■布鲁氏菌感染调节巨噬细胞表型的机制至今尚未完全阐明。本研究以RAW264.7细胞为模型,探讨流产布鲁氏菌对巨噬细胞表型的调控机制。
■RT-qPCR,采用ELISA和流式细胞术检测流产布鲁氏菌感染巨噬细胞M1/M2极化相关的炎症因子产生和表型转化。采用Westernblot和免疫荧光分析核因子κB(NF-κB)信号通路在布鲁氏菌诱导巨噬细胞极化调控中的作用。染色质免疫沉淀测序(Chip-seq),采用生物信息学分析和荧光素酶报告基因法筛选和验证与巨噬细胞极化相关的NF-κB靶基因,并进一步验证其功能。
■结果表明流产芽孢杆菌以时间依赖性方式诱导巨噬细胞表型转换和炎症反应。随着感染时间的增加,流产B感染引起的M1型先增加,在12小时达到峰值,然后下降,而M2型首先下降,12小时时低谷,然后增加。流产芽孢杆菌的细胞内存活趋势与M2型一致。当NF-κB被抑制时,M1型极化得到抑制,M2型得到促进,流产芽孢杆菌的细胞内存活率显著增加。Chip-seq和荧光素酶报告基因测定结果显示NF-κB与谷氨酰胺酶基因(Gls)结合。当NF-κB被抑制时,Gls表达下调。此外,当Gls被抑制时,M1型极化得到抑制,M2型得到促进,流产芽孢杆菌的细胞内存活率显著增加。我们的数据进一步表明NF-κB及其关键靶基因Gls在控制巨噬细胞表型转化中起重要作用。
■放在一起,我们的研究表明,流产芽孢杆菌感染可以诱导巨噬细胞M1/M2表型的动态转化。强调NF-κB是调节M1/M2表型转变的中心途径。首次阐明了通过调节关键基因Gls调节巨噬细胞表型开关和炎症反应的分子机制,受转录因子NF-κB调控。
The mechanism of Brucella infection regulating macrophage phenotype has not been completely elucidated until now. This study aimed to determine the mechanism of Brucella abortus in the modulation of macrophage phenotype using RAW264.7 cells as a model.
RT-qPCR, ELISA and flow cytometry were used to detect the inflammatory factor production and phenotype conversion associated with M1/M2 polarization of macrophages by Brucella abortus infection. Western blot and immunofluorescence were used to analyze the role of nuclear factor kappa B (NF-κB) signaling pathway in regulation of Brucella abortus-induced macrophage polarization. Chromatin immunoprecipitation sequencing (Chip-seq), bioinformatics analysis and luciferase reporter assay were used to screen and validate NF-κB target genes associated with macrophage polarization and further verify its function.
The results demonstrate that B. abortus induces a macrophage phenotypic switch and inflammatory response in a time-dependent manner. With the increase of infection time, B. abortus infection-induced M1-type increased first, peaked at 12 h, and then decreased, whereas the M2-type decreased first, trough at 12 h, and then increased. The trend of intracellular survival of B. abortus was consistent with that of M2 type. When NF-κB was inhibited, M1-type polarization was inhibited and M2-type was promoted, and the intracellular survival of B. abortus increased significantly. Chip-seq and luciferase reporter assay results showed that NF-κB binds to the glutaminase gene (Gls). Gls expression was down-regulated when NF-κB was inhibited. Furthermore, when Gls was inhibited, M1-type polarization was inhibited and M2-type was promoted, the intracellular survival of B. abortus increased significantly. Our data further suggest that NF-κB and its key target gene Gls play an important role in controlling macrophage phenotypic transformation.
Taken together, our study demonstrates that B. abortus infection can induce dynamic transformation of M1/M2 phenotype in macrophages. Highlighting NF-κB as a central pathway that regulates M1/M2 phenotypic transition. This is the first to elucidate the molecular mechanism of B. abortus regulation of macrophage phenotype switch and inflammatory response by regulating the key gene Gls, which is regulated by the transcription factor NF-κB.