METTL3 通过促进 IRF4 介导的浆细胞浸润促进系统性红斑狼疮中的肾脏损伤，通过 m6A 依赖的方式。
METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus.
机构信息
Department of Nephrology, Center of Kidney and Urology, the Seventh Affiliated Hospital, Sun Yat-Sen University, Shenzhen, 518107, China.
Department of Hematology, Guangzhou Women and Children's Medical Center, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou Medical University, Guangzhou, 510623, China.
出版信息
BMC Med. 2024 Nov 5;22(1):511. doi: 10.1186/s12916-024-03735-y.
BACKGROUND
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause. N6-methyladenosine (m6A) is the most common mRNA modification and participates in various immune processes such as interferon production and immune cell regulation. However, the role of m6A in dysregulated immune response of SLE remains unknown.
METHODS
PBMCs from SLE patients were collected to compare the m6A modification profile by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Interferon regulatory factor 4 (IRF4) was identified by combination with MeRIP-seq and RNA-Seq. IRF4 and methyltransferase 3 (METTL3) were detected using qRT-PCR and WB. Clinical significance of IRF4 in SLE patients was explored subsequently. IRF4 expression in B cell subsets of female MRL/lpr mice was detected by flow cytometry. Adeno-associated viruses (AAV) including AAV9-METTL3-OE and/or AAV9-IRF4-sh were treated with female MRL/lpr mice. Autoantibody levels and kidney injury were tested by ELISA, pathological staining, and immunofluorescence. m6A level of IRF4 was detected by MeRIP-qPCR. The downstream effectors of IRF4 contributing to renal pathology were explored by RNA-seq and verified by qRT-PCR.
RESULTS
m6A methylation features were obviously aberrant in SLE patients, and IRF4 was the upregulated gene modified by m6A. METTL3 and IRF4 expressions were elevated in SLE patients and kidney of MRL/lpr mice. Clinical analysis indicated that SLE patients with high IRF4 level were more prone to kidney damage. IRF4 expression was especially increased in plasma cells of MRL/lpr mice. METTL3 induced renal IRF4 expression, plasma creatinine, ANA and urine ALB levels, IgG and C3 deposition, and renal damage and plasma cell infiltration were aggravated in MRL/lpr mice. However, IRF4 depletion could partially reduce METTL3-induced kidney damage. Meanwhile, m6A level of IRF4 elevated with METTL3 overexpression. Also, the expression of Cxcl1, Bcl3, and Fos mRNA were significantly reduced after knockdown of IRF4, which were mainly involved in TNF signaling pathway.
CONCLUSIONS
Our study confirmed that upregulated METTL3 promoting IRF4 expression in an m6A-dependent manner, thus causing plasma cell infiltration-mediated kidney damage of SLE. This provides new evidence for the role of m6A in SLE kidney injury.
背景
系统性红斑狼疮（SLE）是一种病因不明的自身免疫性疾病。N6-甲基腺苷（m6A）是最常见的 mRNA 修饰物，参与干扰素产生和免疫细胞调节等多种免疫过程。然而，m6A 在 SLE 中失调的免疫反应中的作用尚不清楚。
方法
收集 SLE 患者的 PBMCs，通过甲基化 RNA 免疫沉淀测序（MeRIP-seq）比较 m6A 修饰谱。结合 MeRIP-seq 和 RNA-Seq 鉴定干扰素调节因子 4（IRF4）。使用 qRT-PCR 和 WB 检测 IRF4 和甲基转移酶 3（METTL3）。随后探讨了 IRF4 在 SLE 患者中的临床意义。通过流式细胞术检测雌性 MRL/lpr 小鼠 B 细胞亚群中的 IRF4 表达。用携带 METTL3-OE 和/或 IRF4-sh 的腺相关病毒（AAV）处理雌性 MRL/lpr 小鼠。通过 ELISA、病理染色和免疫荧光检测抗自身抗体水平和肾脏损伤。通过 MeRIP-qPCR 检测 IRF4 的 m6A 水平。通过 RNA-seq 探讨并通过 qRT-PCR 验证 IRF4 对肾脏病理的下游效应子。
结果
SLE 患者的 m6A 甲基化特征明显异常，IRF4 是受 m6A 修饰的上调基因。SLE 患者和 MRL/lpr 小鼠的肾脏中 METTL3 和 IRF4 的表达升高。临床分析表明，IRF4 水平高的 SLE 患者更易发生肾脏损伤。IRF4 表达在 MRL/lpr 小鼠的浆细胞中尤其增加。METTL3 诱导肾脏中 IRF4 的表达、血浆肌酐、ANA 和尿 ALB 水平、IgG 和 C3 沉积以及肾脏损伤和浆细胞浸润在 MRL/lpr 小鼠中加重。然而，IRF4 耗竭可部分减轻 METTL3 诱导的肾脏损伤。同时，METTL3 过表达可增加 IRF4 的 m6A 水平。此外，IRF4 敲低后，Cxcl1、Bcl3 和 Fos mRNA 的表达显著降低，主要涉及 TNF 信号通路。
结论
本研究证实，上调的 METTL3 通过 m6A 依赖性方式促进 IRF4 表达，从而导致 SLE 中浆细胞浸润介导的肾脏损伤。这为 m6A 在 SLE 肾损伤中的作用提供了新的证据。
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