Liu Binbin, Yu Jianshun, Zhang Jie, Ye Wei, Yao Jiaming
Department of Digestion, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, No. 453 Tiyuchang Road, Xihu District, Hangzhou, 310007, Zhejiang, China.
Eur J Med Res. 2025 Jul 24;30(1):668. doi: 10.1186/s40001-025-02947-z.
ETS2 is a core modulator of macrophages. This study aims to investigate the effects of ETS2 on macrophage polarization in ulcerative colitis (UC) and the involvement of TLR4/NF-κB pathway.
A dextran sulfate sodium (DSS)-induced acute UC mice model was established, along with a lipopolysaccharide (LPS)/IFN-γ-stimulated RAW264.7 cell model to mimic inflammation. Immunofluorescence was employed to examine the co-localization of ETS2 with M1 macrophage markers (F4/80 and iNOS). Flow cytometry quantified the iNOS/F4/80 M1 macrophage subgroups. Inflammatory cytokine (TNF-α and IL-1β) levels in cell supernatants were detected using enzyme-linked immunosorbent assay. Western blot analyzed the expressions of M1 markers (CD86 and iNOS) and TLR4/NF-κB pathway components (TLR4, p-p65/p65, and p-IκBα/IκBα). An sh-ETS2 lentiviral vector was constructed for ETS2 knockdown in vitro and in vivo. The TLR4 agonist RS 09 was used to rescue macrophage polarization and inflammatory responses.
In DSS-induced UC mice, ETS2 was significantly upregulated in colon tissues and co-localized with F4/80. LPS/IFN-γ-treated RAW264.7 cells also exhibited elevated ETS2 expression, accompanied by increased inflammatory cytokine secretion, expansion of iNOS/F4/80 macrophage subgroups, and activated TLR4/NF-κB pathway. Furthermore, ETS2 deficiency in RAW264.7 cells significantly inhibited the macrophage polarization towards M1 pro-inflammatory phenotype and blocked the TLR4/NF-κB pathway. However, RS 09 counteracted the anti-inflammatory effects of ETS2 knockdown. In vivo silencing of ETS2 attenuated M1 macrophage polarization and inflammatory cytokine production, while ameliorating pathology in UC mice.
ETS2 enhanced the inflammatory response in UC by activating TLR4/NF-κB-mediated M1 macrophage polarization.
ETS2是巨噬细胞的核心调节因子。本研究旨在探讨ETS2对溃疡性结肠炎（UC）中巨噬细胞极化的影响以及TLR4/NF-κB信号通路的参与情况。
建立葡聚糖硫酸钠（DSS）诱导的急性UC小鼠模型，以及脂多糖（LPS）/干扰素-γ刺激的RAW264.7细胞模型以模拟炎症。采用免疫荧光法检测ETS2与M1巨噬细胞标志物（F4/80和诱导型一氧化氮合酶（iNOS））的共定位。流式细胞术定量iNOS/F4/80 M1巨噬细胞亚群。使用酶联免疫吸附测定法检测细胞上清液中炎性细胞因子（肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β））水平。采用蛋白质免疫印迹法分析M1标志物（CD86和iNOS）以及TLR4/NF-κB信号通路成分（TLR4、磷酸化p65/p65和磷酸化IκBα/IκBα）的表达。构建sh-ETS2慢病毒载体用于在体外和体内敲低ETS2。使用TLR4激动剂RS 09来挽救巨噬细胞极化和炎性反应。
在DSS诱导的UC小鼠中，结肠组织中ETS2显著上调并与F4/80共定位。LPS/干扰素-γ处理的RAW264.7细胞也表现出ETS2表达升高，同时伴有炎性细胞因子分泌增加、iNOS/F4/80巨噬细胞亚群扩大以及TLR4/NF-κB信号通路激活。此外，RAW264.7细胞中ETS2缺陷显著抑制巨噬细胞向M1促炎表型的极化并阻断TLR4/NF-κB信号通路。然而，RS 09抵消了ETS2敲低的抗炎作用。体内沉默ETS2可减弱M1巨噬细胞极化和炎性细胞因子产生，同时改善UC小鼠的病理状况。
ETS2通过激活TLR4/NF-κB介导的M1巨噬细胞极化增强UC中的炎症反应。
