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. 2015 Oct 13;16(10):24127-38.
doi: 10.3390/ijms161024127.

Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen

Zhengzhong Xu  1 , Chuang Meng  2 , Bin Qiang  3 , Hongyan Gu  4 , Lin Sun  5 , Yuelan Yin  6 , Zhiming Pan  7 , Xiang Chen  8 , Xinan Jiao  9
Affiliations

Differential Effects of Mycobacterium bovis BCG on Macrophages and Dendritic Cells from Murine Spleen

Zhengzhong Xu et al. Int J Mol Sci. .

Abstract

Macrophages (MΦ) and dendritic cells (DCs) are both pivotal antigen presenting cells capable of inducing specific cellular responses to inhaled mycobacteria, and thus, they may be important in the initiation of early immune responses to mycobacterial infection. In this study, we evaluated and compared the roles of murine splenic DCs and MΦs in immunity against Mycobacterium bovis Bacillus Calmette-Guérin (M.bovis BCG). The number of internalized rBCG-GFP observed was obviously greater in murine splenic MΦs compared with DCs, and the intracellular reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels in MΦs were all higher than in DCs. DCs have a stronger capacity for presenting Ag85A peptide to specific T hybridoma and when the murine splenic MΦs were infected with BCG and rBCG::Ag85A, low level of antigen presenting activity was detected. These data suggest that murine splenic MΦs participate in mycobacteria uptake, killing and inducing inflammatory response, whereas the murine splenic DCs are primarily involved in specific antigen presentation and T cell activation.

Keywords: BCG; dendritic cell; macrophage.

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Figures

Figure 1
Figure 1
Cell sorting of murine splenic MΦs and DCs. In this case, the single spleen cell suspension of C57/BL6 mice was first stained with anti-CD11c Microbeads before autoMACS separation led to a positive cell sample of CD11c+ cells (Q2 and Q3) with purity of 95.3% (A); The negative fraction was further incubated with anti-CD11b Microbeads giving a positive cell fraction containing CD11c−CD11b+ cells (Q1) with purity of 85.3% (B). Q1: CD11c−CD11b+, Q2: CD11c+CD11b+, Q3: CD11c+CD11b−, Q4: CD11c−CD11b−.
Figure 2
Figure 2
Infection of murine splenic MΦs and DCs with rBCG-GFP. The percentage of dendritic cells and macrophages infected by rBCG-GFP was determined. Data depicted represent the mean values ± SEM. Statistical significance was determined by a Student’s t-test (* p < 0.05, ** p < 0.01).
Figure 3
Figure 3
Detection of ROS, iNOS and NO of murine splenic MΦs and DCs. The intracellular ROS (A), iNOS (B) and NO (C) produced in murine splenic MΦs and DCs after M.bovis BCG infection were detected. Data depicted represent the mean values ± SEM. Statistical significance was determined by a Student’s t-test (* p < 0.05).
Figure 4
Figure 4
Kinetics of cytokine production following BCG-infected splenic DCs and MΦs. Splenic macrophages (A) and dendritic cells (B) were infected with BCG at a MOI of 5, and the cell culture supernatants were collected and detected at different time points after infection; Splenic macrophages (C) and dendritic cells (D) were infected with different MOIs of BCG, and the cell culture supernatants were collected and detected at 16 h after infection; IL-6 and IFN-γ production increased significantly in M.bovis BCG-infected MΦs (E) and DCs (F) when the effect of IL-10 was blocked by soluble IL-10R. Data depicted represent the mean values ± SEM. Statistical significance was determined by a Student’s t-test (* p < 0.05, ** p < 0.01).
Figure 5
Figure 5
Detection of antigen presenting activity of murine splenic MΦs and DCs in vitro. Murine splenic macrophages and dendritic cells were treated with serial diluted Ag85A peptide (A), Ag85A protein (B), and infected with BCG (C), rBCG::Ag85A (D), then co-cultured with T hybridoma DE10. The change of cell surface MHCII (E), CD40 (F), CD80 (G) and CD86 (H) of murine splenic macrophages and dendritic cells in control group (Black) and BCG infected group (Red).
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