All animal experiments were performed in strict accordance with the Experimental Animal Regulation Ordinances defined by the China National Science and Technology Commission. The study was approved by the Institutional Committee of Post-Graduate Studies and Research at Shihezi University, China (No. 2012-9). Animals are provided with humane care and healthful conditions. All efforts were made to minimize animal suffering.

Brucella abortus 2308 strain was obtained from the Center of Chinese Disease Prevention and Control (Beijing, China). 2308ΔOmp25 mutant was constructed and kept by our laboratory. Brucella was cultured in tryptic soy agar or tryptic soy broth (TSB) (Sigma, St. Louis, MO, USA) at 37°C with 5% CO₂ (vol/vol). The human trophoblast cell line HPT-8 (obtained from Cell Resource Center, IBMS, CAMS/PUMC, Beijing, China) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies, Rockville, MD, USA) at 37°C with 5% CO₂ (vol/vol).

Six-week-old BALB/c female mice were obtained from the Experimental Animal Center of the Academy of Military Medical Science (Beijing, China). Animals were maintained in barrier housing with filtered inflow air in a restricted-access room in pathogen-limited conditions. All experimental procedures and animal care were performed in compliance with institutional animal care regulations. And all experimental procedures and animal care were performed in Biosafety Level 3 Laboratory.

HPT-8 cells were infected with S2308 and 2308ΔOmp25, as previously described (20). The bacteria for infection studies were prepared before the experiment was executed. S2308 and 2308ΔOmp25 were cultured in TSB at 37°C with 5% CO₂ (vol/vol) until logarithmic growth phase. Then, 2 × 10⁶ cells/well were cultured in 6-well plates for 24 h at 37°C under 5% CO₂, and then infected with S2308 or 2308ΔOmp25 at a multiplicity of infection (MOI) of 100 bacteria per cell. Culture plates were centrifuged at 350 × g for 5 min at room temperature. At 45 min post-infection, the cells were washed thrice with medium without antibiotics and then incubated with 50 µg/mL of gentamicin (Invitrogen, Carlsbad, CA, USA) for 1 h to kill extracellular bacteria. Afterward, the medium was removed and replaced with fresh DMEM with 10% FBS containing 25 µg/mL gentamicin (defined as time 0). Uninfected cells were used as control.

HPT-8 cells were infected with S2308 and 2308ΔOmp25 according to the above description. At 4, 8, 24, and 48 h post-infection, supernatant was collected and filtered with 0.22 µm filter membrane (Millipore, MA, USA). Then, supernatant was centrifuged at 16,000 × g for 15 min at 4°C. The levels of TNF-α, interleukin-1 (IL-1), and IL-10 were measured using an ELISA Quantikine Human Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. All assays were performed in triplicate and the concentration of each cytokine in the cell supernatant was calculated using a linear regression equation obtained from the absorbance values of standards, according to the manufacturer’s protocol. All assays were performed three times.

To confirm the MAPK signal pathway associated with the secretion of TNF-α, HPT-8 cells were pre-treated with p38 inhibitor (10 µM), or JNK inhibitor (10 µM) at 37°C for 1 h and then infected with S2308 or 2308ΔOmp25 at a 100:1 MOI according to the above description. At 4, 8, 24, and 48 h post-infection, supernatant was collected and measured the levels of TNF-α according to the above description. All assays were performed three times.

HPT-8 cells were infected with S2308 and 2308ΔOmp25 according to the above description. The activation of p38, ERK1/2, and JNK was detected in infected cells, as previously described (21). Briefly, at 24 h post-infection, supernatant was discarded and cells were lysed in ice-cold Radio Immunoprecipitation Assay Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min, then centrifuged at 16,000 × g for 30 min at 4°C. The supernatant was collected and concentration was detected with BCA protein assay kit (Sangon Biotech, Shanghai, China). 500 µg protein samples separated by 12% SDS-PAGE and electro-transferred to a nitrocellulose membrane using a Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA) at 200 mA for 1 h. Unbound sites on the membrane were blocked in 5% nonfat milk in Tris-buffered saline Tween-20 (TBST) buffer for 1 h at room temperature. Then, the membrane was washed three times with TBST buffer and incubated with rabbit anti-human anti-p38, anti-ERK, or anti-JNK polyclonal antibody (pAb; diluted 1:1,000; Bioworld, Minneapolis, MN, USA) at room temperature for 1 h. After being washed three times, the membrane was incubated with peroxidase conjugated goat anti-rabbit IgG for 1 h at room temperature. After a further washing step, bound conjugate was visualized with an ECL Plus Western Blotting Substrate kit (Thermo Fisher Scientific, USA). All assays were performed three times.

BALB/c mice were infected with Brucella as previously described (22). Briefly, 6-week-old BALB/c female mice (n = 5 per group) were randomly divided into three groups. Group 1 and 2 were inoculated intraperitoneally (i.p.) with 200 µL phosphate-buffered saline (PBS; Sigma-Aldrich, MO, USA) containing 1 × 10⁶ CFU of S2308 or 2308ΔOmp25, respectively, group 3 was inoculated i.p. with 200 µL PBS as negative control. Serum samples were obtained from peripheral blood of immunized mice 2, 4, 6, 8, and 10 weeks post-immunization (23). Serum samples were diluted with sample diluent buffer (1:5). The level of TNF-α was measured using an ELISA Quantikine Mouse Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. All assays were performed in triplicate and the concentration of each cytokine in the cell supernatant was calculated using a linear regression equation obtained from the absorbance values of standards, according to the manufacturer’s protocol. All assays were performed three times.

BALB/c mice (n = 25 per group) were inoculated with S2308 and 2308ΔOmp25 according to the above description. At 2, 4, 6, 8, and 10 weeks post-immunization, mice were euthanized and uteruses were removed aseptically. The uteruses were collected, formalin-fixed, and paraffin-embedded, as previously described (24).

The immunohistochemistry assay was performed as previously described (25). Briefly, the embedded paraffin will be serial sectioned by slicer, with a thickness of 5 µm and mounted on slide glasses coated with poly-l-lysine (Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, the tissue sections were routine deparaffinized and dehydrated with xylene and ethanol. After three times washed with distilled water, the tissue sections were microwaved in 10 mM citrate buffer (pH 6.0; Sigma-Aldrich, MO, USA) at 95°C for 10 min for antigen retrieval and naturally cooled at room temperature. Then, the endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide buffer (Sigma-Aldrich, MO, USA) for 10 min at room temperature. The tissue sections were incubated overnight at 4°C with primary antibody (rabbit anti-mouse anti-p38 or anti-ERK pAb; dilution 1:200; Bioworld, Minneapolis, MN, USA). Subsequently, the sections were washed three times with PBS, each time for 5 min. The sections were incubated at 37°C for 30 min with the secondary antibody (biotinylated goat anti-rabbit IgG; dilution 1:500; ZSGB-BIO, Beijing, China) followed by washing three times with PBS, each time for 5 min. Finally, the tissue sections were dropwise added 100 µL DAB Plus Substrate buffer (Thermo Fisher Scientific, USA) containing 2% (v/v) DAB Plus Chromogen (Thermo Fisher Scientific, USA), and the nuclei were counterstained with hematoxylin. The tissue sections were dehydrated, transparented, and sealed with ethanol, xylene, and Neutral gum.

Cytokine production was expressed as the mean cytokine concentration ± SD. Statistical analysis was performed with Student’s unpaired t-test. The differences between groups were analyzed by analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post-test, by comparing all the groups to one another. Results expressed as percentages were analyzed by the Fisher test. The differences between groups were analyzed by ANOVA using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P values of <0.05 were considered statistically significant.

To detect the expression level of cytokines, we collected supernatant from HPT-8 cells infected with S2308 and 2308ΔOmp25 and then monitored expression levels of cytokine TNF-α, IL-1, and IL-10 by ELISA. Supernatant from HPT-8 cells infected with S2308 produced higher amounts of TNF-α (Figure 1A), IL-1 (Figure 1B), and IL-10 (Figure 1C) than did supernatant from uninfected cells (P < 0.05) and this difference increased with time. Slightly higher cytokine production levels were observed in 2308ΔOmp25-infected cells than in control cells (Figure 1), but there was no significant difference between 2308ΔOmp25 group and control group (P > 0.05).

We next evaluated which branch associated with secretion of TNF-α. HPT-8 cells were pre-incubated for 1 h with 10 µM p38, 10 µM ERK, or 10 µM JNK inhibitors and then infected with S2308 or 2308ΔOmp25 for 4, 8, 24, and 12 h. The levels of TNF-α were assessed in the supernatants of the S2308 or 2308ΔOmp25-infected p38 inhibitor, ERK inhibitor or JNK inhibitor-treated cells. At 4, 8, 24, and 12 h, the S2308 or 2308ΔOmp25-infected p38 inhibitor-treated cells produced higher levels of TNF-α than S2308 or 2308ΔOmp25-infected cells (P < 0.05; Figure 2A). However, there was no significant difference between S2308 or 2308ΔOmp25-infected ERK inhibitor and JNK inhibitor-treated cells and S2308 or 2308ΔOmp25-infected cells (P > 0.05; Figures 2B,C). These results showed that p38 branch could induce secretion of TNF-α in S2308 or 2308ΔOmp25-infected cells.

To assess activation of p38 and ERK1/2 kinases, HPT-8 cells were infected with the 2308ΔOmp25 mutant and the parental strain S2308 at a MOI of 100. We found that at 24 h post-infection, the activation process triggered by S2308 resulted in a phosphorylation of the p38, ERK1/2, and JNK kinases (Figure 3). Infections with 2308ΔOmp25 induced a markedly weaker stimulation of p38, ERK1/2, and JNK kinases, with S2308 demonstrating a slightly higher capacity of activation than 2308ΔOmp25 mutant (Figure 3). These results show that OMP25 involved in activating of MAPK pathway.

To detect the expression level of TNF-α in animal, we collected sera from mice inoculated with S2308, 2308ΔOmp25, or PBS and then measured expression levels of TNF-α by ELISA. Serum samples from mice inoculated with S2308 produced higher amounts of TNF-α (Figure 4) than did serum samples from mice inoculated with 2308ΔOmp25 or PBS (P < 0.05) and this difference increased with time. Slightly higher cytokine production levels were observed in 2308ΔOmp25 immunized mice than in PBS immunized mice (P > 0.05) (Figure 4). Total TNF-α levels increased with time.

The immune complexes of p38 and ERK phosphorylation proteins were located in the cytoplasm of the uterus tissues, and they were strongly stained as tan or brownish yellow. The phosphorylation proteins in p38 and ERK signal pathways had been detected and found in the uterus tissues of S2308 immunized mice (Figures 5A and 6A). But the immune complexes of phosphorylation proteins in p38 and ERK signal pathways were weakly stained in the uterus tissues of 2308ΔOmp25 immunized mice (Figures 5B and 6B). These results showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK.