Dai Zhen-Zhen, Xu Jing, Zhang Qin, Zhou Han, Liu Xiao-Man, Li Hai
Department of Pediatric Orthopedics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; Shanghai Institute for Pediatric Research, Shanghai, China.
Department of Pediatric Orthopedics, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Free Radic Biol Med. 2025 Feb 16;228:267-280. doi: 10.1016/j.freeradbiomed.2025.01.013. Epub 2025 Jan 8.
Elevated synovial expression of the triggering receptor expressed on myeloid cells 1 (TREM1) has been identified as a significant biomarker for assessing disease activity in rheumatoid arthritis (RA). The upregulated expression of TREM1, induced by inflammatory mediators in infiltrating macrophages, plays a critical role in synovitis and joint destruction in RA. Our previous sequencing data linked TREM1 activation to aberrant mitophagy. Thus, we explored the efficacy of targeting TREM1 in treating experimental arthritis and its regulatory effect on mitophagy. TREM1 signalling activation was assessed via TREM1, DAP12, and p-SYK levels, and mitophagy was measured through PINK1, PARKIN, and LC3A/B levels. In vitro, TREM1-overexpressing RAW264.7 cells were generated, and the differences in expression and pathways were analyzed via RNA-seq. Changes in the number and morphology of mitochondria and mitophagy in TREM1-overexpressing RAW264.7 cells and normal control were observed via transmission electron microscopy, MitoTracker confocal microscopy and mitochondrial membrane potential analysis. The promotion of TOMM40 gene transcription by TREM1-activated E2F1 was determined via ChIP-PCR and E2F1 siRNA. We found that TREM1 was highly expressed and activated in the synovial tissues of CIA mice concomitant with abnormal mitophagy. The mitochondrial outer membrane transporter TOMM40 was upregulated in experimental arthritis, and the protein levels of PINK1 and LC3B were decreased. RNA-seq analysis indicated that mitophagy-related proteins were extensively downregulated and that the transcription factor E2F1 and the mitochondrial outer membrane transporter TOMM40 were significantly upregulated in TREM1-overexpressing cells. ChIP-PCR revealed that TREM1 overexpression significantly promoted the interaction between E2F1 and TOMM40 gene in RAW264.7 cells. E2F1 knockdown markedly reversed TOMM40 upregulation, mitophagy injury and ROS production in TREM1-overexpressing macrophages but not in control cells. Our study provides preliminary evidence that E2F1 regulates TOMM40 transcription and disrupts mitophagy flux in TREM1-activated macrophages. Inhibiting TREM1 effectively mitigated experimental arthritis by restoring macrophage mitophagy and reducing intracellular ROS levels.
髓系细胞触发受体1（TREM1）滑膜表达升高已被确定为评估类风湿关节炎（RA）疾病活动的重要生物标志物。浸润巨噬细胞中的炎症介质诱导TREM1表达上调，在RA的滑膜炎和关节破坏中起关键作用。我们之前的测序数据将TREM1激活与异常线粒体自噬联系起来。因此，我们探讨了靶向TREM1治疗实验性关节炎的疗效及其对线粒体自噬的调节作用。通过TREM1、DAP12和p-SYK水平评估TREM1信号激活，通过PINK1、PARKIN和LC3A/B水平测量线粒体自噬。在体外，构建过表达TREM1的RAW264.7细胞，通过RNA测序分析表达和信号通路的差异。通过透射电子显微镜、MitoTracker共聚焦显微镜和线粒体膜电位分析观察过表达TREM1的RAW264.7细胞和正常对照中线粒体数量和形态以及线粒体自噬的变化。通过染色质免疫沉淀PCR（ChIP-PCR）和E2F1小干扰RNA（siRNA）确定TREM1激活的E2F1对TOMM40基因转录的促进作用。我们发现，TREM1在胶原诱导性关节炎（CIA）小鼠的滑膜组织中高表达并被激活，同时伴有异常的线粒体自噬。实验性关节炎中线粒体外膜转运体TOMM40上调，PINK1和LC3B蛋白水平降低。RNA测序分析表明，线粒体自噬相关蛋白广泛下调，转录因子E2F1和线粒体外膜转运体TOMM40在过表达TREM1的细胞中显著上调。ChIP-PCR显示，TREM1过表达显著促进RAW264.7细胞中E2F1与TOMM40基因的相互作用。E2F1基因敲低显著逆转了过表达TREM1的巨噬细胞中TOMM40上调、线粒体自噬损伤和活性氧（ROS）产生，但对对照细胞无此作用。我们的研究提供了初步证据，表明E2F1调节TOMM40转录并破坏TREM1激活的巨噬细胞中的线粒体自噬通量。抑制TREM1可通过恢复巨噬细胞线粒体自噬和降低细胞内ROS水平有效减轻实验性关节炎。
