediated by METTL3 is implicated in the B-cell autoimmune response in pSS. Inhibition of EZH2 presents a promising therapeutic strategy for pSS treatment. In B cells and salivary gland (SG) tissues from patients with primary Sjögren's syndrome (pSS), METTL3 expression is elevated, resulting in increased m6A methylation of EZH2 mRNA. This modification enhances EZH2 mRNA stability, thereby promoting EZH2 expression. As an epigenetic regulator, EZH2 facilitates the trimethylation of histone H3 at lysine 27 (H3K27me3) on the CDKN1A locus, leading to suppressed CDKN1A expression and reduced B cell apoptosis. This mechanism contributes to the abnormal hyperreactivity of B cells in pSS, leading to the expansion of autoreactive antibody-secreting cells (ASCs), increased autoantibody production, and ultimately, a breakdown in peripheral immune tolerance in pSS B cells [Figure was created By Figdraw ( https://www.figdraw.com ).]. . • EZH2 was elevated in diverse B-cell subsets and SGs from pSS. • EZH2 reduced B-cell apoptosis by increasing H3K27me3 levels at the CDKN1A locus in pSS. • The upregulation of EZH2 mediated by METTL3 was implicated in the B-cell autoimmune response in pSS. • Targeting EZH2 could be a promising therapeutic strategy for pSS.