【背景】牛分枝杆菌(Mycobacterium bovis)是重要的人畜共患病原体，严重危害公共卫生安全。巨噬细胞是牛分枝杆菌感染的主要效应细胞，M1型巨噬细胞极化对于宿主免疫防御M. bovis感染至关重要。【目的】探讨鸟苷酸结合蛋白5 (guanylate-binding protein 5, GBP5)对M. bovis感染的小鼠单核巨噬细胞系RAW264.7细胞M1、M2型极化的影响，以及GBP5在M. bovis感染中的作用。【方法】通过前期转录组学数据分析筛选出差异基因GBP5，通过体内、体外验证M. bovis感染后的GBP5的表达水平。小干扰RNA下调细胞内GBP5表达，分别利用RT-qPCR、Western blotting和流式细胞术检测M1、M2型巨噬细胞表面标志物表达变化；平板菌落计数法检测GBP5对M. bovis在巨噬细胞中复制的影响；双荧光素酶报告基因试验检测GBP5对信号转导及转录激活蛋白3 (signal transducer and activator of transcription 3, STAT3)转录调控；Western blotting检测活性氧(reactive oxygen species, ROS)抑制后STAT3的磷酸化情况。【结果】GBP5在M. bovis感染的RAW264.7细胞和C57BL/6小鼠组织中表达增加；下调GBP5后细胞内细菌CFU在感染后不同时间点均显著增加，ROS释放减少，M1型巨噬细胞表面标志物表达减少，但是对M2型巨噬细胞无显著影响；下调GBP5后抑制了STAT3的转录启动。ROS的产生抑制了STAT3的磷酸化。【结论】GBP5促进ROS的产生，调控ROS/STAT3通路，促进巨噬细胞向M1型极化，从而抑制细胞内牛分枝杆菌的存活。
[Background] Mycobacterium bovis, a major zoonotic pathogen, seriously endangers public health. Macrophages are the main effector cells of M. bovis infection. The polarization of macrophages into M1 type is of great significant for the host immune defense against M. bovis infection. [Objective] To explore the effect of guanylate-binding protein 5 (GBP5) on the M1/M2 polarization of mouse mononuclear macrophage RAW264.7 cells infected with M. bovis, and reveal the role of GBP5 in M. bovis infection. [Methods] GBP5 was screened out as differentially expressed gene by preliminary transcriptomic data analysis. The increased expression level of GBP5 after M. bovis infection was verified in vivo and in vitro. The expression of GBP5 was down-regulated in the cells by siRNA, and then the surface markers of M1 and M2 macrophages were detected by RT-qPCR, Western blotting, and flow cytometry. The effect of GBP5 on M. bovis replication in macrophages was examined via plate colony counting method. The regulation of signal transducer and activator of transcription 3 (STAT3) transcription by GBP5 was verified by dual-luciferase reporter system. Phosphorylation of STAT3 was detected by Western blotting after inhibition on the release of reactive oxygen species. [Results] The expression of GBP5 was up-regulated in RAW264.7 cells and C57BL/6 mouse tissue after M. bovis infection. The down-regulation of GBP5 expression significantly increased the intracellular colony forming units (CFU) of M. bovis at different time points, reduced the release of ROS, and inhibited the surface markers of M1 macrophages. Otherwise, it exerted no significant effect on M2 macrophages. In addition, the down-regulation of GBP5 inhibited the transcription of STAT3. ROS production inhibited the phosphorylation of STAT3. [Conclusion] GBP5 can promote ROS production and regulate ROS/STAT3 pathway to promote the polarization of macrophages into M1 type, thereby inhibiting the survival of M. bovis in cells.