ABSTRACT
A Salmonella vector vaccine expressing the saliva-binding region (SBR) of the adhesin AgI/II of Streptococcus mutans has been shown to induce a mixed Th1/Th2 anti-SBR immune response in mice and to require Toll-like receptor 2 (TLR2), TLR4, and MyD88 signaling for the induction of mucosal anti-SBR antibody responses. Since dendritic cells (DC) are critical in innate and adaptive immunity, the present study assessed the role of SBR expression by the vector vaccine in DC activation. Bone marrow-derived DC from wild-type and TLR2, TLR4, and MyD88 knockout mice were stimulated with Salmonella vector BRD509, the SBR-expressing Salmonella vector vaccine BRD509(pSBRT7), or SBR protein, and the DC responses to different stimuli were compared by assessing costimulatory molecule expression, cytokine production, and signaling pathways. The DC response to both BRD509(pSBRT7) and BRD509 was dependent mainly on TLR4. BRD509(pSBRT7) and BRD509 induced upregulation of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) expression. Lower levels of interleukin-10 (IL-10) and IL-12p40 were produced by BRD509(pSBRT7)-stimulated DC than by BRD509-stimulated DC. Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by DC stimulated with BRD509. However, BRD509(pSBRT7)-treated DC produced a higher level of IL-6 than BRD509-stimulated cells. The low IL-12p40 and high IL-6 cytokine profile expressed by BRD509(pSBRT7)-stimulated DC may represent a shift toward a Th2 response, as suggested by the increased expression in Jagged-1. These results provide novel evidence that a heterologous protein expressed by a Salmonella vector vaccine can differentially affect DC activation.
INTRODUCTION
Streptococcus mutans has been considered to be a major causative agent of human dental caries (15, 30). Initial adherence of S. mutans to tooth surfaces is mediated largely by the surface fibrillar adhesin known as AgI/II (also known as antigen B, P1, SpaP, and PAc) (13, 29). Numerous studies in experimental animal models have shown that immunization with AgI/II can induce specific salivary IgA antibody responses that inhibit S. mutans colonization and protect against dental caries (25, 46, 50). Furthermore, in our previous studies (14) and those of others (12), it was shown that the 42-kDa saliva-binding region (SBR) of AgI/II also exhibits good immunogenicity in inducing specific salivary IgA response.
Attenuated Salmonella mutants are being used to develop live antigen delivery systems for mucosal immunization (6, 24, 53). Following oral immunization, live Salmonella vectors expressing recombinant heterologous antigens can enter the Peyer's patches, an important IgA inductive site, through specialized microfold cells (7, 22). In the Peyer's patches, the Salmonella bacteria replicate and persist while expressing recombinant proteins, thus serving as a source of heterologous antigens for the induction of mucosal immune responses. In our previous studies (16), we derived the Salmonella vector vaccine BRD509(pSBRT7) by introducing a plasmid encoding SBR of AgI/II into the BRD509 strain of Salmonella enterica serovar Typhimurium (aroA aroD mutant) under the control of the inducible T7 promoter and demonstrated the ability of the cloned SBR to induce salivary and serum antibody responses following mucosal immunization with BRD509(pSBRT7). However, it is still not clear how BRD509(pSBRT7) activates antigen-presenting cells (APC), such as dendritic cells (DC), to initiate the immune response.
Adaptive immunity is triggered and profoundly influenced by innate immunity. The most potent APC, DC, play an important role in linking the innate and adaptive immune systems due to a number of characteristics, including their presence at sites of antigen entry, their ability to take up and process antigen, their ability to migrate to secondary lymphoid tissue, and their capability to present antigenic peptides and activate naïve T cells (4, 5, 45, 49). Naive Th cells can acquire at least four distinct phenotypes following activation by DC, including three distinct types of effectors, Th1, Th2, and Th17 cells, and various subsets of regulatory T cells (44, 56). Studies showed that cytokines produced by DC exert an important influence on the differentiation of specific Th effector cells (1, 36, 40, 51). Moreover, there is evidence that distinct Notch ligands expressed on DC also play a role in Th development (3, 39). Therefore, it is important to delineate the signaling molecules involved in DC activation by the Salmonella vector vaccine expressing heterologous SBR protein, BRD509(pSBRT7), by the Salmonella vector, and by the SBR protein, since they may activate DC in different ways, thus influencing the nature of innate immunity and the resultant adaptive immune response.
DC recognize conserved microbial components via pattern recognition receptors, such as the Toll-like receptors (TLRs) (20, 26, 34). Most mammalian species have 10 to 15 TLRs. TLR1, -2, -4, -5, and -6 are located on the cell surface and recognize unique microbial components, whereas TLR3, -7, -8, and -9 are localized in intracellular endosomal compartments and recognize nucleic acids, which are not unique to microorganisms (26, 35). Exposure of DC to microbial stimuli leads to TLR triggering of intracellular signaling pathways. Two TLR signaling pathways that have been well characterized are the MyD88-dependent pathway, which is utilized by all TLRs known except TLR3, and the Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent pathway, which is utilized by TLR3 and TLR4 (2, 52). Activation of these pathways in DC results in the upregulated expression of costimulatory molecules and in antigen presentation, thus providing the respective signal 2 and 1 necessary to T cells for the initiation of adaptive immunity. In terms of a Salmonella vector vaccine expressing heterologous antigens, there are several TLR agonists that could activate DC. In a recent study (47), we investigated the role of TLR2, TLR4, and MyD88 in the antibody response to the Salmonella vaccine BRD509(pSBRT7) and demonstrated a role for TLR4 and MyD88 in the induction of salivary IgA anti-Salmonella antibodies. Furthermore, the involvement of TLR2, TLR4, and MyD88 in the induction of salivary anti-SBR antibody responses following the primary and booster immunizations was shown. However, the role of TLRs in the Salmonella vaccine-induced DC activation and the potential influence on the subsequent adaptive immunity have not been delineated. Therefore, in the present study, we compared the DC responses to the Salmonella vaccine BRD509(pSBRT7) and the Salmonella vector BRD509 in the context of TLR2, TLR4, and MyD88 by the assessment of signaling pathways, costimulatory molecules, and cytokine production.
MATERIALS AND METHODS
Mice.C57BL/6 wild-type (WT) and TLR2, TLR4, and MyD88 knockout (KO) mice (on the C57BL/6 background) were bred and maintained in an environmentally controlled, pathogen-free animal facility at the University of Alabama at Birmingham. The original TLR2, TLR4, and MyD88 KO breeding pairs were obtained under a material transfer agreement from Shizuo Akira (Osaka University, Osaka, Japan). Female mice (8 to 10 weeks of age) were used in this study. All experiments were done according to the guidelines of the National Institutes of Health, and protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
Bacteria and culture conditions.Attenuated Salmonella enterica serovar Typhimurium BRD509, a Salmonella vector strain, and the BRD509(pSBRT7) clone expressing SBR under the control of the T7 promoter were cultured in Luria-Bertani (LB) broth under aerobic conditions at 30°C, overnight. Afterwards, cultures were resuspended in LB medium and grown aerobically at 30°C for 2.5 h. The medium for BRD509(pSBRT7) always contained 50 μg/ml of carbenicillin and 50 μg/ml of kanamycin. The cultures were harvested at mid-log phase by centrifugation, and the cell pellets were suspended in phosphate-buffered saline (PBS). The bacterial concentration was determined by reading the optical density at 600 nm and extrapolating from a standard curve established by plating serial dilutions onto LB plates.
SBR, LPS, and Pam3CSK4.SBR was purified from cell lysates of Escherichia coli BL21(DE3) containing pET20b(+)-SBR using a His-bind resin column (Novagen, Madison, WI), as previously described (55). The purified SBR contained <0.0002% lipopolysaccharide (LPS), as determined by the Limulus amoebocyte assay. Escherichia coli K-12 LPS and Pam3CSK4 {N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lyslg)(3)-lysine} were purchased from InvivoGen (San Diego, CA). LPS served as a TLR4 agonist, and Pam3CSK4 served as a TLR2 agonist.
Generation of DC.Bone marrow-derived DC were generated as previously described (18, 19). Briefly, mice were sacrificed and the femurs and tibias were dissected and flushed with ice-cold PBS to extrude the bone marrow. A cell strainer was used to dissociate the bone marrow to get bone marrow cell suspensions. Then, erythrocytes were lysed using M-Lyse buffer (R&D Systems, Minneapolis, MN), and washed cells were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (50 U/ml), streptomycin (50 μg/ml), l-glutamine (2 mM), β-mercaptoethanol (50 μM), sodium pyruvate (1 mM), sodium bicarbonate (1.5 mg/ml), and HEPES (25 mM). The bone marrow cells were cultured in 24-well plates at a density of 1 × 106 cells/ml/well and incubated at 37°C in a humidified 5% CO2 environment. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D Systems) was added to the cultures at a final concentration of 20 ng/ml. Culture medium and GM-CSF were replaced on days 2 and 4. Additional culture medium with GM-CSF was added on day 6, and cells were harvested on day 7. This protocol routinely yielded >80% CD11c+ cells as determined by flow cytometry.
Flow cytometry.DC derived from WT and TLR2, TLR4, and MyD88 KO mice were cultured in 24-well tissue culture plates containing antibiotic-free RPMI 1640 medium at a density of 1 × 106 cells/ml/well. DC were incubated with BRD509 (multiplicity of infection [MOI] of 0.1), BRD509(pSBRT7) (MOI of 0.1), SBR (40 μg/ml), LPS (100 ng/ml), or Pam3CSK4 (300 ng/ml) for 24 h in a 5% CO2 incubator at 37°C. The DC were then harvested and stained with fluorescence-labeled antibodies against CD80, CD86, major histocompatibility complex class II (MHC II), and CD40 (eBioscience, San Diego, CA) in fluorescence-activated cell sorter (FACS) buffer (PBS supplemented with 2% bovine serum albumin and 0.1% sodium azide). Samples were incubated for 30 min on ice, washed with FACS buffer twice, and assayed on a FACSCalibur instrument (BD Bioscience, San Jose, CA). Data were analyzed using CellQuest software (BD Bioscience).
Cytokine ELISA.DC derived from WT and TLR2, TLR4 and MyD88 KO mice were cultured in 96-well tissue culture plates (2 × 105 cells/well) and stimulated as described above. Culture supernatants were harvested after 24 h and assessed for the levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) (eBioscience) and IL-12p40 and IL-10 (R&D Systems) by enzyme-linked immunosorbent assay (ELISA), according to the manufacturers' instructions. In another experiment, DC derived from WT mice were stimulated with BRD509(pSBRT7) or BRD509 for 48 h, and cytokine production was assayed by ELISA.
Western analysis.To assess the mitogen-activated protein kinases (MAPKs) engaged following stimulation of DC with BRD509 or BRD509(pSBRT7), DC derived from WT and TLR2, TLR4, and MyD88 KO mice were cultured in 24-well tissue culture plates (1 × 106 cells/well) and incubated with BRD509 (MOI of 0.1) or BRD509(pSBRT7) (MOI of 0.1) for 0, 10, 30, 60, or 120 min in a 5% CO2 incubator at 37°C. Cells were harvested, washed twice with ice-cold PBS, and lysed on ice for 10 min in radioimmunoprecipitation assay lysis buffer (Upstate Biotechnology, Lake Placid, NY). The cell lysates were then transferred to tubes, incubated on ice for another 20 min, and then centrifuged at 14,000 × g for 15 min at 4°C. The supernatants were collected and evaluated by Western analysis using specific antibodies against the phosphorylated forms of p38 (Thr180/Tyr182), ERK1/2 (P44/42, Thr202/Tyr204), or SAPK/JNK (Thr183/Tyr1859) (Cell Signaling Technology, Beverly, MA). Equal protein loading was monitored by the assessment of total p38. Blots were evaluated by ECL Western blotting detection reagents, according to the manufacturer's instructions (Amersham Bioscience, Piscataway, NJ). To assess the involvement of the Notch ligands Jagged-1 and Delta-1, DC derived from WT mice were stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), or SBR (40 μg/ml) for 12, 24, and 48 h. The expression of Jagged-1 and Delta-1 was evaluated by Western blot analysis using specific antibodies against Jagged-1 (Cell Signaling Technology) or Delta-1 (R&D Systems). β-Actin served as the loading control. Densitometry scans of the blots were performed using an AlphaImager 2000 documentation and analysis system (Alpha Innotech, San Leandro, CA).
p38 inhibition.To determine if the differential activation of p38 was involved in the differential production of cytokines between BRD509-stimulated DC and BRD509(pSBRT7)-stimulated DC, DC derived from WT mice were pretreated for 1 h with or without 10 μM SB203580 (EMD Biosciences, Inc., La Jolla, CA), a selective inhibitor for p38, and then stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), or SBR (40 μg/ml) for 24 h. After stimulation, culture supernatants were harvested and assessed for the levels of IL-6, TNF-α, IL-12p40, and IL-10 by ELISA.
Statistical analysis.Statistical significance in the cytokine responses to BRD509(pSBRT7), BRD509, and SBR was determined by unpaired analysis of variance (ANOVA), followed by post hoc analysis with the Tukey-Kramer multiple comparison test using GraphPad InStat version 3.0a (GraphPad Software, San Diego, CA). When determining the statistical significance between two groups, an unpaired t test was applied. Differences between groups were considered significant at a P value of <0.05.
RESULTS
TLR4-mediated upregulation in the expression of costimulatory molecules following stimulation with BRD509(pSBRT7) or BRD509.Initial studies to determine the TLR involvement in the induction of costimulatory molecules and MHC II expression on DC following BRD509(pSBRT7) and BRD509 stimulation revealed that BRD509(pSBRT7) and BRD509 induced an evident upregulation of CD80, CD86, and CD40 but only a slight upregulation of MHC II expression in DC from WT, TLR2 KO, and MyD88 KO but not TLR4 KO mice (Fig. 1 A). These results indicate that upregulation of these molecules by BRD509(pSBRT7) and BRD509 stimulation occurs mainly through TLR4 signaling via a TRIF-dependent pathway. Upregulation of costimulatory molecules by SBR was also TLR4 and TRIF dependent (Fig. 1A). Interestingly, MHC II expression following stimulation of DC derived from MyD88 KO mice was higher than that observed with WT DC (Fig. 1B), suggesting that MyD88 signaling exerts an inhibitory effect on MHC II expression.
Upregulation of costimulatory molecules and MHC II on DC. DC derived from WT and TLR2, TLR4, and MyD88 KO mice were stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), SBR (40 μg/ml), LPS (100 ng/ml), or Pam3CSK4 (300 ng/ml) for 24 h. Cells were stained with fluorescence-labeled antibodies against CD80, CD86, CD40, or MHC II, followed by FACS analysis. (A) Expression of CD80, CD86, CD40, and MHC II on DC from WT and TLR2, TLR4, and MyD88 KO mice following stimulation with BRD509, BRD509(pSBRT7), SBR, or unstimulated control. (B) Increased expression of MHC II on MyD88 KO DC compared to that on WT DC following different stimulation. Numbers represent the mean fluorescence intensity. Data are representative of three independent experiments.
BRD509(pSBRT7)-stimulated DC produce a cytokine profile different from that of BDR509-stimulated DC but similar to that of SBR-stimulated DC.To delineate the influence of TLRs in cytokine production, DC derived from WT and TLR2, TLR4, and MyD88 KO mice were stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), SBR (40 μg/ml), LPS (100 ng/ml), or Pam3CSK4 (300 ng/ml) for 24 h, and the culture supernatants were collected and assayed for IL-12p40, IL-6, TNF-α, and IL-10 by ELISA. BRD509(pSBRT7) induced significantly (P < 0.01) lower levels of IL-12p40 and IL-10 production by WT DC than BRD509; however, comparable levels of TNF-α were observed (Fig. 2A). Although a slightly higher level of IL-6 was seen with BRD509(pSBRT7)-stimulated DC than with BRD509-stimulated DC, the difference was not statistically significant (Fig. 2A). SBR stimulation resulted in higher IL-6 levels but lower IL-12p40, IL-10, and TNF-α levels than did BRD509(pSBRT7) and BRD509 stimulation of WT DC (Fig. 2A). TLR4 deficiency resulted in a marked decrease in the production of IL-12p40, TNF-α, and IL-10 following BRD509(pSBRT7), BRD509, or SBR stimulation (Fig. 2B). However, a differential regulation of IL-6 was observed, in that while stimulation of TLR4 KO DC with BRD509 showed increased IL-6 levels over those observed with WT DC, albeit not statistically significant, stimulation of TLR4 KO DC with BRD509(pSBRT7) or SBR decreased or completely abrogated the IL-6 response, respectively (Fig. 2B). In TLR2 KO DC, BRD509(pSBRT7) and BRD509 induced levels of IL-6 and TNF-α similar to those seen with WT DC. Moreover, higher levels of IL-12p40 and IL-10 were seen in TLR2 KO DC than in WT DC (Fig. 2B). This finding is supported by a previous study (48) showing that production of IL-12p40, IL-10, and TNF-α by macrophages from TLR2-deficient mice following stimulation with Salmonella enterica serovar Enteritidis was no less than that by WT macrophages. MyD88 deficiency resulted in an overall decrease in cytokine production (Fig. 2B), suggesting that MyD88 signaling plays an important role in the production of IL-12p40, TNF-α, and IL-10 following BRD509(pSBRT7) and BRD509 stimulation of DC. Interestingly, IL-6 production by BRD509(pSBRT7) but not by BRD509 was significantly lower in MyD88 KO DC than in WT DC, suggesting that MyD88 exerts a regulatory effect on the induction of IL-6 by BRD509(pSBRT7) stimulation. Furthermore, IL-6 levels following BRD509(pSBRT7) stimulation of WT DC were higher than those induced by BRD509 stimulation (Fig. 2A). SBR induction of IL-12p40 and IL-10 by WT-, TLR2 KO-, and MyD88 KO-derived cells was barely detectable, and although that of TNF-α was in- creased, it did not reach the levels induced by BRD509 or BRD509(pSBRT7) stimulation. Yet, the level of IL-6 induced by SBR was higher than that elicited by BRD509 and BRD509(pSBRT7) stimulation of WT- and TLR2 KO- but not MyD88 KO-derived cells. Thus, these observations suggest that the decreased IL-6 levels seen with MyD88 KO cells following BRD509(pSBRT7) stimulation were related to the SBR-encoding plasmid.
Cytokine production by DC. DC derived from WT and TLR2, TLR4, and MyD88 KO mice were incubated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), SBR (40 μg/ml), LPS (100 ng/ml), or Pam3CSK4 (300 ng/ml) for 24 h. Culture supernatants were collected and assayed for IL-12p40, IL-10, TNF-α, and IL-6 by ELISA. Results are expressed as the means ± standard deviations (SD) of triplicate cultures from one of three independent experiments. (A) Cytokine production by WT DC. *, P < 0.05; **, P < 0.01. (B) Comparison of cytokine production by WT and TLR2, TLR4, and MyD88 KO DC. *, P < 0.05; †, P < 0.01 (compared with WT DC).
BRD509(pSBRT7) induces significantly higher IL-6 production than BRD509 after prolonged stimulation.Although BRD509(pSBRT7) induced higher IL-6 production than BRD509 in WT DC after 24 h of stimulation, the difference was not statistically significant. Therefore, we questioned whether a longer stimulation would affect IL-6 levels. Indeed, when WT DC were incubated with BRD509(pSBRT7) or BRD509 for 48 h and supernatants were assessed for cytokine levels, we still observed much lower levels of IL-12p40 and IL-10 in BRD509(pSBRT7) cultures than in BRD509 cultures and comparable levels of TNF-α. However, IL-6 levels induced by BRD509(pSBRT7) were significantly higher than those induced by BRD509 stimulation (Fig. 3).
Cytokine production by BRD509- and BRD509(pSBRT7)-stimulated DC after 48 h of stimulation. DC from WT mice were stimulated with BRD509 (MOI of 0.1) or BRD509(pSBRT7) (MOI of 0.1). Forty-eight hours later, culture supernatants were collected and assayed for IL-12p40, IL-10, TNF-α, and IL-6 by ELISA. Results are expressed as the means ± SD of triplicate cultures from one of three independent experiments. **, P < 0.01 compared with the BRD509 group.
Decreased p38 phosphorylation by BRD509(pSBRT7) stimulation.MAPKs, including ERK1/2, JNK, and p38, have been shown to play important roles in TLR-induced cytokine production (37). Thus, we next assessed the activation of the MAPK pathway following BRD509(pSBRT7) and BRD509 stimulation by Western analysis. Stimulation of DC derived from WT and TLR2 KO mice with BRD509(pSBRT7) or BRD509 induced phosphorylation of p38 and ERK1/2 but not JNK (Fig. 4). Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by BRD509 stimulation. No difference was seen in ERK1/2 phosphorylation between the two groups. In DC derived from TLR4 and MyD88 KO mice, no obvious phosphorylation of ERK1/2, JNK, or p38 was seen following either BRD509 or BRD509(pSBRT7) stimulation (data not shown), indicating that activation of MAPKs by both BRD509 and BRD509(pSBRT7) was TLR4 and MyD88 dependent.
Stimulation of DC with BRD509(pSBRT7) results in decreased p38 phosphorylation. DC derived from WT and TLR2, TLR4, and MyD88 KO mice were stimulated with BRD509 (MOI of 0.1) or BRD509(pSBRT7) (MOI of 0.1) for 0, 10, 30, 60, and 120 min. Western blot analysis of p38, ERK1/2, and JNK phosphorylation (p-) in whole-cell lysates. Total p38 served as a loading control. Results are from WT (A) and TLR2 KO (B) DC. Data are representative of three independent experiments with similar results.
MAPK p38 plays a role in the induction of cytokine production by SBR and the Salmonella vectors.Next, we determined if the observed differential phosphorylation of p38 was involved in the distinct cytokine profiles induced by BRD509- and BRD509(pSBRT7)-stimulated DC. DC were thus treated with SB203580, a p38 inhibitor, prior to stimulation with BRD509, BRD509(pSBRT7), or SBR. Supernatants were harvested 24 h later and assessed for cytokine production by ELISA. Inhibition of p38 resulted in a downregulation of IL-12p40, IL-10, and IL-6 following BRD509(pSBRT7) or SBR stimulation, whereas DC stimulation with BRD509 resulted in decreased levels of IL-12p40 and IL-10 but not IL-6 (Fig. 5).
Inhibition of p38. DC derived from WT mice were pretreated with or without the selective p38 inhibitor (SB203580, 10 μM) for 1 h and then stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), or SBR (40 μg/ml) for 24 h. The levels of IL-12p40, IL-10, TNF-α, and IL-6 in culture supernatants were determined by ELISA. Results are expressed as the means ± SD of triplicate cultures from one of three independent experiments. *, P < 0.05; **, P < 0.01 (compared to “no inhibitor” group).
BRD509(pSBRT7)-stimulated DC showed increased Jagged-1 expression over that induced by BRD509-stimulated DC.The Notch ligands Delta-1 and Jagged-1 have been shown to selectively induce Th1 or Th2 differentiation (3, 33, 38); thus, we wanted to determine if signaling via these molecules could shed some light on the cytokine pattern. Induction of Jagged-1 expression occurred after 12, 24, and 48 h of stimulation of DC with BRD509(pSBRT7) or BRD509 (Fig. 6A). Yet, stimulation with BRD509(pSBRT7) showed higher levels of Jagged-1 than those observed with BRD509 stimulation at all experimental times. The level of Jagged-1 expression by SBR-stimulated DC peaked at 12 h but declined by 24 and 48 h. Conversely, BRD509(pSBRT7) and BRD509 induced an increase in Jagged-1 expression at all time points tested. The induction of Delta-1 was delayed; however, by 48 h, Delta-1 expression was robust, and levels were comparable among the various experimental conditions (Fig. 6B). The differential regulation of Jagged-1 was not due to loading differences, as can be seen by the β-actin control (Fig. 6C).
Jagged-1 and Delta-1 expression in stimulated DC. DC derived from WT mice were incubated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), or SBR (40 μg/ml) for 12, 24, and 48 h. Cells were lysed, and whole-cell lysates were assessed for Jagged-1 (A) and Delta-1 (B) expression by Western blot analysis; β-actin served as a loading control (C). The number below each band represents the fold increase over baseline, quantitated by densitometry. Results are representative of three independent experiments with similar results.
DISCUSSION
In the present study, we investigated the difference in the immune responses induced by the SBR-expressing Salmonella vaccine BRD509(pSBRT7) and the Salmonella vector BRD509 in murine bone marrow-derived DC cultures. Specifically, we determined the involvement of TLRs in this process. Since upregulation of costimulatory and MHC molecules is an important indicator of DC maturation, we first assessed the induction of CD80, CD86, CD40, and MHC II expression in stimulated DC. Upregulation of CD80, CD86, CD40, and MHC II by both BRD509(pSBRT7) and BRD509 was TLR4 dependent, indicating that SBR expression by the Salmonella vector BRD509 did not influence the upregulation pattern and TLR dependency compared to those of the Salmonella vector. Since TLR4 can utilize the MyD88 and the TRIF pathways to deliver signaling, the finding that MyD88 deficiency did not abolish upregulation of CD80, CD86, CD40, and MHC II molecules suggests that TRIF participated in the modulation of DC maturation. This is in line with previous reports demonstrating that a MyD88-independent, but TRIF-dependent signaling pathway was responsible for the surface expression of MHC II and costimulatory molecules in LPS-activated DC (17, 23). In the present study, we saw a higher expression of MHC II in MyD88 KO DC than in WT DC after BRD509(pSBRT7), BRD509, SBR, LPS, or Pam3CSK4 stimulation, suggesting that MyD88 signaling exerts an inhibitory effect on MHC II expression. Yao et al. (54) also found that MyD88 signaling mediated the inhibitory effect of LPS on MHC II expression on DC and macrophages, by demonstrating that MyD88-dependent activation of MAPKs by LPS stimulation downregulated the CIITA, a master regulator for MHC II expression, thus leading to a decrease in MHC II expression. Therefore, although MHC II expression on DC depends mainly on TRIF signaling, MyD88 signaling can also play a regulatory role in this process through downstream MAPK signaling.
A comparison of the cytokine responses induced by BRD509(pSBRT7) and BRD509 revealed that SBR expression in the Salmonella vector influenced the IL-12p40, IL-10, and IL-6 cytokine responses, whereas no effect on the production of TNF-α was seen. Since the IL-12 produced by DC is a strong promoter of naïve T cell differentiation along the Th1 lineage (21, 32), the result that BRD509(pSBRT7) induced much smaller amounts of IL-12p40 than BRD509 suggests that SBR expression in the Salmonella vector dampens the Th1 response. This is in line with the lack of IL-12p40 following stimulation with SBR purified protein but not after LPS stimulation. Conversely, IL-6 secreted by DC can play a critical role in promoting Th2 and limiting Th1 responses, likely participating in early Th1/Th2 control during CD4+ T cell activation (8, 9, 27). A comparison of BRD509- and BRD509(pSBRT7)-stimulated DC showed lower IL-12p40 and higher IL-6 levels in the latter, and although the IL-6 levels were not statistically significant until 48 h of stimulation, the trend could suggest a Th2-stimulatory capacity. Furthermore, SBR induced only a slight amount of IL-12p40, whereas the amount of IL-6 produced was significantly increased over that seen with BRD509 but not BRD509(pSBRT7) stimulation. Production of IL-12p40, IL-10, and TNF-α was dependent on TLR4 and MyD88 signaling; however, the induction of IL-6 production by BRD509 did not seem to depend on TLR2, TLR4, or MyD88 signaling, suggesting that other TLR or adaptor molecules may play a role in BRD509-induced IL-6 production (10). BRD509(pSBRT7) showed a greater dependency on TLR4 and MyD88 than BRD509 in inducing IL-6 production, probably due to the capacity of BRD509(pSBRT7) to express SBR, a protein stimulating IL-6 production through TLR4 via the MyD88 pathway.
Since MAPKs, including ERK1/2, JNK, and p38, play important roles in TLR-induced cytokine production (37), we compared activation of the MAPKs in BRD509(pSBRT7)- and BRD509-stimulated DC. Neither BRD509(pSBRT7) nor BRD509 induced the phosphorylation of ERK1/2, JNK, or p38 in DC from TLR4 and MyD88 KO mice; however, phosphorylated p38 and ERK1/2 were seen in DC from WT and TLR2 KO mice following stimulation with BRD509(pSBRT7) or BRD509. Moreover, a higher level of phosphorylated p38 was observed in BRD509-stimulated DC than in BRD509(pSBRT7)-stimulated DC from WT and TLR2 KO mice. Activation of p38 MAPK through MKK3 is required for the production of IL-12 by macrophages and DC (31). Inhibition of the p38 pathway blocks IL-12p40 and IL-10 promoter activity, resulting in suppressed IL-12p40 and IL-10 transcription (43). In our study, inhibition of p38 activity downregulated IL-12p40 and IL-10 production in DC stimulated with BRD509 and BRD509(pSBRT7), suggesting the involvement of p38 in IL-12p40 and IL-10 production. Thus, it is possible that the decreased p38 phosphorylation observed in BRD509(pSBRT7)-stimulated DC is responsible for the reduced IL-12p40 and IL-10 levels compared to those obtained following BRD509 stimulation. Activation of p38 contributed to the IL-6 production in BRD509(pSBRT7)- and SBR-stimulated DC but not in BRD509-stimulated DC, suggesting that p38 involvement in the production of IL-6 following DC stimulation with BRD509(pSBRT7) is due to the SBR.
Notch proteins are membrane-bound receptors that regulate diverse cell fate decisions in multicellular organisms. The Notch signaling pathway consists of four receptors, Notch1 to Notch4, and their ligands Delta-1, -3, and -4 and Jagged-1 and -2. The major biological role of Notch signaling is to control the developmental fate of cells and to make cells different from one another (28, 42). Recent studies have suggested that distinct Notch ligands expressed on APC might regulate Th1 and Th2 fate choice. In this regard, it was shown that Delta-1 biased naïve CD4+ T cells toward the Th1 fate (33), whereas Jagged-1 biased toward Th2 (3, 38), suggesting that pathogens drive distinct Th fate choices through the induction of alternative Notch ligands on APC. Since BRD509(pSBRT7)-stimulated DC produced less IL-12p40 and more IL-6 than BRD509-stimulated DC, we wondered if there could be a difference in Notch ligand expression between BRD509(pSBRT7)- and BRD509-stimulated DC. Western analysis revealed a notably higher expression of Jagged-1 in BRD509(pSBRT7)-stimulated cells than in BRD509-stimulated cells and similar expression levels of Delta-1. This result, along the lines of the cytokine data, suggests an increased Th2-stimulatory capacity of BRD509(pSBRT7) over BRD509. The expression of SBR by BRD509(pSBRT7) may be responsible for the increased Jagged-1 expression in BRD509(pSBRT7)-stimulated DC since SBR was also shown to upregulate Jagged-1 expression. Although Jagged-1 expression induced by SBR was not sustained, expression of SBR by the plasmid in BRD509(pSBRT7) likely contributed to the expression of Jagged-1 over time. In a recent study by Foldi et al. (11), TLR induction of Notch ligand expression in primary macrophages revealed that TLRs can induce Jagged-1 expression rapidly, and conversely, elevated Jagged-1 expression augmented TLR-induced IL-6 production. In our study, although at 24 h BRD509(pSBRT7)-stimulated DC produced a level of IL-6 comparable to that induced by BRD509 stimulation, BRD509(pSBRT7) induced significantly higher IL-6 levels than BRD509 by 48 h. The higher IL-6 production in BRD509(pSBRT7)-stimulated DC may be due to the greater upregulation of Jagged-1 expression than that observed with BRD509-stimulated DC.
The differential responses induced by the two Salmonella vector vaccines can perhaps be explained based on the evidence that in vitro BRD509(pSBRT7) continuously expresses the recombinant SBR (16), which contributes to the activation of DC. Our results extend the findings of others (41) who reported a difference in induction of a proinflammatory cytokine response by macrophages infected with a Salmonella vector vaccine expressing a heterologous recombinant protein from enterotoxigenic Escherichia coli compared to that seen with the isogenic Salmonella vector. They proposed that the presence of a recombinant protein may alter the way in which the Salmonella vaccine vector is recognized and processed by the innate immune system. In our present study, we showed that Salmonella-expressed recombinant protein can regulate the innate immune response through MAPKs and Notch signaling. It is important to keep in mind that since the target inductive site for an oral Salmonella vector vaccine is the gut, differences may exist between gut DC and bone marrow-derived DC used in this study, such as the number of expressed TLRs. Nevertheless, methods of processing by the various DC are basically similar.
In summary, SBR expression by the Salmonella vector BRD509 differentially regulates the immune response of DC via p38 and Jagged-1 regulation. These findings provide new insight on how a heterologous antigen expressed by a Salmonella vector vaccine influences DC-mediated innate immunity, which should be helpful for the development of improved Salmonella vector vaccines. However, since Salmonella vector vaccines often differ based on the way in which the Salmonella is rendered avirulent and the nature of the expressed heterologous protein, it is of importance to determine how different characteristics of a Salmonella vector vaccine can alter the nature of the host response.
ACKNOWLEDGMENTS
We thank Gregory Harber for his technical assistance.
This work was supported by grant DE09081 from the National Institutes of Health.
FOOTNOTES
- Received 5 May 2011.
- Returned for modification 26 May 2011.
- Accepted 28 June 2011.
- Accepted manuscript posted online 11 July 2011.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.
