Abstract:[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.
