Background/Purpose: Enhancer of zeste homolog 2 (EZH2) has been shown to regulate early B cell development and the differentiation of antibody secreting cells (ASCs). We have previously demonstrated increased EZH2 expression in peripheral blood mononuclear cells isolated from lupus patients, and that pharmacological inhibition of EZH2 alleviates lupus-like disease in mouse models. The goal of this study was to evaluate the role of B cell EZH2 overexpression in lupus pathogenesis.

Methods: Using CRISPR/Cas9 technology, we generated an MRL/lpr mouse with floxed Ezh2, which was crossed with CD19-Cre mice to examine the effect of EZH2 deficiency in B cells in MRL/lpr lupus-prone mice. Plasma anti-double-stranded DNA (anti-dsDNA) antibody levels and proteinuria were measured by ELISA. Kidney sections were stained with hematoxylin and eosin (H&E) to evaluate nephritis. Differentiation of B cells was assessed by flow cytometry. Single cell RNA sequencing and single cell B cell receptor sequencing were used to investigate compositional and functional changes of B cell subsets. In vitro B cell culture with/without an XBP1 inhibitor was performed. EZH2 and XBP1 mRNA levels in CD19⁺ B cells isolated from SLE patients and healthy controls were analyzed.

Results: We show that Ezh2 deletion in B cells significantly decreased anti-dsDNA antibody production and improved glomerulonephritis in MRL/lpr mice. B cell development and differentiation were altered in the bone marrow and spleen in EZH2-deficient mice. Differentiation of germinal center (GC) B cells and ASCs was impaired. Single-cell RNA sequencing showed that XBP1, a key transcription factor in B cell development, was downregulated in the absence of EZH2, and that inhibiting XBP1 in vitro impairs ASC development similar to EZH2-deficient mice. Further, single cell B cell receptor RNA sequencing revealed defective immunoglobulin class switch recombination in EZH2-deficient mice. IGHM (immunoglobulin heavy constant mu) which defines the IgM isotype was significantly higher, and IGHGs (immunoglobulin heavy constant gamma) including IGHG1, IGHG2C, and IGHG3 which encodes IgG1, IgG2c and IgG3 respectively showed a trend to be lower in the plasma cells from CD19 Cre-Ezh2^fl/fl mice compared to Ezh2^fl/fl controls. In lupus patient B cells, we observed a strong correlation between EZH2 and XBP1 mRNA expression levels.

Conclusion: Our results suggest that EZH2 overexpression in B cells contributes to disease pathogenesis in lupus. EZH2 enhances GC B cell development and the differentiation of ASCs, at least in part via upregulating XBP1. Inhibiting B cell EZH2 expression impairs B cell development and immunoglobulin class switch recombination, and might provide a novel therapeutic approach in lupus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ezh2-knockout-in-b-cells-impairs-plasmablast-differentiation-and-ameliorates-lupus-like-disease-in-mrl-lpr-mice/