METTL3通过介导IRF8的m6A甲基化激活TLR4/NF-kB通路，参与m.tb诱导的THP-1巨噬细胞损伤和炎症

IF 2.9 3区医学 Q3 IMMUNOLOGY

Tuberculosis Pub Date : 2025-06-13 DOI:10.1016/j.tube.2025.102667

Yunhua Chen , Xiaolin Tang , Chao He , Chong Xiao , Ziping Zhao

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引用次数: 0

摘要

巨噬细胞在细胞内细菌感染的免疫应答中起着核心作用，包括结核分枝杆菌（M.tb）。甲基转移酶样3 （Methyltransferase-like 3, METTL3）参与了TB巨噬细胞的调控，本研究拟通过m.tb感染THP-1巨噬细胞建立体外模型，探讨METTL3与干扰素调节因子-8 （interferon regulatory factor-8, IRF8）在TB中的分子机制。方法采用srt - qpcr和Western blot分别分析mRNA和蛋白的表达。通过细胞计数试剂盒-8法、EdU法和流式细胞术/TUNEL法检测细胞活力、增殖和凋亡。采用酶联免疫吸附法检测炎症因子。甲基化RNA免疫沉淀（MeRIP）， RIP和Co-IP评估基因之间的相互作用。结果sirf8敲低可减轻结核分枝杆菌感染THP-1巨噬细胞的损伤和炎症反应。METTL3通过诱导m6A甲基化增强IRF8 mRNA的稳定性。IGF2BP1作为m6A读取器影响IRF8的m6A甲基化。METTL3在m.tb诱导的THP-1巨噬细胞中的作用归因于IRF8的正调控。IRF8结合TLR4和METTL3可以通过靶向IRF8调控TLR4的表达。IRF8/TLR4轴促进m.tb诱导的THP-1细胞损伤和炎症。TLR4/NF-kB通路被mettl3介导的IRF8激活。结论METTL3通过诱导IRF8的m6A甲基化激活TLR4/NF-kB通路，加速了m.tb感染的THP-1巨噬细胞的细胞损伤和炎症反应。

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METTL3 contributes to M.tb-induced injury and inflammation in THP-1 macrophages by mediating m6A methylation of IRF8 to activate TLR4/NF-kB pathway

Background

Macrophages play central roles in the immunity response to infection of intracellular bacteria, including Mycobacterium tuberculosis (M.tb) in tuberculosis (TB). Methyltransferase-like 3 (METTL3) has been implicated in the macrophage regulation in TB, and this study intended to investigate the molecular mechanism of METTL3 with interferon regulatory factor-8 (IRF8) in TB using in vitro model established by M.tb-infected THP-1 macrophages.

Methods

RT-qPCR and Western blot were utilized to analyze mRNA and protein expression, respectively. Cell viability, proliferation, and apoptosis were examined through cell counting kit-8 assay, EdU assay, and flow cytometry/TUNEL assay. Inflammatory cytokines were detected via enzyme-linked immunosorbent assay. Methylated RNA Immunoprecipitation (MeRIP), RIP and Co-IP were performed to assess the interaction between genes.

Results

IRF8 knockdown alleviated injury and inflammation in M.tb-infected THP-1 macrophages. METTL3 enhanced IRF8 mRNA stability by inducing m⁶A methylation. IGF2BP1 functioned as an m⁶A reader to affect m⁶A methylation of IRF8. The function of METTL3 in M.tb-induced THP-1 macrophages was attributed to the positive regulation of IRF8. IRF8 bound to TLR4 and METTL3 could regulate TLR4 expression via targeting IRF8. IRF8/TLR4 axis promoted M.tb-induced THP-1 cell injury and inflammation. TLR4/NF-kB pathway was activated by METTL3-mediated IRF8.

Conclusion

These findings revealed that METTL3 expedited cell injury and inflammatory reaction in M.tb-infected THP-1 macrophages by inducing m⁶A methylation of IRF8 to activate TLR4/NF-kB pathway.

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来源期刊

Tuberculosis 医学-呼吸系统

CiteScore

4.60

自引率

3.10%

发文量

审稿时长

49 days

期刊介绍： Tuberculosis is a speciality journal focusing on basic experimental research on tuberculosis, notably on bacteriological, immunological and pathogenesis aspects of the disease. The journal publishes original research and reviews on the host response and immunology of tuberculosis and the molecular biology, genetics and physiology of the organism, however discourages submissions with a meta-analytical focus (for example, articles based on searches of published articles in public electronic databases, especially where there is lack of evidence of the personal involvement of authors in the generation of such material). We do not publish Clinical Case-Studies. Areas on which submissions are welcomed include: -Clinical TrialsDiagnostics- Antimicrobial resistance- Immunology- Leprosy- Microbiology, including microbial physiology- Molecular epidemiology- Non-tuberculous Mycobacteria- Pathogenesis- Pathology- Vaccine development. This Journal does not accept case-reports. The resurgence of interest in tuberculosis has accelerated the pace of relevant research and Tuberculosis has grown with it, as the only journal dedicated to experimental biomedical research in tuberculosis.