Establishment of a mouse model for Mycobacterium tuberculosis persistent infection and its immunological characteristics
XU Cheng-Ming1, KANG Jiang1, WANG Li-Mei1, HAN Wen-Dong2, SUN Zhi-Ping2, DING Yue-Na2, QU Di2,3, BAI Ying-Lan1, XU Zhi-Kai1
1. Department of Microbiology, The Fourth Military Medical University, Xi’an 710032, China; 2. Biosafety Level 3 Laboratory, Fudan University, Shanghai 200032, China; 3. Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, School of Basic Medicine, Fudan University, Shanghai 200032, China
Abstract：To establish a persistent infection mouse model and study its immunological characteristics, female C57BL/6 mice were infected with Mycobacterium tuberculosis (M. tuberculosis) H37Rv at a dose of 1×105 cfu by tail vein injection. Four weeks post infection, one group of mice was treated with isoniazid and rifampicin in drinking water for 8 weeks. At 4, 8 and 12 weeks after infection, 6 mice were euthanized to examine bacterial burden in the lungs and spleens. The homogenate of each organ was serially diluted and plated on plates for bacterial count after 21-day incubation. Mice sera were separated and anti-M. tuberculosis IgG and subclasses were analyzed by enzyme-linked immunosorbent assay (ELISA). CD4+ splenocytes were cultured with purified protein derivative (PPD) stimulation, and the percentage of interferon γ (IFN-γ), interleukin 2 (IL-2), IL-4, and tumor necrosis factor α (TNF-α) expressing cells were then identified by intracellular cytokine staining and flowcytometry. The results showed that the lg CFU of bacterial loads in lungs and spleens reached to 3.67±0.25 and 3.54±0.24, respectively 4 weeks after infection and the level retained for at least 8 weeks. The chemotherapy reduced the bacterial load of organs significantly. 12 weeks after infection, M. tuberculosis specific total IgG level increased significantly in the untreated group (P<0.01), especially IgG1 (P<0.01). The IgG level in the chemotherapy-treated group also increased（P<0.05）but lower than that in the untreated group (P <0.01). Compared with the untreated group, the chemotherapy-treated group had a lower IgG1 level (P <0.01) but a higher IgG2a level (P <0.05). The untreated group had a significantly higher percentage of IFN-γ and IL-2 expressing CD4+ splenocytes (P<0.01, P<0.001) and lower percentage of IL-4 expressing CD4+ splenocytes compared with the control group (P <0.01). The percentages of IL-2 and IL-4 expressing CD4+ splenocytes were both lower after chemotherapy compared with untreated group (P<0.05, P<0.01). A persistent infection mouse model of M. tuberculosis was established, and its immunological characteristics were identified in the study. It may provide certain applications in new vaccine and drug screening study against tuberculosis.
