IL-10 Dampens the Th1 and Tc Activation through Modulating DC Functions in BCG Vaccination

Abstract

BCG, the only registered vaccine against Mycobacterial Tuberculosis (TB) infection, has been questioned for its protective efficacy for decades. Although lots of efforts were made to improve the BCG antigenicity, few studies were devoted to understand the role of host factors in the variability of the BCG protection. Using the IL-10KO mice and pulmonary tuberculosis infection model, we have addressed the role of IL-10 in the BCG vaccination efficacy. The data showed that IL-10-deficient dendritic cells (DCs) could promote the immune responses through upregulation of the surface costimulatory molecule expression and play an orchestra role through activating CD4⁺T cell. IL-10-deficient mice had higher IFN γ, TNF α, and IL-6 production after BCG vaccination, which was consistent with the higher proportion of IFN γ ⁺CD3⁺, IFN γ ⁺CD4⁺, and IFN γ ⁺CD8⁺ T cells in the spleen. Particularly, the BCG-vaccinated IL-10KO mice showed less inflammation after TB challenge compared to WT mice, which was supported by the promoted Th1 and Tc, as well as the downregulated Treg responses in IL-10 deficiency. In a conclusion, we demonstrated the negative relationship between Th1/Tc responses with IL-10 production. IL-10 deficiency restored the type 1 immune response through DC activation, which provided better protection against TB infection. Hence, our study offers the first experimental evidence that, contrary to the modulation of BCG, host immunity plays a critical role in the BCG protective efficacy against TB.

Figure 1

The chart of BCG vaccination and challenge to establish the BCG vaccine/challenge mouse model. The IL-10KO and WT mice were housed for 2 weeks after arriving. For vaccination, the mice were intravenously injected with 5 × 10⁵ CFU BCG in 200 μL PBS; the control group was given PBS only. For challenge, at D21 after vaccination, the mice were infected intranasally with 5 × 10⁶ CFU BCG. The immune responses were detected after 21 days.

Figure 2

The pathological changes in vaccinated IL-10KO and WT mice following intranasally (i.n.) challenge infection after BCG vaccination. Pretreated mice were challenged intranasally with 5 × 10⁶ CFUs BCG and analyzed for histopathological changes in the lungs at day 21 postchallenge infection. Lung tissue sections (6 μm) were stained with H&E for inflammation. (a) Low magnification (×10), (b) High magnification (×40). The representative of three independent experiments (n = 5) with similar results was shown.

Figure 3

IL-10KO mice showed higher IFN γ production after BCG challenge in the lungs. The mice were treated as described in the legend of Figure 4. The lungs were digested as described in Materials and Methods. The intracellular IFN γ-positive T cells were analyzed by intracellular cytokine staining as described previously [29]. (a) Gating strategy: firstly, the major cells were gated for further gated based on the CD3_Ɛ expression. Then, the CD3_Ɛ⁺ cells were gated on the CD4 and CD8 expression for CD4⁺ and CD8⁺ T cells. (b) The figures from FAC analysis for the IFN-γ⁺ expression were shown. (c) Summary of the percentage of the IFN-γ⁺CD3_Ɛ⁺, CD4⁺, and CD8⁺ T cells in the lungs, and Student's t test was used for analysis. The representative of three independent experiments (n = 5) with similar results was shown. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 4

IL-10KO mice showed higher IFN γ production after BCG challenge in dLN. The mice were treated as described in the legend. The single cells from dLN were stained, and the intracellular IFN γ-positive T cells were analyzed by intracellular cytokine staining as described previously [29]. (a) Representative figures from FAC analysis were shown. (b) Summary of percentage of the IFN γ⁺CD3_Ɛ⁺, CD4⁺, and CD8⁺ T cells in dLN. Data are shown as the representative of three independent experiments with similar results (n = 5) and Student's t test was used for analysis. ^∗∗p < 0.01.

Figure 5

IL-10KO mice had higher IFN γ production after BCG vaccination in the spleen. The spleen was digested with enzyme as described in Materials and Methods. The cells were stained with FITC anti-CD3_Ɛ, PE anti-CD4, PerCy5.5 anti-CD8, and APC anti-IFN γ. The intracellular IFN γ-positive cells were analyzed by intracellular cytokine staining when cells were gated on CD3_Ɛ, CD4, and CD8 as described previously [29]. (a) Representative figures from FAC analysis were shown. (b) Summary of percentage of the IFN γ⁺CD3_Ɛ⁺, CD4⁺, and CD8⁺ T cells in the spleen, and Student's t test was used for analysis. ^∗p < 0.05, ^∗∗p < 0.01. The representative of three independent experiments (n = 5) with similar results was shown.

Figure 6

Cytokine levels after BCG vaccination in IL-10KO mice compared to WT mice. IL-10KO and WT mice were immunized with BCG as described in Materials and Methods. At d 21 after vaccination, the single cells prepared from the spleen, lungs, and dLN were cultured for 72 h stimulated with HK-BCG (5 × 10⁵ CFUs/mL) before supernatant collection. The cytokines were measured by the ELISA. Data were summarized and representative of three independent experiments with similar results were shown. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 7

Cytokine production from DC-CD4⁺T cell coculture system. Spleen DCs (7.5 × 10⁵ cells/mL) from WT and IL-10KO BCG-vaccinated mice were cocultured with BCG-educated CD4⁺T cells (7.5 × 10⁶ cells/mL) as described in Materials and Methods. Cell supernatants were collected at 72 h for IFN γ and IL-4 detection by ELISA. Data were summarized as mean ± SD (n = 5) and representative of three independent experiments with similar results were shown. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 8

Lung Foxp3⁺ Treg cells were analyzed after BCG challenged in IL-10KO and WT mice. The mice were treated as described in the legend of Figure 7. Lung single cells were stained with FITC anti-CD3_Ɛ, PerCy5.5 anti-CD4, APC anti-CD25, and PE-Foxp3. The Foxp3 expression was analyzed by flow cytometry when cells were gated on CD3_Ɛ⁺CD4⁺T cells. (a) The representative pictures of Foxp3⁺CD3_Ɛ⁺CD4⁺CD25⁺Tregs were shown. (b) Summary of the frequency of Foxp3⁺CD25⁺CD4⁺T cells in total T cells in the lungs. Data are shown as mean ± SD (n = 5) and are representative of three independent experiments with similar results. ^∗∗p < 0.01.