J Vet Sci.  2016 Dec;17(4):505-513. 10.4142/jvs.2016.17.4.505.

Mycobacterium vaccae induces a strong Th1 response that subsequently declines in C57BL/6 mice

• Zhang L
• Jiang Y
• Cui Z
• Yang W
• Yue L
• Ma Y
• Shi S
• Wang C
• Wang C
• Qian A

• KMID: 2412606
• DOI: http://doi.org/10.4142/jvs.2016.17.4.505

Abstract

Mycobacterium (M.) vaccae is a fast-growing species of saprophytic bacteria that is widely distributed. To understand the host immune responses induced by M. vaccae isolated from bovine submaxillary lymph nodes, C57BL/6 mice were infected with reference strain M. vaccae Bacillus Calmette-Guérin (BCG) and isolated M. vaccae using intraperitoneal injections. Comparison of the bacterial replication and organ pathology between M. vaccae and M. vaccae BCG revealed that M. vaccae was more malignant than M. vaccae in mice. We also demonstrated that serum from the M. vaccae-infected mice contained a higher expression level of gamma-interferon (IFN-γ), tumor necrosis factor alpha, monocyte chemoattractant protein-1, interleukin (IL)-4, IL-12, IL-10 and transforming growth factor beta than did the other groups, especially after week 4. Furthermore, when the numbers of CD3⁺CD4⁺IFN-γ⁺ and CD3⁺CD4⁺IL4⁺ cells in the infected mice were observed by flow cytometry, we found that a powerful T helper 1 (Th1) response was induced by M. vaccae infection, which was associated with the emergence of CD3⁺CD4⁺IFN-γ⁺ cells. However, the Th1 response declined over time, which was associated with appearance of the CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD152⁺Treg cell reaction. In addition, a strong Th2 response was found. Finally, we found that M. vaccae infection increased the production of type I IFNs, which was associated with a reduced Th1 response.

Keyword

Mycobacterium bovis Bacillus Calmette-Guérin; Mycobacterium vaccae; Th1 cells response; regulatory T cell

MeSH Terms

Animals
Cattle
Cytokines/*genetics/metabolism
Disease Models, Animal
Female
Mice
Mice, Inbred C57BL
Mycobacterium/*immunology
Mycobacterium Infections/*immunology
Mycobacterium bovis/physiology
Specific Pathogen-Free Organisms
Th1 Cells/*immunology
Tuberculosis, Bovine/immunology/microbiology
Cytokines

Figure

• 

  Fig. 1 Lung histology of Mycobacterium (M.) vaccae and M. bovis Bacillus Calmette-Guérin (BCG)-infected mice. (A) Bacterial counts in the lungs at weeks 2, 4, 6 and 8 after infection of mice infected with M. vaccae or M. bovis BCG. Student's t-test vs. the M. bovis BCG group, **p < 0.01, ***p < 0.001. (B) Representative lung acid fast staining bacilli two weeks after M. vaccae or M. bovis BCG infection. (C) Representative lung histopathology in infected animals on weeks 2, 4, 6 and 8 of the infection. H&E stain. 800× (B), 40× (C).

• 

  Fig. 2 Early infiltration of CD4+, CD8+ T cells, CD3+CD4+ IFN-γ+ cells and CD3+ CD4+ IL4+ cells in the lungs of mice infected with M. vaccae and M. bovis BCG. (A) Total number of cells expressing CD3+CD4+ surface markers. (B) CD3+CD8+ surface markers in the lungs of mice infected with various strains. (C) Number of CD3+ and CD4+ IFN-γ+ cells in M. vaccae-infected mice. (D) Number of CD3+ and CD4+ IL4+ cells in M. vaccae-infected mice. The results are expressed as the average ± SEM (n = 5 mice) of CD3+CD4+, CD3+CD8+ T cells, CD3+CD4+ IFN-γ+ cells or CD3+ and CD4+ IL4+ cells in the lungs. Student's t-test vs. the normal group, *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 3 Differential production of IFN-γ, TNF-α, MCP-1, IL-12p40, IL-10 and TGF-β cytokines in the serum of C57BL/6 mice infected with various bacterial strains (M. vaccae and M. bovis BCG). The levels of IFN-γ, TNF-α, MCP-1, IL-12p40, IL-10 and TGF-β were measured by ELISA. The results are expressed as the means ± SEM (n = 5 mice) in the serum. Student's t-test vs. the normal group, *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 4 Early infiltration of DCs and pDCs in the lungs of mice infected with the M. vaccae isolate and M. bovis BCG. Lung cells obtained from C57BL/6 mice infected with M. vaccae or M. bovis BCG and normal mice were assayed for the total number of pDCs (A) and DCs (B) using flow cytometry. The results are expressed as the average ± SEM of (n = 5 mice) pDCs or DCs in the lungs. Student's t-test vs. the normal group, *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 5 CD4+CD25+Foxp3+ cells in infected mice. Mice infected with M. vaccae showed a substantial increase in CD4+CD25+Foxp3+ regulatory T cells in the lungs relative to the M. bovis BCG and normal groups (A). Mice infected with M. vaccae showed a significant increase in CD4+CD25+CD152+T cells in the lungs relative to the M. bovis BCG and normal groups of mice at 3 weeks, whereas a larger number was observed in M. bovis BCG-infected mice beginning at 3 weeks (B). The results are expressed as the mean number ± SEM of CD4+CD25+Foxp3+ or CD4+CD25+CD152+ T cells (n = 5 mice) in the lungs. Student's t-test vs. the normal group, *p < 0.05, **p < 0.01, ***p < 0.001.

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