Coxiella burnetii Antigen-Stimulated Dendritic Cells Mediated Protection against Coxiella burnetii in BALB/c Mice

Abstract

Coxiella burnetii is the etiological agent of human Q fever. In this study, adaptive transfer of mouse bone marrow–derived dendritic cells (BMDCs) stimulated with C. burnetii antigen, phase I whole-cell antigen (PIAg), lipopolysaccharide (LPS)–removed PIAg (PIIAg), protein antigen Com1, or SecB significantly reduced coxiella burden in recipient mice compared with control mice. Mice that received PIIAg-pulsed BMDCs displayed substantially lower coxiella burden than recipient mice of PIAg-pulsed BMDCs after C burnetii challenge. The protection offered by the antigen-activated BMDCs was correlated with the increased proliferation of helper T (T_H) T_H1 CD4⁺ cells, preferential development of T_H17 cells, and impaired expansion of regulatory T lymphocytes. Our results suggest that PIIAg is far superior to PIAg in activating BMDCs to confer protection against C. burnetii in vivo, whereas Com1 and SecB are protective antigens because Com1- or SecB-pulsed BMDCs confer partial protection.

Coxiella burnetii, a gram-negative obligate intracellular bacterium, is the etiological agent of human Q fever [1]. Though overt disease is not apparent in most hosts of C. burnetii, humans may become infected and develop clinical symptoms after inhalation of C. burnetii–contaminated aerosols. Often misdiagnosed, the disease presents in the acute form as a flu-like illness with fever, chills, malaise, and a characteristic periorbital pain [2]. Chronic disease, although rare, is a severe illness that usually manifests as endocarditis and occasionally as vascular infection, osteomyelitis, or chronic hepatitis [3]. Epidemiologic studies suggest that some patients who contract acute Q fever progress years later to chronic disease, which most commonly presents as endocarditis or hepatitis [4]. Moreover, aerosol transmission, environmental stability, and a very low infectious dose [5] make C. burnetii a potential biowarfare agent (Centers for Disease Control and Prevention [CDC], category B).

Dendritic cells (DCs), the antigen-presenting cells (APCs) found in the skin, mucosa, and lymphoid tissues, capture and process microbial antigens from epithelia and tissues. DCs then migrate to draining lymph nodes to start a primary immune response by activating T lymphocytes and secreting cytokines [6, 7]. DCs are the most potent APCs that link the innate and adaptive immune responses and are critical factors in the host defense system against many pathogens because of their ability to effectively activate naïve CD4⁺ and CD8⁺ T lymphocytes [8, 9]. However, the role of DCs, particularly of DC subsets, in triggering immune responses during intracellular bacterial infection has been poorly understood. Earlier studies demonstrated that DCs play an important role in regulation of host responses against intracellular bacteria such as Listeria monocytogenes and Candida albicans [10, 11]. For rickettsiae, Jordan et al found that the DCs activated by Rickettsia conorii could offer protective immunity to naïve mice by their vigorous proinflammatory response [12]. In addition, Fang et al first reported that mouse bone marrow-derived dendritic cells (BMDCs) isolated from a mouse strain resistant to rickettsial infection exhibited higher expression of major histocompatibility complex class II (MHC-II), showed elevated production of interleukin-12 (IL-12) p40 upon rickettsial challenge, and were more potent in priming naïve CD4⁺ T cells to produce γ interferon (IFN-γ) compared with BMDCs isolated from a mouse strain susceptible to rickettsial infection [13]. For C. burnetii, Shannon and colleagues found that infection with the virulent phase I strain that produces full-length LPS did not result in human DC maturation, whereas infection with the avirulent phase II strain that produces truncated LPS resulted in a Toll-like receptor 4 (TLR 4)–independent DC maturation and much more IL-12 and tumor necrosis factor (TNF) production. This inability to induce efficient maturation of DCs is due to the presence of full-length LPS in virulent C. burnetii that masks Toll-like receptor ligands from innate immune recognition by DCs [14].

To further investigate the roles of DCs in initiating and orchestrating the immune response against C. burnetii, we stimulated BMDCs isolated from BALB/c mice with C. burnetii whole-cell or protein antigens. After maturation in vitro, the antigen-pulsed BMDCs were adaptively transferred into naïve BALB/c mice, followed with C burnetii challenge. The activated BMDCs’ mediated protection against C. burnetii was measured and the initial interactions between antigen-pulsed BMDCs and helper helper T (T_H )– and regulatory T (Treg)–cell subsets (T_H1, T_H2, T_H17, and Treg) had been characterized.

MATERIALS AND METHODS

Mice

Male BALB/c mice (aged 6–8 wk) were purchased from the laboratory animal center of Beijing, China. The animal usage was approved by the Beijing administrative committee for laboratory animals, and the animal care met the standard of the committee.

Purification and Culture of C. burnetii

Virulent phase I C. burnetii (Xinqiao strain) isolated from ticks in China [15] was propagated in embryonated eggs and inactivated with formalin. The cells were purified by renografin density centrifugation as described elsewhere [16, 17]. The purified cells were used as a phase I antigen (PIAg). The purified cells were extracted with trichloroacetic acid (TCA) to remove LPS, and the TCA-extracted cells were recognized as artificial phase II Ag (PIIAg) [18].

Recombinant Protein Antigens

The gene-encoding protein antigens Com1 (outer membrane protein 1), SecB (putative protein-export protein), and EnhA (enhanced entry protein A) were amplified with primer pairs (Table 1) designed based on gene sequences of RSA 493 strain (GenBank: NC002971). The gene fragments were cloned into plasmid pET32a(+) systems and expressed in Escherichia coli BL21 (DE3) (Novagen), using a method that was described elsewhere [19]. The His-tagged recombinant proteins were purified by affinity chromatography with Ni-NTA resin (Qiagen GmbH, Hilden) according to the manufacture's protocols (Figure 1A). The purified recombinant proteins were analyzed by immunoblotting assay with sera that were collected from Xinqiao strain–infected mice at day 28 after infection (Figure 1B). The LPS levels of the recombinant proteins were <.06 endotoxin units (EU)/mL determined by Limulus amoebocyte lysate assay (Sigma-Aldrich).

Table 1.

Three Pairs of Primers Designed for Expression of enhA, secB, and com1 in E. coli

Gene ID	Gene Name	Primers	Restriction Sites	Production (bp)
CBU0053	enhA	5′-GCTGAATTCTCGATGCTTGTTCTGCTTGC-3′	EcoRI	693
		5′-CATCTCGAGCACGATGATCTGAGTACCGAC-3′	XhoI
CBU1519	secB	5′-GAAGGATCCCGAACAAATACGCCAGAT-3′	BamHI	414
		5′-CGTCTCGAGTTCAAACAAGGCATCAAAG-3′	XhoI
CBU1910	com1	5′-ATCGGATCCTTAGCCGGAACCTTGACC-3′	BamHI	657
		5′-GCCCTCGAGTAACGCTTTATTACCAATGACG-3′	XhoI

Figure 1.

Analysis of recombinant protein antigens of C. burnetii. A, SDS-PAGE analysis of the purified recombinant proteins fused with 17 kDa tagged-region from pET32a(+) expression plasmid. B, Immunoblotting assay of the recombinant protein antigens with sera from C. burnetii–infected mice.

Generation of Dendritic Cells from Mouse Bone Marrow

We isolated BMDCs (CD11c⁺) from bone marrow of BALB/c mice according to the protocol described elsewhere [20]. We prepared a single-cell suspension from bone marrow from the mouse femurs and then cultured it in complete Roswell Park Memorial Institute 1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 50 μM 2-mercaptoethanol, 100 μg/mL streptomycin sulfate, and 100 U/mL penicillin. Approximately 1 × 10⁶ cells were added to each well of a 6-well plate, and granulocyte-macrophage colony-stimulating factor (GM-CSF; 20 ng/mL; PeproTech) and IL-4 (10 ng/mL; PeproTech) were added to the culture medium. After 6 days of culture, nonadherent cells were collected and stimulated with C. burnetii antigens.

BMDC Activation Assay

We pulsed GM-CSF plus IL-4-treated BMDCs (1 × 10⁶ cells/well) with .01 mL of whole-cell antigen (1 × 10⁶ cells of PIAg or PIIAg), protein antigen (10 ng of Com1, SecB, or EnhA), E. coli LPS (50 ng; Sigma), or .01 mL of antigen dilution buffer. We used the LPS-pulsed BMDCs and buffer-pulsed BMDCs (mock-pulsed BMDCs) as positive and negative controls, respectively. Antigen-pulsed cells were incubated at 37°C and 5% CO₂, and we collected the supernatants at 5, 10, and 22 h during incubation for detection of IL-6, IL-10, and IL-12p40 production by enzyme-linked immunosorbent assay (ELISA) kit (eBioscience). In addition, we harvested the antigen-pulsed BMDCs after 24 h of incubation and stained with the combination of antibodies (FITC-conjugated anti-CD11c; PE-conjugated anti-CD11b; PE/Cy5-conjugated anti-CD40, anti-CD80, anti-CD86, and anti-MHC-II; BioLegend) according to the manufacturer's protocol. We determined stained cells with a FACScalibur flow cytometer (BD Biosciences) and analyzed data with CellQuest software (version 5.2; BD Biosciences).

BMDC Adaptive Transfer and Mouse Challenge

After 24 h of antigen stimulation, we resuspended nonadherent antigen-pulsed BMDCs in culture media at the concentration of 5 × 10⁶ cells/mL [12]. The antigen-pulsed cells containing >80% CD11c⁺ and CD11b⁺ double-positive cells were intraperitoneally injected into BALB/c mice (5 × 10⁵ cells/mouse). Twenty-four hours after the BMDCs injection, mice were intraperitoneally challenged with Xinqiao strain C. burnetii (1 × 10⁸ cells/mouse) in a biosafety level 3 laboratory. We used the mice receiving mock-pulsed BMDCs as negative controls. We killed the mice on day 6 after challenge and harvested their spleens for detection of C. burnetii by a quantitative PCR analysis [19, 21].

Generation of Antigen-Specific T Cells Induced by Antigen-Pulsed BMDCs

At day 6 after transfer, we prepared splenocytes from recipient mice and cultured them with homologous antigen-pulsed BMDCs in the ratio of 10:1 (splenocyte:BMDC). We collected the supernatants at 5-, 10-, and 22-h coincubation for measurement of IFN-γ, IL-4, and IL-17 by enzyme-linked immunosorbent assay (ELISA) kit (eBioscience). Additionally, after 18 h of co-culture, we analyzed splenocytes for expression of CD69, IFN-γ, IL-17, and Foxp3 on CD4⁺ T cells using flow cytometry. We incubated splenocytes with FITC-conjugated anti-CD4 (eBioscience) for 30 min at 4°C, and we then stained some of splenocytes with PE-conjugated anti-CD69 (eBioscience). For intracellular staining, we added PMA (10 ng/mL) and ionomycin (1 μg/mL) onto the splenocytes at 2 h, and then we added Brefeldin A (1 μg/mL), an inhibitor of intracellular protein transport, at 4 h to contain intracellular cytokines or APC-conjugated anti-CD25 (eBioscience). The cells were stained with antibodies to cell surface molecules and then fixed and permeabilized (IC fixation/permeabilization buffer; eBioscience) for 20 min. We washed the cells with permeabilization buffer and stained them with one of the antibodies (PE-conjugated anti-IL-4, APC-conjugated anti-IFN-γ, PE-conjugated anti-Foxp3, or PE-conjugated anti-IL-17; eBioscience) according to the manufacturer's protocol. We determined stained cells with FACScalibur flow cytometry and analyzed data with CellQuest software.

Statistical Analysis

We performed data analysis with SPSS software (version 10.0; SPSS). We compared groups using the 1-way analysis of variance (ANOVA) test. Values were expressed as means with standard deviations (SDs). P values of <.05 were considered significant.

RESULTS

Effects of C. burnetii Antigens on Maturation of BMDCs

Compared with mock-pulsed BMDCs, the percentages of MHC-II and CD11c⁺ double-positive cells on PIIAg-, PIAg-, Com1-, SecB-, or EnhA-pulsed BMDCs were 1.3–2.9-fold higher after 24-h stimulation (Figure 2). For expression of costimulatory molecules, PIAg- or PIIAg-pulsed BMDCs were higher than Com1-, SecB-, or EnhA-pulsed BMDCs; however, there was no significant difference between Com1-, SecB-, and EnhA-pulsed BMDCs (Figure 2).

Figure 2.

Activation and maturation of bone marrow–derived dendritic cells (BMDCs) induced with C. burnetii antigens. The lipopolysaccharide-pulsed BMDCs were used as positive controls (LPS), and mock-pulsed BMDCs were used as negative controls (Control). Data are representative of 3 independent experiments with similar results, and the percentage of double-positive cells is indicated in the top right corner.

Protection against C. burnetii Mediated by Antigen-Pulsed BMDCs

Compared with mice receiving mock-pulsed BMDCs, mice receiving BMDCs pulsed by PIIAg, PIAg, or Com1 exhibited significantly lower coxiella burden, and mice receiving SecB-pulsed BMDCs displayed a moderate reduction of coxiella burden. However, coxiella burden in mice receiving EnhA-pulsed BMDCs or E. coli LPS-pulsed BMDCs was slightly higher than that in the control group (Figure 3). Mice receiving PIIAg-pulsed BMDCs exhibited much lower coxiella load than mice receiving PIAg- or Com1-pulsed BMDCs (Figure 3).

Figure 3.

Estimation of coxiella burden in recipient mice of antigen-stimulated bone marrow–derived dendritic cells (BMDCs) by quantitative polymerase chain reaction (PCR). C. burnetii 23S IVS DNA copies were determined in 5 mouse samples per group by quantitative real-time PCR, and data were expressed as the average copy number of 5 samples. The mice receiving mock-pulsed BMDCs were used as negative controls (Ctrl). As compared with the control (Ctrl), P < .05 is considered statistically significant and indicated by asterisks. Error bars indicate the standard deviation (SD) of the mean for 5 mice. *P < .05; **P < .01.

Primary Proliferative Responses in Naïve CD4⁺ T Cells Exposed to Antigen-Pulsed BMDCs

After co-culture with antigen-pulsed BMDCs, CD4⁺ T cells in splenocytes from mice receiving PIIAg-, PIAg-, Com1-, or SecB-pulsed BMDCs exhibited significantly higher CD69 expression than that of the control group, whereas splenocytes from mice receiving EnhA-pulsed BMDCs displayed a weak CD69 expression on CD4⁺ T cells (Figure 4A). The percentages of CD4⁺ and IFN-γ double-positive cells in splenocytes from mice receiving BMDCs-pulsed with PIIAg, PIAg, Com1, or SecB were 3.6-, 2.5-, 2.1-, and 1.6-fold higher, respectively, than that in splenocytes from mice receiving EnhA-pulsed BMDCs (Figure 4B). In addition, the percentages of CD4⁺ and IL-17 double-positive cells in splenocytes from mice receiving PIIAg-, PIAg-, Com1-, or SecB-pulsed BMDCs were 5.10%, 4.94%, 2.85%, and 1.98%, respectively, whereas the percentages of CD4⁺ and IL-17 double-positive cells in splenocytes from mice receiving EnhA-pulsed BMDCs was similar to control group percentages (.97% vs .42%) (Figure 4C). However, the percentages of regulatory T lymphocytes (Treg cells, Foxp3-expressing CD25⁺CD4⁺) in splenocytes from mice receiving PIIAg-, PIAg-, Com1-, and SecB-pulsed BMDCs were 5.25%, 1.56%, 3.81%, and 7.12%, respectively, which were substantially lower than those from mice receiving EnhA-pulsed BMDCs (13.05%) or control group (15.55%) (Figure 4D).

Figure 4.

Differential effects of antigen-pulsed bone marrow–derived dendritic cells (BMDCs) on naïve T cells. Expression of CD69 (A), IFN-γ (B), IL-17 (C), or Foxp3/CD25 (D) on CD4⁺ T cells was detected by flow cytometry. Splenocytes from recipient mice of lipopolysaccharide-pulsed BMDCs were used as controls (LPS), and splenocytes from recipient mice of mock-pulsed BMDCs were used as negative controls (Control). Data are representative of 3 independent experiments with similar results. The percentage of double-positive cells is indicated in the top right corner.

Cytokine Secretion of BMDCs and Splenocytes

After antigen stimulation, we detected the cytokines in supernatants of BMDCs using ELISA. BMDCs pulsed with PIIAg, PIAg, Com1, or SecB exhibited a greater amount of IL-6 and IL-12p40 than EnhA-pulsed BMDCs (Figure 5A and 5B); however, EnhA-pulsed BMDCs displayed substantially higher secretion of IL-10 (Figure 5C). In addition, higher levels of IFN-γ and IL-17 and lower levels of IL-4 were determined on splenocytes from mice receiving BMDCs pulsed with PIIAg, PIAg, Com1, or SecB compared with splenocytes from recipient mice of EnhA-pulsed BMDCs (Figure 5D, 5E, and 5F).

Figure 5.

Determination of cytokines from antigen-pulsed bone marrow–derived dendritic cells (BMDCs) and splenocytes of recipient mice of antigen-pulsed BMDCs by ELISA. IL-6 (A), IL-12p40 (B), and IL-10 (C) were determined in supernatants of antigen-pulsed BMDCs. IL-4 (D), IFN-γ (E), and IL-17 (F) were determined in supernatants of mouse splenocytes after coculture with homologous antigen-pulsed BMDCs. Lipopolysaccharide-pulsed BMDCs/splenocytes from recipient mice of lipopolysaccharide-pulsed BMDCs were used as controls (LPS), and mock-pulsed BMDCs/splenocytes from recipient mice of mock-pulsed BMDCs were used as negative controls (Ctrl). Data are expressed as the mean ± SD of 3 independent experiments. As compared with control (Ctrl): *P < .05; **P < .01.

DISCUSSION

Dendritic cells (DCs) serve as immune sentinels that detect the presence of pathogens and orchestrate the host′s immune response to infection [22], and immature DCs may be first encountered by C. burnetii during natural infection. Immature DCs infected ex vivo with virulent phase I organisms of C. burnetii exhibited much lower levels of maturation or inflammatory cytokines compared with that infected with avirulent phase II organisms, suggesting that LPS as a shielding molecule prevented access of C. burnetii surface protein antigens to pattern recognition receptors on DCs [14]. In addition, BMDCs activated with chlamydial organisms induced protection against chlamydial infection of the female genital tract in mice [23] and R. conorii–activated DCs mediated protection against lethal rickettsial challenge [12]. The ability of DCs to induce protection against different infections has prompted investigation of their use as cell-based vaccines. Although the technique of antigen-activated DC transfer may not be practical in humans, the transfer of DCs that induces protection in experimental animals will extend our understanding of the requirements to induce protective immune responses and help us to identify protective antigens for designing molecular vaccines against corresponding infections.

In this study, C. burnetii whole-cell antigens PIAg and PIIAg were used to stimulate BMDCs. Our results showed that the expression of MHC-II on PIIAg-pulsed cells was approximately 2-fold greater than that of PIAg-pulsed BMDCs. In addition, the levels of costimulatory molecules (CD40, CD80, and CD86) of PIIAg-pulsed BMDCs were also higher than those of PIAg-pulsed BMDCs. Our results are consistent with those of a previous study of human monocyte-derived DCs infected with phase I and phase II organisms [14], suggesting that LPS of C. burnetii interfered with maturation and activation of BMDCs.

To study immune protections mediated by antigen-activated BMDCs, we transferred C. burnetii antigen-pulsed BMDCs into naïve mice and then challenged the recipient mice with virulent C. burnetii organisms 24 h later. We analyzed the coxiella burden in spleens from mice by quantitative PCR 6 days after challenge. Our results showed that recipient mice of PIIAg-, PIAg-, Com1-, or SecB-pulsed BMDCs exhibited significantly lower coxiella burden compared with mice receiving mock-pulsed BMDCs, suggesting that these C. burnetii antigens could activate BMDCs to confer protection against C. burnetii. Furthermore, mice receiving PIIAg-pulsed BMDCs exhibited much lower coxiella burden than those receiving PIAg-pulsed BMDCs, indicating that BMDCs pulsed by LPS-removed coxiella cells could induce even greater protection against C. burnetii and that the protection was associated with the bacteria’s surface-exposed protein antigens that had a potent induction of maturation and activation of BMDCs. However, Q fever vaccine prepared with phase I organisms that produce full-length LPS elicited a significantly higher level of protection against virulent C. burnetii infection than did vaccine made by phase II organisms that produce truncated LPS [24]. The generation of robust immunity to C. burnetii could be attributed to full-length LPS of phase I organisms [24]. It is well known that phase I organisms elicit antibodies to phase I and phase II antigens; however, phase II organisms elicit antibodies mainly to phase II antigens. Specific antibodies play important roles in protective immunity against a number of pathogens via a variety of different mechanisms, including direct antibactericidal activity, complement activation, toxin neutralization, opsonizaton/phagocytosis, antibody-dependent cellular cytotoxicity, altered intracellular trafficking of pathogens, and modulation of the immune response through interactions with Fc and complement receptors [25]. Also, phase I antibodies play important roles in immunity to C. burnetii, as in antibody-opsonization of phase I organisms to increase their phagocytosis by macrophages and passive immunization of naïve mice with sera from phase I organism–immunized mice to protect animals against virulent C. burnetii [25]. The precise mechanisms of antibody-mediated immunity against C. burnetii remain to be further investigated.

In this study, coxiella burden in recipient mice of Com1- or SecB-pulsed BMDCs was significantly lower than that in mice receiving mock-pulsed BMDCs; however, there was no significant difference in coxiella burden between mice receiving EnhA-pulsed BMDCs and mice receiving mock-pulsed BMDCs. The result demonstrates that Com1- or SecB-pulsed BMDCs can elicit efficient maturation and activation of BMDCs to confer protection against C. burnetii in mice, although Com1 failed to induce this protection in a previous study [26]. In the previous study, Com1 was used to immunize animals with Titermax adjuvant, which was inefficient to induce a cell-mediated immune response against C. burnetii infection. Because cell-mediated immunity plays a critical role in controlling C. burnetii infection, in this study, Com1 was used to prime BMDCs ex vivo; the Com1-activated BMDCs might elicit a T cell–mediated immune response via direct interaction with T cells in vivo after adaptive transfer into recipient mice. Moreover, Com1 and SecB were both recognized by the sera from C burneti–infected mice but EnhA was not. These data suggest that Com1 and SecB are potent immunogens that can induce a humoral immune response to produce specific antibodies and EnhA is a weak immunogen because of its inability to elicit the humoral immune response.

Although there was no significant difference among Com1-, SecB-, and EnhA-pulsed BMDCs for expression of MHC-II and costimulatory molecules, the protection levels mediated by these antigen-pulsed BMDCs in vivo were dramatically different in this study, suggesting that differential function effectors may be induced by different antigen-pulsed BMDCs.

Our results showed that PIIAg-, PIAg-, Com1-, or SecB-pulsed BMDCs efficiently activate syngeneic CD4⁺ T cells leading to higher expression of an early activation marker CD69, whereas EnhA-pulsed BMDCs had a weak effect on T cell activation. Furthermore, splenocytes from mice receiving BMDCs pulsed with these antigens also exhibited greater percentages of CD4⁺ and IFN-γ double-positive cells compared with splenocytes from recipient mice of EnhA-pulsed BMDCs. The data demonstrate that BMDCs pulsed with these C. burnetii antigens except EnhA have ability to activate CD4⁺ T cells to promote their clonal expansion and acquisition of effector function, such as IFN-γ production. Our data also showed that the percentages of CD4⁺ and IFN-γ double-positive cells in splenocytes from recipient mice of PIIAg-, PIAg-, Com-, or SecB-pulsed BMDCs were well correlated with their protection levels, suggesting that IFN-γ produced by CD4⁺ T cells played an important role in this specific protection.

It is well accepted that cells of the monocyte/DC lineage, particularly activated DCs, dictate adaptive T cell response toward a T_H1 or T_H2 pattern by secreting the cross-regulatory cytokines IL-12 and IL-10 [27]. IL-12 is the most crucial cytokine that drives the development of naïve T cells into T_H1 cells that produce high levels of IFN-γ in vitro and in vivo [28]. In contrast, IL-10 is involved in down-regulating DC antigen-presenting function and inducing T cell tolerance [29]. In addition, IL-10 can inhibit IL-12 release and IL-12 effect on T cells, thus down-regulating T_H1 responses [30]. A previous study of human DCs showed that the T_H2 cytokine IL-4 enhanced DC1 maturation but killed precursor DC2, and this IL-4 effect was blocked by IFN-γ [31]. Furthermore, IL-6 is recognized as a cytokine having ability to promote the generation and development of IL-17-producing CD4⁺ T cells and suppress the development of Treg cells [32].

To define the influence of antigen-pulsed BMDCs on subsets of CD4⁺ T cells, we analyzed the cytokines (IFN-γ, IL-4, IL-6, IL-10, IL-12p40, and IL-17) in culture supernatant of antigen-pulsed BMDCs or splenocytes from recipient mice of antigen-pulsed BMDCs. Our results showed that BMDCs stimulated by PIIAg, PIAg, Com1, or SecB secreted more IL-6 and IL-12p40 and less IL-10 compared with EnhA-pulsed BMDCs. Higher levels of IFN-γ and IL-17 and lower levels of IL-4 were secreted by splenocytes from mice receiving BMDCs activated by PIIAg, PIAg, Com1, or SecB compared with splenocytes from recipient mice of EnhA-pulsed BMDC. Additionally, higher percentages of CD4⁺ and IL-17 double-positive cells and lower percentages of Treg cells were observed in splenocytes from mice receiving BMDCs pulsed by PIIAg, PIAg, Com1, or SecB, but the opposite results were observed in splenocytes from recipient mice of EnhA-pulsed BMDCs. These data suggest that BMDCs activated by these antigens except EnhA can stimulate the production of proinflammatory cytokines IL-6 and IL-12, which in turn stimulates CD4⁺ T-cell differentiation into T_H1 (IFN-γ) and T_H17 cells and inhibits generation of T_H2 (IL-4) and Treg cells. Thus, the protection mediated by these antigen-pulsed BMDCs can be attributed to the advantageous transformation of T_H1/T_H17.

In this study, the protection against C. burnetii was induced in vivo by PIIAg-, PIAg-, Com1-, or SecB-activated BMDCs, and this protection was associated with proliferation of T_H1 CD4⁺ T cells, preferential development of T_H17 cells, and impaired expansion of Treg cells. Our results suggest that PIIAg possesses a substantially greater ability to induce a protective immune response against C. burnetii through antigen-activated BMDCs compared with PIAg, and Com1 and SecB are protective antigens because Com1- or SecB-activated BMDCs can mediate partial protection against C. burnetii. Additionally, analysis of protection against C. burnetii mediated by antigen-pulsed BMDCs in mice provides a valuable tool for screening of protective antigens that may be more suitable candidates for preparing subunit vaccines against Q fever.

Funding

This work was supported by the National Natural Science Foundation of China (grants 30670101 and 30700744) and the National Basic Research Program of China (grants 2010CB530200 and 2010CB530205).