Inactivation of the Type IV Secretion System Reduces the Th1 Polarization of the Immune Response to Brucella abortus Infection

Bacterial strains, media, and culture conditions.Bacterial strains used in this study were Brucella abortus wild-type strain 2308 and its isogenic mutant strain BA41 (17), which has an insertion of mTn5Km2 at nucleotide 1232 of the B. abortus virB locus (GenBank accession number AF226278). This insertion is located 59 bp downstream of the virB1 gene and is polar upon the expression of downstream genes in the virB operon (39). Strains were cultured on tryptic soy agar (Difco/Becton-Dickinson, Sparks, MD) or in tryptic soy broth at 37°C on a rotary shaker. Bacterial inocula for infection of mice were cultured on tryptic soy agar plus 5% blood (2). For cultures of strain BA41, kanamycin (Km) was added to the culture medium at 100 mg/liter. All work with live B. abortus cells was performed at biosafety level 3.

Infection of mice.Female C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were used at the age of 6 to 8 weeks. Mice were held in microisolator cages with sterile bedding and water and irradiated feed in a biosafety level 3 facility. Groups of 5 to 10 mice were inoculated intraperitoneally (i.p.) with 0.1 ml of phosphate-buffered saline (PBS) containing 5 × 10⁵ cells of a B. abortus wild-type strain or an isogenic virB mutant. All animal experiments were approved by the Texas A&M University and University of California Laboratory Animal Care and Use Committees and were conducted in accordance with institutional guidelines.

Multiplex cytokine assays.Detection of specific cytokines in the serum or spleens of C57BL/6 mice was performed using Multi-Plex cytokine assays (4) (Bio-Rad, Hercules, CA). Groups of five C57BL/6 mice were infected i.p. with 5 × 10⁵ cells of either the wild type or the virB mutant, and serum was obtained from the saphenous vein at days 1, 4, and 7 postinfection. Cytokine detection was performed according to the instructions provided by the kit's manufacturer. Briefly, serum was filtered twice through a 0.2-μm filter and then diluted 1:3 in Multi-Plex sample diluent buffer, added to the plate, and incubated at room temperature for 30 min. The samples were washed several times, and 25 μl of the detection antibody solution was added to each well. Washes were monitored by incubation at room temperature for 30 min. The samples were washed, and 50 μl of streptavidin-phycoerythrin was added and incubated at room temperature for 10 min. The samples were washed and resuspended in 125 μl of assay buffer (proprietary formula) and read using the Luminex 100 instrument (Bio-Rad).

For the detection of specific cytokines in the spleen, groups of five C57BL/6 mice were infected i.p. with 5 × 10⁵ cells of either wild-type or virB mutant B. abortus, and spleens were obtained at days 1, 4, and 7 postinfection. Spleens were homogenized in 3 ml of PBS and passed through a 100-μm cell strainer. The cells were centrifuged for 10 min at 1,000 rpm. The supernatant was passed twice through a 0.2-mm filter and cultured to ensure that no viable bacteria were present. Cytokines were concentrated by using Millipore 5K filter devices and stored at −80°C. Cytokine detection was performed as described above.

ELISA.The presence of antibody specific for Brucella abortus in the serum samples from 10 C57BL/6 mice infected with B. abortus 2308 and 10 mice infected with the B. abortus virB mutant was determined by indirect enzyme-linked immunosorbent assay (ELISA). MaxiSorp plates from Qiagen (Valencia, CA) were coated with 100 μl formalin-killed whole B. abortus cells (1 μg/ml) in carbonate buffer (pH 9.6), and plates were incubated at 4°C overnight. After washing with PBS and 0.05% Tween 20, the serum samples were diluted 1:100 in PBS containing 1% bovine serum albumin and incubated at room temperature for 1 h. After washing with PBS and 0.05% Tween 20, the reactivity was measured using horseradish peroxidase-conjugated anti-mouse immunoglobulin M (IgM), IgG, IgG1, IgG2a, or IgG3 (1:1,000; BD Pharmingen, San Diego, CA) by incubating the plates at 37°C for 1 h. The reaction was developed with Sigma Fast o-phenylenediamine dihydrochloride tablet sets. The resulting color was read at 410 nm with an ELISA microplate reader (MR5000; Dynatech). Data points are the averages of duplicate dilutions, with each measurement being performed twice.

RNA isolation.RNA was isolated using Tri reagent (Molecular Research Centre, Inc., Cincinnati, OH). Spleen samples were homogenized in 1 ml of Tri reagent and incubated for 5 min at room temperature. RNA was extracted by adding 0.2 ml of chloroform and centrifuging the samples at 10,000 rpm for 15 min at 4°C. RNA was precipitated with 0.5 ml of isopropanol and resuspended in H₂O.

cDNA.One microgram of RNA was transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Branchburg, NJ). The RNA was mixed with a solution containing 5 μl of 10× buffer, 11 μl of 20 mM MgCl₂, 10 μl of deoxynucleoside triphosphates, 2.5 μl of random hexamers, 1 μl of reverse transcriptase, and H₂O in a final volume of 50 μl. Samples were incubated at 25°C for 10 min, followed by reverse transcription at 48°C for 30 min and inactivation at 95°C for 5 min.

Real-time PCR.Reverse-transcribed cDNA was amplified with previously reported primer sets (26) for mouse IFN-γ (forward primer TCA AGT GGC ATA GAT GTG GAA GAA and reverse primer TGG CTC TGC AGG ATT TTC ATG) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward primer TGT AGA CCA TGT AGT TGA GGT CA and reverse primer AGG TCG GTG TGA ACG GAT TTG) using Sybr green PCR master mix (Applied Biosystems) and an ABI Prism 7900HT detection system (Applied Biosystems) according to the manufacturer's instructions. Induction of mRNA was determined from the threshold cycle (C_T) values normalized for GAPDH expression and then normalized to the value derived from the naive controls (23).

Intracellular cytokine staining.Intracellular staining for IFN-γ was performed in splenocytes from groups of four C57BL/6 mice infected for 3 days with B. abortus 2308 or a virB mutant. Splenocytes from each mouse were analyzed individually using four-color flow cytometry. Briefly, after passing the spleen cells through a 100-μm cell strainer and treating the samples with ACK buffer (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM Na₂EDTA [pH 7.2]) to lyse red blood cells, splenocytes were washed with PBS (Gibco) containing 1% bovine serum albumin (fluorescence-activated cell sorter [FACS] buffer). Cells were incubated for 6 h in RPMI 1640-5% fetal bovine serum with 10 μg/ml brefeldin A (Sigma). Cells were stained individually or with a cocktail of Pacific Blue-conjugated rat anti-mouse CD4 (L3T4), Alexa Fluor 700-conjugated rat anti-mouse CD8a (Ly-2), and allophycocyanin II-Alexa Fluor 750 rat anti-mouse CD3e (eBioscience). The cells were washed with FACS buffer and fixed with 4% formaldehyde for 30 min at 4°C, followed by permeabilization for 30 min using a saponin-based buffer and staining with phycoerythrin-conjugated anti-mouse IFN-γ for 30 min at 4°C. Finally, samples were washed in FACS buffer containing saponin. Flow cytometry analysis was performed using an LSRII apparatus (Becton Dickinson, San Diego, CA), and data were collected for over 1 million cells/mouse. The CD3 cells were gated (R1) based on side scatter and CD3 expression. CD4⁺ or CD8⁺ T cells were gated (R2) based on CD3 expression and CD4 or CD8 expression, respectively.

Statistical analysis.For the determination of statistical significance between experimental groups, a Student's t test was performed on the data after logarithmic conversion. A P value of <0.05 was considered to be significant.

The T4SS mutant and the wild type elicit different antibody responses in infected mice.To compare host responses to wild-type B. abortus and a virB mutant, anti-B. abortus IgG levels in serum from infected mice were measured by ELISA. The results of this experiment (Fig. 1A) revealed that between days 7 and 21, the virB mutant elicited a higher B. abortus-specific IgG response than strain 2308, while after day 56, mice infected with wild-type B. abortus exhibited higher IgG titers. This trend was also observed when IgM was assayed in the same serum samples (data not shown). The differences in titer early during infection may reflect increased killing and presentation of antigen from the virB mutant, which started to be cleared from spleens of infected mice by 5 days after infection, while CFU of the wild-type strain did not decline (31). To determine whether these IgG responses are qualitatively similar, we assayed the same serum samples for IgG1, IgG2a, and IgG3 isotypes. Both strains elicited similar levels of IgG1 (Fig. 1B) and IgG3 (Fig. 1D), but levels of IgG2a (Fig. 1C) were significantly higher in mice infected with B. abortus 2308 between 35 and 70 days postinfection. In an identical experiment performed using BALB/c mice (data not shown), we obtained the same results, showing that this difference is not related to differences between the two mouse strains in their responses to Brucella infection (25).

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FIG. 1.

Measurement of antibody levels in sera from mice infected with B. abortus 2308 or the virB mutant by ELISA. Samples from groups of 10 mice inoculated i.p. with 5 × 10⁵ cells of either B. abortus 2308 or the virB mutant were taken at different times postinoculation. The significance of differences between arithmetic means of data for wild-type and mutant mice at each time point was determined using a Student's t test. Asterisks indicate significant differences between immunoglobulin levels in mice infected with 2308 and those in mice infected with the virB mutant (P < 0.05).

The virB mutant elicits reduced levels of circulating IFN-γ and IL-12 in comparison with those of wild-type B. abortus.In order to determine whether the higher levels of IgG2a elicited by wild-type B. abortus reflected a greater Th1 polarization of the early immune response, we determined the levels of the Th1-associated cytokines IFN-γ and IL-12 after infection with wild-type B. abortus or the virB mutant. For these experiments, mice were infected with either wild-type B. abortus or the virB mutant, and serum was obtained at days 1, 4, and 7 postinfection. The detection of IFN-γ and IL-12p40 was performed using BioPlex cytokine assays (Fig. 2). Since the cytokine levels in naive serum varied among mice, the increase in the cytokine concentration in infected serum samples over that in uninfected serum samples was calculated (Fig. 2). Results of the cytokine assays showed that on days 4 and 7 postinfection, levels of IFN-γ and IL-12p40 remained unchanged in the sera of mice infected with B. abortus virB, whereas in mice infected with the B. abortus wild-type strain, the levels of IL-12p40 and IFN-γ increased significantly above preinfection levels.

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FIG. 2.

Quantification of cytokine levels in sera of mice infected with B. abortus 2308 or the virB mutant. Samples from groups of 10 mice infected i.p. with either B. abortus 2308 or the virB mutant were taken at different times postinoculation, and cytokine concentrations were determined by Multi-Plex assays. Data shown are increases in cytokine concentrations over preinfection levels. The significance of differences for wild-type and mutant mice at each time point was determined using a paired Student's t test. Error bars represent standard deviations. Asterisks indicate significant differences between the wild type and the mutant (P < 0.05).

Splenocytes of mice infected with the Brucella wild-type strain produce higher levels of IFN-γ than do splenocytes of mice infected with the T4SS mutant.Since B. abortus grows and persists in tissues of the reticuloendothelial system, we determined whether the difference in the level of circulating IFN-γ correlates with changes in IFN-γ production in splenic tissue when the CFU levels of 2308 and the virB mutant are similar. RNA from spleens isolated from mice infected with B. abortus 2308 or the virB mutant was used to quantify IFN-γ transcripts by quantitative real-time PCR. The results shown in Fig. 3A are normalized to levels of GAPDH and to levels in uninfected mice using the 2^−ΔΔCT formula. Figure 3B (bottom) shows increases in IFN-γ protein levels measured by a BioPlex assay in concentrated supernatants of spleen homogenates. Significantly greater increases in both IFN-γ transcripts and protein levels were observed at day 4 postinfection in mice infected with B. abortus 2308 than in mice infected with the virB mutant, which is in agreement with the levels of IFN-γ measured in the serum (Fig. 2).

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FIG. 3.

Quantification of IFN-γ levels in spleens of mice infected with B. abortus 2308 or the virB mutant. (A) Transcript levels in spleens from groups of five mice infected with either B. abortus 2308 (black bars) or the virB mutant (white bars) were assayed by real-time reverse transcription-PCR at different times postinoculation. Increases (2^−ΔΔCT) in transcript levels were calculated by normalizing C_T values for IFN-γ levels to C_T values for GAPDH levels and to C_T values for IFN-γ levels measured in a group of mock-infected mice. (B) Cytokine measurement of IFN-γ in splenic tissue by Multi-Plex assay was performed with groups of five mice infected with either B. abortus 2308 or the virB mutant at different times postinoculation. Data shown are increases in cytokine concentrations over preinfection levels. The significance of differences for wild-type and mutant mice at each time point was determined using a paired Student's t test. Error bars represent standard deviations. Asterisks indicate significant differences between the wild type and the mutant (P < 0.05). Results shown at the top and bottom are from two independent experiments.

Splenic CD4⁺ T cells, but not CD8⁺ T cells, produce more IFN-γ when mice are infected with wild-type B. abortus than when they are infected with the virB mutant.We decided to determine the main source of IFN-γ in splenic T cells after infection with wild-type Brucella or the virB mutant (Fig. 4 and 5). Three days after infection, when the bacterial burdens of wild-type B. abortus and the virB mutant were similar (31, 32; data not shown), mice infected with wild-type B. abortus had more numerous IFN-γ-producing CD4⁺ T cells (9.7% ± 0.6%) than did virB mutant-infected mice (2.3% ± 0.5%) (Fig. 6). However, no difference in the frequencies of IFN-γ-producing CD8⁺ T cells in wild-type and mutant-infected mice was observed (2.7% ± 0.6% for 2308 and 2.6% ± 0.5% for the virB mutant) (Fig. 6). This result shows that CD4⁺ T cells are the source of increased levels of IFN-γ elicited by B. abortus and that infection with the virB mutant elicits reduced IFN-γ production by splenic CD4⁺ T cells.

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FIG. 4.

IFN-γ-producing CD4⁺ T cells in spleens of mice after infection with B. abortus 2308 or the virB mutant. C57BL/6 mice (four mice per group) were injected i.p. with PBS or 5 × 10⁵ CFU of B. abortus 2308 or the virB mutant. Mice were sacrificed 3 days after infection, and splenocytes were stained with a cocktail of Pacific Blue-conjugated anti-mouse CD4 (L3T4), Alexa Fluor 700-conjugated anti-mouse CD8a (Ly-2), APC-Alexa Fluor 750 anti-mouse CD3e, and phycoerythrin-conjugated anti-mouse IFN-γ. One million cells/sample were acquired with an LSRII apparatus. Numbers indicate the percentages of cells in each gate. Data for one mouse per group are shown as a representative of the group. SSC, side scatter.

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FIG. 5.

IFN-γ-producing CD8⁺ T cells in spleens of mice after infection with B. abortus 2308 or the virB mutant. C57BL/6 mice (four mice per group) were injected i.p. with PBS or 5 × 10⁵ CFU of B. abortus 2308 or the virB mutant and sacrificed 3 days after infection. Splenocytes were stained as described in the legend of Fig. 4. One million cells/sample were acquired with an LSRII apparatus. Numbers indicate the percentages of cells in each gate. Data for one mouse per group are shown as a representative of the group. SSC, side scatter.

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FIG. 6.

Percentage of splenic CD4⁺ T cells or CD8⁺ T cells secreting IFN-γ after infection with B. abortus 2308 or the virB mutant. C57BL/6 mice (four mice per group) were injected i.p. with PBS or 5 × 10⁵ CFU of B. abortus 2308 or the virB mutant. Mice were sacrificed 3 days after infection, and cells/mouse were stained for IFN-γ-producing cells. One million cells/sample were acquired with an LSRII apparatus. The asterisk represents statistical significance determined using a Student's t test on data after logarithmic conversion.