The immune mechanism of Mycoplasma hyopneumoniae 168 vaccine strain through dendritic cells

Abstract

Background: Mycoplasma hyopneumoniae (Mhp) causes porcine enzootic pneumonia, a disease that cause major economic losses in the pig industry. Dendritic cells (DCs), the most effective antigen-presenting cells, are widely distributed beneath respiratory epithelium, DCs uptake and present antigens to T cells, to initiate protective immune responses in different infections. In this study, we investigated the role of porcine DCs in vaccine Mhp-168 exposure.

Results: The antigen presenting ability of DCs were improved by vaccine Mhp-168 exposure. DCs could activate T-cell proliferation by up-regulating the antigen presenting molecule MHCII expression and co-stimulatory molecule CD80/86. However, the up-regulation of IL-10 and accompany with down-regulation of IFN-γ gene level may account for the limitation of attenuated Mhp-168 strain use as vaccine alone.

Conclusion: These findings are benefit for exploring the protection mechanisms and the possible limitations of this attenuated Mhp-168 vaccine.

Keywords: Vaccine Mhp-168 strain, Dendritic cells, Immune protection.

Fig. 1

Generation of BMDCs. Porcine BMDCs were cultured in RPMI 1640 medium supplemented with GM-CSF and IL-4. a The clusters of BMDCs on day 5, as viewed by optical microscopy at 100 ×magnification. b Morphology of cells after 6 days of culture, dendritic processes could be observed using optical microscopy at 400 × magnification. c Porcine BMDCs were cultured in RPMI 1640 medium supplemented without GM-CSF and IL-4 as control

Fig. 2

Expression of antigen presenting molecules and co-stimulatory molecules on DCs after vaccine Mhp-168 exposure at 24 h.p.i. Immature BMDCs were exposed to vaccine Mhp-168 (40/dendritic cell) or LPS (10 ng/ml) as positive control for 24 h. Dot plots show the percentage of CD1a⁺SWC3a⁺DCs, CD80/86⁺SWC3a⁺ DCs and MCHII⁺SWC3a⁺ DCs. a Untreated BMDCs. b Mhp 168 treated DCs. c LPS treated DCs. d Bar graphs show the percentage of CD1a⁺SWC3a⁺, CD80/86⁺SWC3a⁺, MHCII⁺SWC3a⁺ cells in BMDCs populations after vaccine Mhp-168 exposure. Data show the means ± SEM (n = 3 per group). All experiments were performed independently three times. Statistical significance was assessed by Student’s t-test. Differences were considered significant at (*) 0.01 < p < 0.05, (**) p < 0.01

Fig. 3

Cytokines mRNA expression levels of DCs after vaccine Mhp-168 exposure. a IL-10; b IL-12; c IFN-γ. BMDCs were treated with vaccine Mhp-168 or LPS (10 ng/mL) for 8 h, 12 h and 24 h. The mRNA expression levels of IL-10, IL-12, IFN-γ were detected by real-time quantitative PCR (RT-qPCR). Data are expressed as means ± SD (n = 3 per group). All experiments were performed independently three times. Statistical significance was assessed by Student’s t-test. Differences were considered significant at (*) 0.01 < p < 0.05, (**) p < 0.01

Fig. 4

Vaccine Mhp-168 treated DCs increased the capacity of T lymphocytes stimulation. Pretreatment DCs with vaccine Mhp-168 for 24 h. Vaccine Mhp-168-treated DCs were co-cultured with T lymphocytes (ratios 1:1, 1:5) for another 5 days (a, b). DCs were stimulated with LPS or remained unstimulated then co-cultured with T lymphocytes as positive or control group. T lymphocytes proliferation was evaluated using CFSE. Data are presented as mean ± SEM (n = 3 per group). All experiments were performed independently three times. Statistical significance was assessed by Student’s t-test. Differences were considered significant at (*) 0.01 < p < 0.05, (**) p < 0.01