The recombinant four different proteins of B. melitensis (OMP25, RomA, prpA, and wadC) were purified and analyzed by SDS-PAGE. The OMP25, RomA, prpA, and wadC proteins were approximately 25 kDa, 31 kDa, 30 kDa, and 27 kDa in size (Fig. 1A). Western blotting was used to confirm the specificity of the expressed proteins (Fig. 1B).
The total RNA of RAW264.7 cells and DCs was extracted after stimulation with four recombinants proteins, followed by cDNA synthesis using the total RNA template. The mRNA expression of cytokines and apoptosis-related genes was confirmed using real-time PCR. Generally, the different patterns of cytokine expression were induced after stimulation with different doses of recombinant proteins. In RAW264.7 cells, the expression of apoptotic-related genes (Bax and Bcl-2) was not significantly affected by treatment with the OMP25 protein, except for the upregulation of the Bax gene after stimulation with 30 µg OMP25 (p < 0.05) (Fig. 2A and 2I). IL-4 gene was significantly downregulated in a dose-dependent way after stimulation with 30 µg and 50 µg of OMP25 protein (p < 0.05, p < 0.01) (Fig. 2E and 2I). The IFN-beta (IFN-β) and IL-12p40 genes were also downregulated after stimulation with 30 µg or 50 µg of OMP25, although not significantly (Fig. 2C, 2G, and 2I). The IFN-gamma (IFN-γ) gene was dose-dependently upregulated after stimulation with 30 µg or 50 µg of OMP25 protein (p < 0.01) (Fig. 2D and 2I). The IL-10 and TNF-alpha (TNF-α) genes were efficiently induced in the cells treated with 30 µg and 50 µg of the OMP25 protein, but not in a dose-dependent way (Fig. 2F, 2H, and 2I). However, most cytokines and apoptotic-related genes were not greatly affected by stimulation with the OMP25 protein in DCs. Only the IL-12p40 gene was induced significantly by treatment with 30 µg of OMP25 protein, and the IL-10 gene was induced efficiently in a dose-dependent way. Additionally, the TNF-α gene expression was inhibited by stimulation with 50 µg of OMP25 protein in DCs (p < 0.05, p < 0.01) (Additional file 1F, 1G, 1H, and 1I).
Interestingly, the Bax gene was downregulated significantly in the RAW264.7 cells after treatment with 30 µg of prpA protein (p < 0.01), whereas it was greatly induced by stimulation of 50 µg of prpA (p < 0.01) (Fig. 3A and 3I). Although 50 µg of prpA protein was also able to induce higher Bcl-2 gene expression, there was no statistical difference in contrast to the PBS group. IL-10 gene expression was induced efficiently when the cells were stimulated with 30 µg and 50 µg of prpA protein (p < 0.01, p < 0.001) (Fig. 3F and 3I), and the TNF-α gene was also upregulated significantly by stimulation with 50 µg of prpA (p < 0.05) (Fig. 3H and 3I). However, the expression of IFN-β, IL-4, and IL-12p40 genes were significantly inhibited dose-dependently after stimulation with prpA protein (Fig. 3C, 3E, 3G, and 3I). Intriguingly, the expression of the IFN-β, IL-4, and IL-12p40 genes was significantly upregulated in the DCs treated with 50 µg of prpA protein (p < 0.05) (Additional file 2C, 2E, 2G, and 2I). In the DCs, only 50 µg of prpA protein-induced Bax and Bcl-2 gene expression (p < 0.05) (Additional file 2A, 2B, and 2I). The expression of IL-10, TNF-α, and IFN-γ was not affected in the DCs by stimulation with the prpA protein.
After stimulation of the RAW264.7 cells with the wadC protein, the Bax gene was significantly induced by the treatment with the 30 ug and 50 µg of protein (p < 0.05) (Fig. 4A and 4I), whereas the Bcl-2 and IFN-γ gene expressions were not influenced by the wadC protein. The IL-10 gene expression was markedly inhibited when the cells were stimulated with 30 µg and 50 µg of wadC protein (p < 0.001, p < 0.0001) (Fig. 4F and 4I). IL-12p40 and IL-4 gene expressions were also downregulated, but there was no statistical difference between the PBS and treatment groups. However, the IFN-β and TNF-α expressions were greatly upregulated after stimulation with the wadC protein (p < 0.05, p < 0.05) (Fig. 4C, 4H, and 4I). When the DCs were stimulated by the wadC protein, on the contrary, Bax and Bcl-2 were both significantly downregulated (p < 0.01, p < 0.01) (Additional file 3A, 3B, and 3I). IFN-β and TNF-α were greatly inhibited in the DCs in contrast to the RAW264.7 cells (Additional file 3C, 3H, and 3I). However, the expression of IFN-γ and IL-4 was induced by the treatment with 50 µg of wadC protein (p < 0.05) (Additional file 3D, 3E, and 3I). Interestingly, the expression of IL-10 and IL-12p40 was significantly downregulated in both the DCs and RAW264.7 cells. (p < 0.05, p < 0.01) (Additional file 3F, 3G, and 3I).
In the case of RomA protein stimulation, Bax gene expression was significantly downregulated in the RAW264.7 cells stimulated with 30 µg of protein (p < 0.01); however, Bcl-2 gene expression was upregulated by the treatment with 50 µg of protein (p < 0.001) (Fig. 5A, 5B, and 5I). There were no meaningful changes in the gene expression of IFN-β and IL-12p40 in RAW264.7 cells (Fig. 5C, 5G, and 5I). The IFN-γ gene expression was markedly induced in RAW264.7 cells stimulated with 30 µg and 50 µg of RomA (p < 0.01, p < 0.01) (Fig. 5D and 5I). The IL-4 gene expression was not affected by the RomA protein stimulation, whereas the IL-10 and TNF-α gene expressions in RAW264.7 cells were significantly downregulated by the treatment of the RomA protein (p < 0.0001, p < 0.001) (Fig. 5F, 5H, and 5I). In the DCs, both of the two apoptotic-related genes were upregulated by the stimulation of RomA protein (p < 0.01, p < 0.01) (Additional file 4A, 4B, and 4I). However, the expression of IFN-β and IL-4 was efficiently induced after the treatment with RomA in the DCs, but not in the RAW264.7 cells (Additional 4C, 4E, and 4I). TNF-α gene expression was also significantly inhibited in the DCs treated with 50 µg of RomA protein (p < 0.01) (Additional file 4G and 4I). IFN-γ, IL-10, and IL-12p40 gene expressions were not affected in the DCs after stimulation with RomA protein (Additional 4D, 4F, 4G, and 4I).