Expression of cytokine and apoptosis-related genes in bovine peripheral blood mononuclear cells stimulated with Brucella abortus recombinant proteins

Abstract

Brucellosis is a clinically and economically important disease. Therefore, eradication programs of the disease have been implemented in several countries. One hurdle in these programs is the detection of infected animals at the early stage. Although the protein antigens as diagnostic antigens have recently received attention, the exact mechanisms at the beginning of immune responses are not yet known. Therefore, genes encoding five B. abortus cellular proteins were cloned and the expressed recombinant proteins were purified. The expression of several cytokine genes (IL-1β, IL-4, IL-6, IL-12p40, IFN-γ, TNF-α, and iNOS) was analyzed in bovine peripheral blood mononuclear cells (bPBMC) after stimulation with the recombinant proteins. Three apoptosis-related genes, Bax, Bcl-2, and TLR4, were also included in the analysis to find out the adverse effects of the proteins to the cells. Each protein induced different patterns of cytokine expression depending on the stimulation time and antigen dose. Expression of IL-6, IL-12p40, and IFN-γ was induced with all of the proteins while IL-1β, IL-4, TNF-α, and iNOS gene expression was not. Expression of apoptosis-related genes was not altered except TLR4. These results suggest that the cellular antigens of B. abortus induce both humoral and cellular immunity via the production of IL-6, IL-12p40, and IFN-γ in bPBMC without exerting any adverse effects on the cells.

Figure 1

Analysis of the purified recombinant proteins. SDS-PAGE (A) and Western blotting (B) with an anti-His antibody. Lane M: molecular weight markers (Life Technologies, USA), lane 1: outer membrane protein 28, lane 2: malate dehydrogenase, lane 3: elongation factor, lane 4: arginase, lane 5: metal-dependent hydrolase.

Figure 2

Gene expression of inducible nitric oxide synthease (iNOS) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 3

Gene expression of interleukin-1β (IL-1β) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 4

Gene expression of interleukin-4 (IL-4) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 5

Gene expression of interleukin-6 (IL-6) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 6

Gene expression of interleukin-12 p40 (IL-12p40) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 7

Gene expression of interferon-γ (IFN-γ) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 8

Gene expression of tumor necrosis factor-α (TNF-α) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 9

Gene expression of Bax in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 10

Gene expression of Bcl-2 in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.

Figure 11

Gene expression of Toll-like receptor 4 (TLR4) in bovine PBMC. Bovine PBMC were stimulated with 5 μg (A) and 10 μg (B) of five different recombinant proteins (OMP28, mdh, tsf, rocF, and 0628) of Brucella abortus at 0, 12, and 24 h. Gene expression was analyzed by real-time quantitative RT-PCR and normalized by the expression of β-actin.