Downregulation of Mitogen-Activated Protein Kinases by the Bordetella bronchiseptica Type III Secretion System Leads to Attenuated Nonclassi
http://www.100md.com
感染与免疫杂志 2005年第1期
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-B in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.
INTRODUCTION
Bordetellae are small, aerobic, gram-negative coccobacilli associated with respiratory infections in mammals. Bordetella pertussis and Bordetella parapertussis are human pathogens (34), whereas Bordetella bronchiseptica has a wide host range and may represent their evolutionary progenitor (42). B. bronchiseptica infections are frequently chronic or even asymptomatic (6). Therefore, it serves as a good model to study mechanisms employed by pathogens to downregulate host immune responses. The virulence and colonization factors expressed by B. bronchiseptica include filamentous hemagglutinin (8), fimbriae (29), adenylate cyclase toxin (CyaA) (17), dermonecrotic toxin (44), and a type III secretion system (TTSS) (46). Type III secretion systems allow gram-negative bacteria to modulate the host response by translocating effector molecules into the plasma membrane or cytoplasm of host cells (5, 12, 19).
Host reactions to bacterial infection include a wide spectrum of inflammatory and anti-inflammatory responses. These require the coordinate induction of multiple signaling pathways, including three major mitogen-activated protein kinase (MAPK) pathways, extracellular signal-regulated kinases (ERKs) 1 and 2, p38 proteins (p38 , , , and ), and Jun amino-terminal kinases (JNK) 1 and 2, and also the NF-B pathway. These pathways regulate the expression of genes encoding cytokines, adhesion molecules, and costimulatory molecules that coordinate various aspects of immune functions (40). For example, interleukin- (IL-)12 production is regulated by the MAPK kinase kinase kinase (MKK3)-p38 pathway (9, 28), whereas the specific kinetics of activation of the ERK pathway lead to either macrophage activation or proliferation (41). Thus, these signal transduction pathways are critical in determining the activation state of macrophages and dendritic cells, i.e., classically versus alternative and type II-activated macrophages (30) and semimature versus fully mature dendritic cells (26). It is therefore of significant interest to analyze these signal transduction pathways in dendritic cells and macrophages that interact with respiratory pathogens in the initial stages of infection.
In Yersinia, Salmonella, Shigella, and Pseudomonas spp., type III-secreted factors are known to interact with the cytoskeleton and various intracellular signaling cascades (including MAPK pathways) of target cells (20, 21, 22, 24, 31, 38, 48). Depending on the bacterial species, the target cells can respond in different, sometimes opposite, ways. Yop proteins encoded by the Yersinia TTSS are translocated into a wide range of cell types, and the action of these Yop effectors is not cell type specific (2). The Yop effectors are postulated to contribute to the suppression of inflammation, phagocytosis, and host immune responses (4). On the other hand, type III-secreted factors from Salmonella and Shigella promote host inflammatory responses and uptake by macrophages (35, 38).
In B. bronchiseptica, the TTSS plays a role in allowing persistent colonization of the host. Cytological studies showed that type III-secreted effectors mediated cytotoxicity in several cell types, the aberrant aggregation of the transcription factor NF-B in epithelial cells, and differentiation of dendritic cells into a semimature state (39, 46, 47). In vivo infection studies revealed that a functional type III secretion system is required for long-term persistent colonization in the trachea and for the downregulated production of anti-Bordetella immunoglobulins (47).
In this study, we investigated the role of the Bordetella type III secretion system in the modulation of host MAPK signal transduction pathways and cytokine expression with in vitro cell culture models. The activation of ERK-1/2, p38 proteins, JNK1/2, and the expression of cytokines in primary cell cultures of bone marrow-derived dendritic cells (BMDC) and bone marrow-derived macrophages (BMM) as well as a macrophage-like cell line (RAW 264.7) in response to B. bronchiseptica infection was analyzed by intracellular staining followed by flow cytometry, immunoblotting, and real-time reverse transcription-PCR analysis. The observed differences are discussed in the context of the possible role of specific cytokines in pathogen clearance.
MATERIALS AND METHODS
Cell cultures, media, and bacterial strains. RAW 264.7 murine macrophage-like cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco). BMM and BMDC were generated from bone marrow isolated from the femurs of C57BL/6 mice as previously described (25). Briefly, cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine and 50 μM 2-mercaptoethanol with 20 ng of macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) per ml for BMM and BMDC, respectively. All media were supplemented with 10% heat-inactivated fetal calf serum (HyClone), penicillin at 100 IU/ml, and streptomycin at 100 μg/ml. At day 9 BMM were trypsinized and transferred into new medium containing 20 ng of GM-CSF per ml (13, 37) and incubated for another 24 h. For MAPK analysis, BMM and 10-day-old BMDC were serum and growth factor deprived in RPMI for 1.5 h prior to infection to reduce basal levels of phosphorylated MAPK (32). Serum deprivation of RAW 264.7 cells was performed for 16 h. For all other assays, cells were transferred to new medium containing 5 ng of GM-CSF per ml and serum.
The Bordetella bronchiseptica type III secretion-defective mutant containing an in-frame deletion in the bscN gene (which is proposed to encode an ATPase required for the secretion process) and the CyaA-defective mutant were described previously (17, 46). The wild-type and mutant bacteria were cultured and used for infection of cultured cells as previously described (6, 7, 27, 45). Infections were performed at a multiplicity of infection of 10 except for RAW 264.7 cells, for which a multiplicity of infection of 50 was chosen due to the reduced cytotoxic effect mediated by wild-type bacteria. By this means, cell death rates were standardized to approximately 20% dead cells at 45 min postinfection as determined by the vital dye TO-PRO-3 (Molecular Probes) (46). Anisomycin (10 μM) and lipopolysaccharide (LPS, 25 μg/ml, purified from E. coli serotype O127:B8) (Sigma) were used as positive controls for MAPK activation and cytokine induction, respectively.
Preparation of cell lysates. After the indicated incubation times, the cells were washed once on ice with phosphate-buffered saline containing 1 mM Na3VO4. Washed cells were scraped and boiled with 150 μl of 1x Laemmli sample buffer (containing 100 mM dithiothreitol) for analysis of ERK1 and -2, p38, and JNK phosphorylation or lysed for 15 min on ice with 0.5 ml of lysis buffer for ERK1 and -2 and p38 kinase (Cell Signaling). The lysate was sonicated and centrifuged at 20,000 x g for 10 min at 4°C. The protein content of the supernatant was determined with the Bio-Rad DC protein assay.
Immunoprecipitation of phosphorylated ERK1/2 and p38. Samples containing 200 μg of total protein were incubated with immobilized phosphospecific monoclonal antibodies for ERK1/2 and p38 (Cell Signaling), incubated overnight at 4°C, immunoprecipitated, and subjected to the kinase assay with ELK-1 for ERK1/2 and ATF-2 for p38 as the substrate according to the manufacturer's protocol.
Western blotting. Equal volumes (50 μl) of whole-cell lysates and immunoprecipitates were subjected to electrophoresis on a sodium dodecyl sulfate-10% polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride membranes (Immobilon-F, Millipore) and incubated overnight with phosphospecific polyclonal antibodies to ERK1/2, p38, JNK, ELK-1, and ATF-2 and standard antibodies to ERK1/2, p38, and JNK (all from Cell Signaling except for phosphospecific monoclonal antibody to JNK, which was obtained from Santa Cruz Biotechnology). Membranes were the subsequently processed according to the manufacturer's protocol (Cell Signaling), and the presence of the proteins was revealed with horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibodies (Amersham) and visualized by the enhanced chemiluminescence detection system (Pierce). Results shown are representative of at least three independent experiments.
mRNA preparation and cytokine expression analysis. Total RNAs were extracted 1.5 h postinfection with the RNeasy mini kit (Qiagen). Reverse transcription and real-time PCR were performed as described elsewhere (14) with the following modifications: reverse transcription-PCR was performed with 1 μg of total cell mRNA, gene-specific oligonucleotide primers were used for the reverse transcription reaction, IL-12p40 primers were modified as follows: upstream primer, 5'-CGG CAG CAG AAT AAA TAT GAG AAC-3'; downstream primer, 5'-GAA GTA GGA ATG GGG AGT GCT C-3', PCR amplifications were performed with 1 μl of cDNA sample, 400 nM primers, 400 nM TaqMan probe (VIC as the reporter dye and TAMRA as the quencher), and TaqMan Universal PCR Master Mix (Applied Biosystems). As an external standard, purified specific PCR products of reverse-transcribed RNA from unstimulated BMM were used. Initial differences in the amount of RNA subjected to reverse transcription were corrected by calculating the ratios of mRNA expression of the investigated gene to mRNA expression of the glyceraldehhyde-3-phosphate dehydrogenase housekeeping gene (TaqMan rodent glyceraldehhyde-3-phosphate dehydrogenase control reagents; Applied Biosystems). All real-time reverse transcription-PCR amplifications were performed in triplicate with no template controls for each cytokine investigated. Results represent mean values of three independent experiments.
Intracellular staining of MAPKs and cytokines. For intracellular staining and subsequent fluorescent-activated cell sorting (FACS) analysis of MAPK activation, cells were processed according to a previously described method (10) with Fix and Perm cell permeabilization reagents (Caltag). All phosphospecific antibodies (Cell Signaling) were diluted in Caltag B. Secondary Alexa Fluor 647-conjugated anti-rabbit immunoglobulin antibodies were diluted in phosphate-buffered saline containing 0.5 mM EDTA, 3% fetal bovine serum, 1 mM Na3VO4, 5 μg of leupeptin per ml, and protease inhibitor cocktail tablet (Roche). Prior to intracellular staining, the cells were incubated with unlabeled FcII/III (clone 2.4G2) (BD Pharmingen) antibody to block Fc receptor binding.
BMM and BMDC were identified by surface staining with F4/80 (clone CI:A3-1) (Caltag) antibody and CD11c (clone HL3) (BD Pharmingen) antibody, respectively. To assay intracellular cytokine levels, Golgiplug (BD Pharmingen) was added 2 h postinfection to accumulate cytokines for 3.5 h. Cells were stained as described for MAPKs except that the fixation step was 15 min and the methanol step was omitted. The following antibodies were used: phycoerythrin-conjugated IL-6 (clone MP5-20F3), allophycocyanin-conjugated IL-10 (clone JES5-16E3), allophycocyanin-conjugated IL-12p40/p70 (clone C15.6), fluorescein isothiocyanate-conjugated tumor necrosis factor alpha (TNF-) (clone MP6-XT22) (BD Pharmingen). FACS analysis was performed on a FACSCalibur machine (Becton Dickinson) with Cellquest software and analyzed with FlowJo software (TreeStar). Data are representative of at least three independent experiments of 106 cells per sample, and 50,000 to 100,000 events were collected.
RESULTS
Type III-secreted factors downregulate ERK1/2 and p38 phosphorylation in RAW 264.7 cells after initial type III secretion-independent activation. Type III-secreted factors are necessary for persistent colonization of the murine respiratory tract, and components of both the innate and adaptive immune systems of the host are suggested to be targets of type III-secreted products (47). Thus, we aimed to investigate the influence of these factors on professional antigen-presenting cells with in vitro infection studies with wild-type and type III secretion-defective mutant strains of B. bronchiseptica.
We first analyzed their role in the induction of MAPK signaling pathways due to the importance of these pathways in inflammatory and other immune responses. Figures 1A and C show that only low levels of phosphorylated ERK1/2 and p38 were observed in uninfected RAW 264.7 cells (lane 1). Robust ERK1/2 and some p38 phosphorylation was detected in macrophages at 20 min postinfection with the wild type or type III secretion mutant (Fig. 1A and C, lane 3). At 45 min postinfection, activation of both ERK1/2 and p38 was downregulated in macrophages infected with the wild-type strain (Fig. 1A and C, lane 4), but they remained activated in macrophages infected with the mutant (lane 6).
A control immunoblot probed with standard ERK1/2 and p38 antibodies demonstrated equal loading of ERK1/2 or p38 in each lane (Fig. 1B and D). Due to the cytotoxic effect of the wild-type bacteria on host cells, it is possible that the observed downregulation was due to cell death. Therefore, we additionally analyzed ERK1/2 and p38 phosphorylation in these cells with flow cytometry, which allowed us to focus on live cells (gates in Fig. 2A) according to cell viability assays (data not shown). The flow cytometry analysis (Fig. 2B) was consistent with the data obtained in the immunoblots. Therefore, the downregulation of ERK1/2 and p38 can also be observed in live cells. Analysis of JNK in infected cells did not reveal any activation at 10, 20, or 45 min postinfection (data not shown).
To determine whether the observed downregulation of MAPK phosphorylation also downregulated the corresponding MAPK activity, we used a MAPK pulldown assay and incubated the enzymes with known substrates, i.e., ELK for ERK 1 and 2 and ATF-2 for p38. The phosphorylated products were then detected by Western immunoblot. As shown in Fig. 3, the downregulation of MAPK phosphorylation almost completely abolished MAPK activity in vitro (Fig. 3A and B, lane 3).
Type III-secreted factors and adenylate cyclase toxin (CyaA) contribute to the downregulation of ERK 1 and 2 and p38 phosphorylation in BMM. Since the relevance of MAPK data obtained on an immortalized cell line may be limited, we performed similar experiments to analyze MAPK activation in primary cell cultures of BMM. In addition, previous studies on dendritic cells showed that the non-type III-secreted virulence factor CyaA specifically downregulated p38 MAPK activity (39). We therefore also determined the possible role of CyaA on the MAPK activation profile in BMM.
Figure 4 shows that at 45 min postinfection, ERK1/2 and p38 activation was downregulated in macrophages infected with the wild-type strain (Fig. 4A and C, lane 4), but they remained activated in macrophages infected with either one of the two deletion mutants, bscN or cyaA (lane 6 and 8). However, the robust ERK phosphorylation observed in RAW 264.7 cells 20 min after infection could not be detected in BMM (Fig. 4C, lanes 3, 5, and 7), and this might be due to the higher multiplicity of infection used in RAW 264.7 cells. Again, we additionally analyzed ERK1/2 and p38 phosphorylation in these cells with flow cytometry, which allowed us to focus on live cells. The data were consistent with the data obtained from the immunoblots, showing that the downregulation of ERK 1 and 2 and p38 by wild-type bacteria can also be observed in live cells (Fig. 5). In all cells analyzed, no activation of JNK was found at 20 or 45 min postinfection (data not shown).
Activation of ERK phosphorylation in BMDC by type III-secreted factors is transient. Dendritic cells are very effective inducers of T-cell immunity and T-cell tolerance. Thus, it was of particular interest to study the effect of type III-secreted factors on dendritic cells and compare to macrophages. LPS stimulation of BMDC resulted in strong activation of ERK1/2 and p38 after 45 min (Fig. 6D and H). In contrast, similar periods of incubation with wild-type bacteria or the type III secretion mutant resulted in no activation of ERK1/2 or p38 (Fig. 6B and F). At 20 min postinfection, however, a transient activation of ERK1/2 was found in wild-type-infected BMDC (Fig. 6E). In all cells analyzed, no activation of JNK was found at 20 or 45 min postinfection (data not shown).
Type III-secreted factors downregulate IL-6 and IL-10 production in macrophages but not in BMDC. To investigate whether the observed modulation of MAPKs in BMM and BMDC correlates with changes in cytokine and chemokine expression, we performed real-time reverse transcription-PCR studies on various pro- and anti-inflammatory cytokines and one chemokine. Because ERK and p38 can regulate cytokine production at both the transcriptional and posttranscriptional levels (11, 18), we also measured cytokine production with intracellular staining. In BMM, IL-6 and IL-10 mRNA levels were downregulated in wild-type-infected cells compared to cells infected with the type III secretion-deficient mutant, whereas IL-12p40, TNF-, and the chemokine macrophage chemoattractant protein 1 (MCP-1) were expressed at relatively low levels in both wild-type and type III secretion-deficient mutant strains infected cells (Fig. 7A). A similar cytokine transcription pattern was found in RAW 264.7 cells with TTSS-mediated downregulation of IL-6 and IL-10 and to a lesser extent TNF- mRNA levels (Fig. 7B).
When we examined cytokine production with intracellular staining, the downregulation of IL-6 and IL-10 was also found at the posttranscriptional level in wild-type-infected BMM when we focused on the live cell population (Fig. 8A). Intracellular staining for TNF- revealed the presence of some TNF- in both wild-type- and type III secretion-deficient mutant-infected BMM, whereas the low production of the Th-1-polarizing cytokine IL-12p40/p70 was in accordance to the real-time reverse transcription-PCR data (Fig. 8B). Our results also show that in BMM, CyaA is required for the downregulation of IL-12 and TNF- as well as the upregulation of IL-10 (Fig. 8), an observation that is consistent with another investigation (36). In contrast to the observations made on macrophages where type III-secreted factors mediated downregulation of IL-6 and IL-10, the IL-6 and IL-10 mRNA and protein levels were not altered by type III-secreted factors in BMDC (Fig. 7C and 8A). This suggests a macrophage-specific TTSS-dependent downregulation of IL-6 and IL-10 that appears to attenuate nonclassical macrophage activation.
DISCUSSION
MAPKs are evolutionarily conserved signal transduction pathways that play important roles in the transduction of signals in the innate immune responses of plants, insects, and mammals (3). The present study shows that in different antigen-presenting cells, the MAPK signaling pathways are differentially regulated upon infection by the same bacteria. Whereas specific MAPK signaling molecules are transiently activated in BMDC in a TTSS-dependent manner, MAPKs in BMM are downregulated. Thus, cells that have differentiated into distinct immunoregulatory functions react differently upon stimulation by the same bacterial species. These findings are possibly due to a cell subset-specific recognition of pathogen-associated molecular patterns.
It is likely that the perception of type III-secreted molecules, together with other pathogen-associated molecular patterns like lipopolysaccharide, define the final MAPK signaling profile. Epithelial cells, for example, typically respond poorly to LPS, and this is believed to prevent chronic inflammation of the epithelium that would result from the presence of nonpathogenic gram-negative bacteria in the epithelial flora. Therefore, proinflammatory responses in epithelial cells are expected to be triggered by stimuli other than LPS (43).
A recent study showed that HeLa epithelial cells responded to a specific type III-secreted protein, YopB, of Yersinia pseudotuberculosis, with activation of the ERK1/2 and JNK pathways. This activation was counteracted by multiple other type III-secreted effector proteins (43). Interestingly, a YopB homolog, BopB, was found in B. bronchiseptica (23, 33, 47), suggesting that BopB might be involved in activation of the MAPK signaling pathway in BMDC. However, in the case of B. bronchiseptica infections, p38 stimulation in BMDC was counteracted by another non-type III-secreted virulence factor, CyaA, the adenylate cyclase toxin, which led to downregulation of IL-12 production (39). In the present study, we show that CyaA is required to downregulate IL-12 and TNF- production but promotes IL-10 production in BMM. These observations are also in good accordance with previous findings in J774 macrophages, where CyaA appeared to downregulate IL-12 and TNF- and synergized with LPS to promote IL-10 production (36).
In BMM, the transient type III secretion-dependent activation of ERK found in BMDC was not observed, suggesting a missing perception of the type III-secreted signal in this cell subset. In RAW 264.7 cells, this activation could have been masked by a very rapid response of MAPKs to bacterial stimulation. However, the equal levels of MAPK phosphorylation observed in RAW 264.7 cells infected by both the wild-type and type III secretion-deficient mutant strains within 20 min argue against an early type III secretion-dependent activation. When cells were infected with the type III secretion-deficient mutant, the MAPK pathway was activated in both BMM and RAW 264.7 cells. This suggests that certain pathogen-associated molecular patterns, probably including LPS, trigger MAPK signaling in these cells. The TTSS-dependent downregulation of ERK and p38 in BMM is similar to the observations made in RAW 264.7 cells, where a significant downregulation was also observed. In addition to the downregulation of ERK and p38 mediated by the TTSS, we found that BMM infected by a CyaA deletion mutant also had a similar phenotype. Hence, in macrophages, specific type III-secreted factors may synergize with CyaA to modulate the ERK and p38 phosphorylation cascades.
Searching for downstream targets of the downregulation of MAPK pathways by the B. bronchiseptica TTSS in macrophages, we found that the secretion of IL-6 and IL-10 was impaired. Guo et al. (15) showed that the production of IL-6 in BMM is linked to p38 activation with an SB 203580 (an inhibitor of p38)-resistant kinase. Recent work on CD40 signaling in peritoneal macrophages showed a reciprocal association between p38 and ERK where p38 induced IL-12 production and ERK induced IL-10 production (28). Unexpectedly, in our experiments the activation of p38 in macrophages infected with the type III secretion-deficient mutant did not result in increased production of IL-12. In contrast, the production of IL-12 could be restored in the absence of CyaA. This indicates that CyaA may target additional signal transduction pathways in BMM and possible differences in the regulation of IL-12 expression by various signal transduction pathways in different cell types.
Taken together, the cytokine and chemokine expression profile (large amounts of IL-6 and IL-10, small amounts of IL-12 and TNF-) that was triggered by the type III secretion-deficient mutant is driven by the virulence factor CyaA and possibly other pathogen-associated molecular patterns like LPS. This profile suggests a nonclassical, i.e., alternative or type II, state of macrophage activation (30). Since type II activated macrophages have the ability to preferentially induce Th2 adaptive immune responses (1), the TTSS might attenuate a macrophage response that may otherwise be driven towards a Th2-mediated adaptive immune response. In dendritic cells, transient activation of the ERK pathway did not lead to any changes in cytokine profiles. However, recent work on BMDC indicated that ERK activation was essential in Fas-induced phenotypic maturation of BMDC, leading to caspase 1 activation, IL-1 secretion, upregulation of major histocompatibility complex class II, and costimulatory molecules CD86 and CD40 (16). We have evidence to suggest that the Bordetella TTSS may facilitate the activation of dendritic cells towards tolerogenic functions (39; unpublished results), and we speculate that activation of MAPK pathways may be the underlying mechanism for this activation. In macrophages, our results suggest that both ERK and p38 play important roles in the induction of nonclassical macrophage activation. Therefore, the downregulation of these pathways may be important for pathogen establishment.
We have previously shown that the NF-B signal transduction pathway can be downregulated by the Bordetella TTSS in an epithelial cell line (47). As NF-B is an important component of the proinflammatory cytokine signaling network (40), some of the phenotypes that we have presented might also be due to interactions of type III-secreted factors directly with NF-B. We have not yet been able to demonstrate a direct inhibitory effect of the Bordetella TTSS on the NF-B pathway in macrophages or dendritic cells. This is not surprising because at least some cytokine pathways are still activated to various extents by wild-type bacteria in these cell types, ruling out a mechanism of complete indiscriminant suppression of the NF-B system by the Bordetella TTSS in these cells. However, as there is significant cross talk between the NF-B and MAPK pathways, it is possible that more subtle modulations of the NF-B (direct or indirect) may still be mediated by Bordetella type III-secreted factors.
The differences observed in the responses of dendritic cells and macrophages to B. bronchiseptica infections suggest that the bacterium has evolved to modulate specific host immune response functions in accordance with the specialization of host cell types in the generation of immune responses. While B. bronchiseptica may suppress specific MAPK pathways in macrophages to suppress macrophage activation, it may interact with the same pathways in dendritic cells in other specific ways to modulate their interactions with T cells. The identification of Bordetella type III-secreted effectors and how they interact with specific members of the MAPK pathways in different cell types will allow the elucidation of the mechanisms of cell type-specific modulation of signaling pathways by the Bordetella type III secretion system.
ACKNOWLEDGMENTS
We thank the members of the Yuk laboratory for critical reading of the manuscript and encouraging discussions. We also thank Nigel Fraser and his laboratory for sharing technical expertise in real-time reverse transcription-PCR analysis.
This work was supported by grants from the NIH (AI049346) and Philip Morris Research Management Group.
REFERENCES
1. Anderson, C. F., and D. M. Mosser. 2002. A novel phenotype for activated macrophages: the type II activated macrophage. J. Leukoc. Biol. 72:101-106.
2. Boyd, A. P., N. Grosdent, S. Ttemeyer, C. Geuijen, S. Bleves, M. Iriarte, I. Lambermont, J.-N. Octave, and G. R. Cornelis. 2000. Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins. Eur. J. Cell Biol. 79:659-671.
3. Chang, L., and M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature 410:37-40.
4. Cornelis, G. R. 2002. The Yersinia Ysc-Yop ‘type III’ weaponry. Nat. Rev. Mol. Cell. Biol. 3:742-752.
5. Cornelis, G. R., and F. Van Gijsegem. 2000. Assembly and function of type III secretory systems. Annu. Rev. Microbiol. 54:735-774.
6. Cotter, P. A., and J. F. Miller. 1994. BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model. Infect. Immun. 62:3381-3390.
7. Cotter, P. A., and J. F. Miller. 1997. A mutation in the Bordetella bronchiseptica bvgs gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens. Mol. Microbiol. 24:671-685.
8. Cotter, P. A., M. H. Yuk, S. Mattoo, B. J. Akerley, J. Boschwitz, D. A. Relman, and J. F. Miller. 1998. Filamentous hemagglutinin of Bordetella bronchiseptica is required for efficient establishment of tracheal colonization. Infect. Immun. 66:5921-5929.
9. Dong, C., Davis, R. J., and R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55-72.
10. Fleisher, T. A., S. E. Dorman, J. A. Anderson, M. Vail, M. R. Brown, and S. M. Holland. 1999. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin. Immunol. 90:425-430.
11. Frevel, M. A. E., Bakheet, T., Silva, A. M., Hissong, J. G., Khabar, K. S. A., and B. R. G. Williams. 2003. p38 mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts. Mol. Cell. Biol. 23:425-436.
12. Galán, J. E., and A. Collmer. 1999. Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284:1322-1328.
13. Germann, T., F. Mattner, A. Partenheimer, E. Schmitt, A. B. Reske-Kunz, H.-G. Fischer, and E. Rüde. 1992. Different accessory function of TH1 cells of bone marrow derived macrophages cultured in granulocyte macrophage colony stimulating factor or macrophage colony stimulating factor. Int. Immunol. 4:755-764.
14. Giulietti, A., L. Overbergh, D. Valckx, B. Decallonne, R. Bouillon, and C. Mathieu. 2001. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386-401.
15. Guo, X., Gerl, R. E., and J. W. Schraders. 2003. Defining the involvement of p38 MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase. J. Biol. Chem. 278:22237-22242.
16. Guo, Z., M. Zhang, H. An, W. Chen, S. Liu, J. Guo, Y. Yu, and X. Cao. 2003. Fas ligation induces IL-1-dependent maturation and IL-1-independent survival of dendritic cells: different roles of ERK and NF-B signaling pathways. Blood 102:4441-4447.
17. Harvill, E. T., P. A. Cotter, M. H. Yuk, and J. F. Miller. 1999. Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity. Infect. Immun. 67:1493-1500.
18. Hoffmeyer, A., Grosse-Wilde, A., Flory, E., Neufeld, B., Kunz, M., Rapp, U. R., and S. Ludwig. 1999. Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor gene expression in T lymphocytes. J. Biol. Chem. 274:4319-4327.
19. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.
20. Jendrossek, V., H. Grassme, I. Mueller, F. Lang, and E. Gulbins. 2001. Pseudomonas aeruginosa-induced apoptosis involves mitochondria and stress-activated protein kinases. Infect. Immun. 69:2675-2683.
21. Jia, J., M. Alaoui-El-Azher, M. Chow, T. C. Chambers, H. Baker, and S. Jin. 2003. c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis. Infect. Immun. 71:3361-3370.
22. Juris, S. J., F. Shao, and J. E. Dixon. 2002. Yersinia effectors target mammalian signalling pathways. Cell. Microbiol. 4:201-211.
23. Kuwae, A., M. Ohishi, M. Watanabe, M., Nagai, and A. Abe. 2003. BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells. Cell. Microbiol. 5:973-983.
24. Lin, S. L., T. X. Le, and D. S. Cowen. 2003. SptP, a Salmonella typhimurium type III-secreted protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Ras activation. Cell. Microbiol. 5:267-275.
25. Lutz, M. B., N. Kukutsch, A. L. J. Ogilvie, S. Rner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77-92.
26. Lutz, M. B., and G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity Trends Immunol. 9:445-449.
27. Martinez de Tejada, G., J. F. Miller, and P. A. Cotter. 1996. Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica. Mol. Microbiol. 22:895-908.
28. Mathur, R. K., Awasthi, A., Wadhone, P., Ramanamurthy, B., and B. Saha. 2004. Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. Nat. Med. 10:540-544.
29. Mattoo, S., J. F. Miller, and P. A. Cotter. 2000. Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response. Infect. Immun. 68:2024-2033.
30. Mosser, D. M. 2003. The many faces of macrophage activation. J. Leukoc. Biol. 73:209-212.
31. Murli, S., R. O. Watson, and J. E. Galán. 2001. Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of salmonella with host cells. Cell. Microbiol. 3:795-810.
32. Mynott, T. L., B. Crossett, and S. R. Prathalingam. 2002. Proteolytic inhibition of Salmonella enterica serovar typhimurium-induced activation of the mitogen-activated protein kinases ERK and JNK in cultured human intestinal cells. Infect. Immun. 70:86-95.
33. Nogawa, H., Kuwae, A., Matsuzawa, T., and A. Abe. 2004. The type III secreted protein BopD in Boretella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane. J. Bacteriol. 186:3806-3813.
34. Parton, R. 1999. Review of the biology of Bordetella pertussis. Biologicals 27:71-76.
35. Rosenberger, C. M., and B. B. Finlay. 2003. Phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens. Nat. Rev. Mol. Cell. Biol. 4:385-396.
36. Ross, P. J., Lavelle, E. C., Mills, K. H., and A. P. Boyd. 2004. Adenylate cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells. Infect. Immun. 72:1568-1579.
37. Rutherford, M. S., and L. B. Schook. 1992. Differential immunocompetence of macrophages derived using macrophage or granulocyte-macrophage colony-stimulating factor. J. Leukoc. Biol. 51:69-76.
38. Sansonetti, P. 2001. Phagocytosis of bacterial pathogens: implications in the host response. Semin. Immunol. 13:381-390.
39. Skinner, J. A., Reissinger, A., Shen, H., and M. H. Yuk. 2004. Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state. J. Immunol. 173:1934-1940.
40. Tato, C. M., and C. A. Hunter. 2002. Host-pathogen interactions: subversion and utilization of the NF-B pathway during infection. Infect. Immun. 70:3311-3317.
41. Valledor, A., Comalada, M., Xaust, J., and A. Celada. 2000. The differential time-course of extracellular-regulated kinase activity correlates with the macrophage response toward proliferation or activation. J. Biol. Chem. 10:7403-7409.
42. van der Zee, A., F. Mooi, J. Van Embden, and J. Musser. 1997. Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences. J. Bacteriol. 179:6609-6617.
43. Viboud, G. I., S. Shu Kin So, M. B. Ryndak, and J. B. Bliska. 2003. Proinflammatory signalling stimulated by the type III translocation factor YopB is counteracted by multiple effectors in epithelial cells infected with Yersinia pseudotuberculosis. Mol. Microbiol. 47:1305-1315.
44. Walker, K. E., and A. A. Weiss. 1994. Characterization of the dermonecrotic toxin in members of the genus Bordetella. Infect. Immun. 62:3817-3828.
45. Wells, A. D., Gudmundsdottir, H., and L. A. Turka. 1997. Following the fate of individual T cells throughout activation and clonal expansion. J. Clin. Investig. 100:3173-3183.
46. Yuk, M. H., E. T. Harvill, and J. F. Miller. 1998. The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica. Mol. Microbiol. 28:945-959.
47. Yuk, M. H., E. T. Harvill, P. A. Cotter, and J. F. Miller. 2000. Modulation of host immune responses, induction of apoptosis and inhibition of NF-B activation by the Bordetella type III secretion system. Mol. Microbiol. 35:991-1004.
48. Zhou, D., M. S. Mooseker, and J. E. Galán. 1999. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283:2092-2095.(Annette Reissinger, Jason)
ABSTRACT
Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-B in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.
INTRODUCTION
Bordetellae are small, aerobic, gram-negative coccobacilli associated with respiratory infections in mammals. Bordetella pertussis and Bordetella parapertussis are human pathogens (34), whereas Bordetella bronchiseptica has a wide host range and may represent their evolutionary progenitor (42). B. bronchiseptica infections are frequently chronic or even asymptomatic (6). Therefore, it serves as a good model to study mechanisms employed by pathogens to downregulate host immune responses. The virulence and colonization factors expressed by B. bronchiseptica include filamentous hemagglutinin (8), fimbriae (29), adenylate cyclase toxin (CyaA) (17), dermonecrotic toxin (44), and a type III secretion system (TTSS) (46). Type III secretion systems allow gram-negative bacteria to modulate the host response by translocating effector molecules into the plasma membrane or cytoplasm of host cells (5, 12, 19).
Host reactions to bacterial infection include a wide spectrum of inflammatory and anti-inflammatory responses. These require the coordinate induction of multiple signaling pathways, including three major mitogen-activated protein kinase (MAPK) pathways, extracellular signal-regulated kinases (ERKs) 1 and 2, p38 proteins (p38 , , , and ), and Jun amino-terminal kinases (JNK) 1 and 2, and also the NF-B pathway. These pathways regulate the expression of genes encoding cytokines, adhesion molecules, and costimulatory molecules that coordinate various aspects of immune functions (40). For example, interleukin- (IL-)12 production is regulated by the MAPK kinase kinase kinase (MKK3)-p38 pathway (9, 28), whereas the specific kinetics of activation of the ERK pathway lead to either macrophage activation or proliferation (41). Thus, these signal transduction pathways are critical in determining the activation state of macrophages and dendritic cells, i.e., classically versus alternative and type II-activated macrophages (30) and semimature versus fully mature dendritic cells (26). It is therefore of significant interest to analyze these signal transduction pathways in dendritic cells and macrophages that interact with respiratory pathogens in the initial stages of infection.
In Yersinia, Salmonella, Shigella, and Pseudomonas spp., type III-secreted factors are known to interact with the cytoskeleton and various intracellular signaling cascades (including MAPK pathways) of target cells (20, 21, 22, 24, 31, 38, 48). Depending on the bacterial species, the target cells can respond in different, sometimes opposite, ways. Yop proteins encoded by the Yersinia TTSS are translocated into a wide range of cell types, and the action of these Yop effectors is not cell type specific (2). The Yop effectors are postulated to contribute to the suppression of inflammation, phagocytosis, and host immune responses (4). On the other hand, type III-secreted factors from Salmonella and Shigella promote host inflammatory responses and uptake by macrophages (35, 38).
In B. bronchiseptica, the TTSS plays a role in allowing persistent colonization of the host. Cytological studies showed that type III-secreted effectors mediated cytotoxicity in several cell types, the aberrant aggregation of the transcription factor NF-B in epithelial cells, and differentiation of dendritic cells into a semimature state (39, 46, 47). In vivo infection studies revealed that a functional type III secretion system is required for long-term persistent colonization in the trachea and for the downregulated production of anti-Bordetella immunoglobulins (47).
In this study, we investigated the role of the Bordetella type III secretion system in the modulation of host MAPK signal transduction pathways and cytokine expression with in vitro cell culture models. The activation of ERK-1/2, p38 proteins, JNK1/2, and the expression of cytokines in primary cell cultures of bone marrow-derived dendritic cells (BMDC) and bone marrow-derived macrophages (BMM) as well as a macrophage-like cell line (RAW 264.7) in response to B. bronchiseptica infection was analyzed by intracellular staining followed by flow cytometry, immunoblotting, and real-time reverse transcription-PCR analysis. The observed differences are discussed in the context of the possible role of specific cytokines in pathogen clearance.
MATERIALS AND METHODS
Cell cultures, media, and bacterial strains. RAW 264.7 murine macrophage-like cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco). BMM and BMDC were generated from bone marrow isolated from the femurs of C57BL/6 mice as previously described (25). Briefly, cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine and 50 μM 2-mercaptoethanol with 20 ng of macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) per ml for BMM and BMDC, respectively. All media were supplemented with 10% heat-inactivated fetal calf serum (HyClone), penicillin at 100 IU/ml, and streptomycin at 100 μg/ml. At day 9 BMM were trypsinized and transferred into new medium containing 20 ng of GM-CSF per ml (13, 37) and incubated for another 24 h. For MAPK analysis, BMM and 10-day-old BMDC were serum and growth factor deprived in RPMI for 1.5 h prior to infection to reduce basal levels of phosphorylated MAPK (32). Serum deprivation of RAW 264.7 cells was performed for 16 h. For all other assays, cells were transferred to new medium containing 5 ng of GM-CSF per ml and serum.
The Bordetella bronchiseptica type III secretion-defective mutant containing an in-frame deletion in the bscN gene (which is proposed to encode an ATPase required for the secretion process) and the CyaA-defective mutant were described previously (17, 46). The wild-type and mutant bacteria were cultured and used for infection of cultured cells as previously described (6, 7, 27, 45). Infections were performed at a multiplicity of infection of 10 except for RAW 264.7 cells, for which a multiplicity of infection of 50 was chosen due to the reduced cytotoxic effect mediated by wild-type bacteria. By this means, cell death rates were standardized to approximately 20% dead cells at 45 min postinfection as determined by the vital dye TO-PRO-3 (Molecular Probes) (46). Anisomycin (10 μM) and lipopolysaccharide (LPS, 25 μg/ml, purified from E. coli serotype O127:B8) (Sigma) were used as positive controls for MAPK activation and cytokine induction, respectively.
Preparation of cell lysates. After the indicated incubation times, the cells were washed once on ice with phosphate-buffered saline containing 1 mM Na3VO4. Washed cells were scraped and boiled with 150 μl of 1x Laemmli sample buffer (containing 100 mM dithiothreitol) for analysis of ERK1 and -2, p38, and JNK phosphorylation or lysed for 15 min on ice with 0.5 ml of lysis buffer for ERK1 and -2 and p38 kinase (Cell Signaling). The lysate was sonicated and centrifuged at 20,000 x g for 10 min at 4°C. The protein content of the supernatant was determined with the Bio-Rad DC protein assay.
Immunoprecipitation of phosphorylated ERK1/2 and p38. Samples containing 200 μg of total protein were incubated with immobilized phosphospecific monoclonal antibodies for ERK1/2 and p38 (Cell Signaling), incubated overnight at 4°C, immunoprecipitated, and subjected to the kinase assay with ELK-1 for ERK1/2 and ATF-2 for p38 as the substrate according to the manufacturer's protocol.
Western blotting. Equal volumes (50 μl) of whole-cell lysates and immunoprecipitates were subjected to electrophoresis on a sodium dodecyl sulfate-10% polyacrylamide gel. The proteins were transferred to polyvinylidene difluoride membranes (Immobilon-F, Millipore) and incubated overnight with phosphospecific polyclonal antibodies to ERK1/2, p38, JNK, ELK-1, and ATF-2 and standard antibodies to ERK1/2, p38, and JNK (all from Cell Signaling except for phosphospecific monoclonal antibody to JNK, which was obtained from Santa Cruz Biotechnology). Membranes were the subsequently processed according to the manufacturer's protocol (Cell Signaling), and the presence of the proteins was revealed with horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibodies (Amersham) and visualized by the enhanced chemiluminescence detection system (Pierce). Results shown are representative of at least three independent experiments.
mRNA preparation and cytokine expression analysis. Total RNAs were extracted 1.5 h postinfection with the RNeasy mini kit (Qiagen). Reverse transcription and real-time PCR were performed as described elsewhere (14) with the following modifications: reverse transcription-PCR was performed with 1 μg of total cell mRNA, gene-specific oligonucleotide primers were used for the reverse transcription reaction, IL-12p40 primers were modified as follows: upstream primer, 5'-CGG CAG CAG AAT AAA TAT GAG AAC-3'; downstream primer, 5'-GAA GTA GGA ATG GGG AGT GCT C-3', PCR amplifications were performed with 1 μl of cDNA sample, 400 nM primers, 400 nM TaqMan probe (VIC as the reporter dye and TAMRA as the quencher), and TaqMan Universal PCR Master Mix (Applied Biosystems). As an external standard, purified specific PCR products of reverse-transcribed RNA from unstimulated BMM were used. Initial differences in the amount of RNA subjected to reverse transcription were corrected by calculating the ratios of mRNA expression of the investigated gene to mRNA expression of the glyceraldehhyde-3-phosphate dehydrogenase housekeeping gene (TaqMan rodent glyceraldehhyde-3-phosphate dehydrogenase control reagents; Applied Biosystems). All real-time reverse transcription-PCR amplifications were performed in triplicate with no template controls for each cytokine investigated. Results represent mean values of three independent experiments.
Intracellular staining of MAPKs and cytokines. For intracellular staining and subsequent fluorescent-activated cell sorting (FACS) analysis of MAPK activation, cells were processed according to a previously described method (10) with Fix and Perm cell permeabilization reagents (Caltag). All phosphospecific antibodies (Cell Signaling) were diluted in Caltag B. Secondary Alexa Fluor 647-conjugated anti-rabbit immunoglobulin antibodies were diluted in phosphate-buffered saline containing 0.5 mM EDTA, 3% fetal bovine serum, 1 mM Na3VO4, 5 μg of leupeptin per ml, and protease inhibitor cocktail tablet (Roche). Prior to intracellular staining, the cells were incubated with unlabeled FcII/III (clone 2.4G2) (BD Pharmingen) antibody to block Fc receptor binding.
BMM and BMDC were identified by surface staining with F4/80 (clone CI:A3-1) (Caltag) antibody and CD11c (clone HL3) (BD Pharmingen) antibody, respectively. To assay intracellular cytokine levels, Golgiplug (BD Pharmingen) was added 2 h postinfection to accumulate cytokines for 3.5 h. Cells were stained as described for MAPKs except that the fixation step was 15 min and the methanol step was omitted. The following antibodies were used: phycoerythrin-conjugated IL-6 (clone MP5-20F3), allophycocyanin-conjugated IL-10 (clone JES5-16E3), allophycocyanin-conjugated IL-12p40/p70 (clone C15.6), fluorescein isothiocyanate-conjugated tumor necrosis factor alpha (TNF-) (clone MP6-XT22) (BD Pharmingen). FACS analysis was performed on a FACSCalibur machine (Becton Dickinson) with Cellquest software and analyzed with FlowJo software (TreeStar). Data are representative of at least three independent experiments of 106 cells per sample, and 50,000 to 100,000 events were collected.
RESULTS
Type III-secreted factors downregulate ERK1/2 and p38 phosphorylation in RAW 264.7 cells after initial type III secretion-independent activation. Type III-secreted factors are necessary for persistent colonization of the murine respiratory tract, and components of both the innate and adaptive immune systems of the host are suggested to be targets of type III-secreted products (47). Thus, we aimed to investigate the influence of these factors on professional antigen-presenting cells with in vitro infection studies with wild-type and type III secretion-defective mutant strains of B. bronchiseptica.
We first analyzed their role in the induction of MAPK signaling pathways due to the importance of these pathways in inflammatory and other immune responses. Figures 1A and C show that only low levels of phosphorylated ERK1/2 and p38 were observed in uninfected RAW 264.7 cells (lane 1). Robust ERK1/2 and some p38 phosphorylation was detected in macrophages at 20 min postinfection with the wild type or type III secretion mutant (Fig. 1A and C, lane 3). At 45 min postinfection, activation of both ERK1/2 and p38 was downregulated in macrophages infected with the wild-type strain (Fig. 1A and C, lane 4), but they remained activated in macrophages infected with the mutant (lane 6).
A control immunoblot probed with standard ERK1/2 and p38 antibodies demonstrated equal loading of ERK1/2 or p38 in each lane (Fig. 1B and D). Due to the cytotoxic effect of the wild-type bacteria on host cells, it is possible that the observed downregulation was due to cell death. Therefore, we additionally analyzed ERK1/2 and p38 phosphorylation in these cells with flow cytometry, which allowed us to focus on live cells (gates in Fig. 2A) according to cell viability assays (data not shown). The flow cytometry analysis (Fig. 2B) was consistent with the data obtained in the immunoblots. Therefore, the downregulation of ERK1/2 and p38 can also be observed in live cells. Analysis of JNK in infected cells did not reveal any activation at 10, 20, or 45 min postinfection (data not shown).
To determine whether the observed downregulation of MAPK phosphorylation also downregulated the corresponding MAPK activity, we used a MAPK pulldown assay and incubated the enzymes with known substrates, i.e., ELK for ERK 1 and 2 and ATF-2 for p38. The phosphorylated products were then detected by Western immunoblot. As shown in Fig. 3, the downregulation of MAPK phosphorylation almost completely abolished MAPK activity in vitro (Fig. 3A and B, lane 3).
Type III-secreted factors and adenylate cyclase toxin (CyaA) contribute to the downregulation of ERK 1 and 2 and p38 phosphorylation in BMM. Since the relevance of MAPK data obtained on an immortalized cell line may be limited, we performed similar experiments to analyze MAPK activation in primary cell cultures of BMM. In addition, previous studies on dendritic cells showed that the non-type III-secreted virulence factor CyaA specifically downregulated p38 MAPK activity (39). We therefore also determined the possible role of CyaA on the MAPK activation profile in BMM.
Figure 4 shows that at 45 min postinfection, ERK1/2 and p38 activation was downregulated in macrophages infected with the wild-type strain (Fig. 4A and C, lane 4), but they remained activated in macrophages infected with either one of the two deletion mutants, bscN or cyaA (lane 6 and 8). However, the robust ERK phosphorylation observed in RAW 264.7 cells 20 min after infection could not be detected in BMM (Fig. 4C, lanes 3, 5, and 7), and this might be due to the higher multiplicity of infection used in RAW 264.7 cells. Again, we additionally analyzed ERK1/2 and p38 phosphorylation in these cells with flow cytometry, which allowed us to focus on live cells. The data were consistent with the data obtained from the immunoblots, showing that the downregulation of ERK 1 and 2 and p38 by wild-type bacteria can also be observed in live cells (Fig. 5). In all cells analyzed, no activation of JNK was found at 20 or 45 min postinfection (data not shown).
Activation of ERK phosphorylation in BMDC by type III-secreted factors is transient. Dendritic cells are very effective inducers of T-cell immunity and T-cell tolerance. Thus, it was of particular interest to study the effect of type III-secreted factors on dendritic cells and compare to macrophages. LPS stimulation of BMDC resulted in strong activation of ERK1/2 and p38 after 45 min (Fig. 6D and H). In contrast, similar periods of incubation with wild-type bacteria or the type III secretion mutant resulted in no activation of ERK1/2 or p38 (Fig. 6B and F). At 20 min postinfection, however, a transient activation of ERK1/2 was found in wild-type-infected BMDC (Fig. 6E). In all cells analyzed, no activation of JNK was found at 20 or 45 min postinfection (data not shown).
Type III-secreted factors downregulate IL-6 and IL-10 production in macrophages but not in BMDC. To investigate whether the observed modulation of MAPKs in BMM and BMDC correlates with changes in cytokine and chemokine expression, we performed real-time reverse transcription-PCR studies on various pro- and anti-inflammatory cytokines and one chemokine. Because ERK and p38 can regulate cytokine production at both the transcriptional and posttranscriptional levels (11, 18), we also measured cytokine production with intracellular staining. In BMM, IL-6 and IL-10 mRNA levels were downregulated in wild-type-infected cells compared to cells infected with the type III secretion-deficient mutant, whereas IL-12p40, TNF-, and the chemokine macrophage chemoattractant protein 1 (MCP-1) were expressed at relatively low levels in both wild-type and type III secretion-deficient mutant strains infected cells (Fig. 7A). A similar cytokine transcription pattern was found in RAW 264.7 cells with TTSS-mediated downregulation of IL-6 and IL-10 and to a lesser extent TNF- mRNA levels (Fig. 7B).
When we examined cytokine production with intracellular staining, the downregulation of IL-6 and IL-10 was also found at the posttranscriptional level in wild-type-infected BMM when we focused on the live cell population (Fig. 8A). Intracellular staining for TNF- revealed the presence of some TNF- in both wild-type- and type III secretion-deficient mutant-infected BMM, whereas the low production of the Th-1-polarizing cytokine IL-12p40/p70 was in accordance to the real-time reverse transcription-PCR data (Fig. 8B). Our results also show that in BMM, CyaA is required for the downregulation of IL-12 and TNF- as well as the upregulation of IL-10 (Fig. 8), an observation that is consistent with another investigation (36). In contrast to the observations made on macrophages where type III-secreted factors mediated downregulation of IL-6 and IL-10, the IL-6 and IL-10 mRNA and protein levels were not altered by type III-secreted factors in BMDC (Fig. 7C and 8A). This suggests a macrophage-specific TTSS-dependent downregulation of IL-6 and IL-10 that appears to attenuate nonclassical macrophage activation.
DISCUSSION
MAPKs are evolutionarily conserved signal transduction pathways that play important roles in the transduction of signals in the innate immune responses of plants, insects, and mammals (3). The present study shows that in different antigen-presenting cells, the MAPK signaling pathways are differentially regulated upon infection by the same bacteria. Whereas specific MAPK signaling molecules are transiently activated in BMDC in a TTSS-dependent manner, MAPKs in BMM are downregulated. Thus, cells that have differentiated into distinct immunoregulatory functions react differently upon stimulation by the same bacterial species. These findings are possibly due to a cell subset-specific recognition of pathogen-associated molecular patterns.
It is likely that the perception of type III-secreted molecules, together with other pathogen-associated molecular patterns like lipopolysaccharide, define the final MAPK signaling profile. Epithelial cells, for example, typically respond poorly to LPS, and this is believed to prevent chronic inflammation of the epithelium that would result from the presence of nonpathogenic gram-negative bacteria in the epithelial flora. Therefore, proinflammatory responses in epithelial cells are expected to be triggered by stimuli other than LPS (43).
A recent study showed that HeLa epithelial cells responded to a specific type III-secreted protein, YopB, of Yersinia pseudotuberculosis, with activation of the ERK1/2 and JNK pathways. This activation was counteracted by multiple other type III-secreted effector proteins (43). Interestingly, a YopB homolog, BopB, was found in B. bronchiseptica (23, 33, 47), suggesting that BopB might be involved in activation of the MAPK signaling pathway in BMDC. However, in the case of B. bronchiseptica infections, p38 stimulation in BMDC was counteracted by another non-type III-secreted virulence factor, CyaA, the adenylate cyclase toxin, which led to downregulation of IL-12 production (39). In the present study, we show that CyaA is required to downregulate IL-12 and TNF- production but promotes IL-10 production in BMM. These observations are also in good accordance with previous findings in J774 macrophages, where CyaA appeared to downregulate IL-12 and TNF- and synergized with LPS to promote IL-10 production (36).
In BMM, the transient type III secretion-dependent activation of ERK found in BMDC was not observed, suggesting a missing perception of the type III-secreted signal in this cell subset. In RAW 264.7 cells, this activation could have been masked by a very rapid response of MAPKs to bacterial stimulation. However, the equal levels of MAPK phosphorylation observed in RAW 264.7 cells infected by both the wild-type and type III secretion-deficient mutant strains within 20 min argue against an early type III secretion-dependent activation. When cells were infected with the type III secretion-deficient mutant, the MAPK pathway was activated in both BMM and RAW 264.7 cells. This suggests that certain pathogen-associated molecular patterns, probably including LPS, trigger MAPK signaling in these cells. The TTSS-dependent downregulation of ERK and p38 in BMM is similar to the observations made in RAW 264.7 cells, where a significant downregulation was also observed. In addition to the downregulation of ERK and p38 mediated by the TTSS, we found that BMM infected by a CyaA deletion mutant also had a similar phenotype. Hence, in macrophages, specific type III-secreted factors may synergize with CyaA to modulate the ERK and p38 phosphorylation cascades.
Searching for downstream targets of the downregulation of MAPK pathways by the B. bronchiseptica TTSS in macrophages, we found that the secretion of IL-6 and IL-10 was impaired. Guo et al. (15) showed that the production of IL-6 in BMM is linked to p38 activation with an SB 203580 (an inhibitor of p38)-resistant kinase. Recent work on CD40 signaling in peritoneal macrophages showed a reciprocal association between p38 and ERK where p38 induced IL-12 production and ERK induced IL-10 production (28). Unexpectedly, in our experiments the activation of p38 in macrophages infected with the type III secretion-deficient mutant did not result in increased production of IL-12. In contrast, the production of IL-12 could be restored in the absence of CyaA. This indicates that CyaA may target additional signal transduction pathways in BMM and possible differences in the regulation of IL-12 expression by various signal transduction pathways in different cell types.
Taken together, the cytokine and chemokine expression profile (large amounts of IL-6 and IL-10, small amounts of IL-12 and TNF-) that was triggered by the type III secretion-deficient mutant is driven by the virulence factor CyaA and possibly other pathogen-associated molecular patterns like LPS. This profile suggests a nonclassical, i.e., alternative or type II, state of macrophage activation (30). Since type II activated macrophages have the ability to preferentially induce Th2 adaptive immune responses (1), the TTSS might attenuate a macrophage response that may otherwise be driven towards a Th2-mediated adaptive immune response. In dendritic cells, transient activation of the ERK pathway did not lead to any changes in cytokine profiles. However, recent work on BMDC indicated that ERK activation was essential in Fas-induced phenotypic maturation of BMDC, leading to caspase 1 activation, IL-1 secretion, upregulation of major histocompatibility complex class II, and costimulatory molecules CD86 and CD40 (16). We have evidence to suggest that the Bordetella TTSS may facilitate the activation of dendritic cells towards tolerogenic functions (39; unpublished results), and we speculate that activation of MAPK pathways may be the underlying mechanism for this activation. In macrophages, our results suggest that both ERK and p38 play important roles in the induction of nonclassical macrophage activation. Therefore, the downregulation of these pathways may be important for pathogen establishment.
We have previously shown that the NF-B signal transduction pathway can be downregulated by the Bordetella TTSS in an epithelial cell line (47). As NF-B is an important component of the proinflammatory cytokine signaling network (40), some of the phenotypes that we have presented might also be due to interactions of type III-secreted factors directly with NF-B. We have not yet been able to demonstrate a direct inhibitory effect of the Bordetella TTSS on the NF-B pathway in macrophages or dendritic cells. This is not surprising because at least some cytokine pathways are still activated to various extents by wild-type bacteria in these cell types, ruling out a mechanism of complete indiscriminant suppression of the NF-B system by the Bordetella TTSS in these cells. However, as there is significant cross talk between the NF-B and MAPK pathways, it is possible that more subtle modulations of the NF-B (direct or indirect) may still be mediated by Bordetella type III-secreted factors.
The differences observed in the responses of dendritic cells and macrophages to B. bronchiseptica infections suggest that the bacterium has evolved to modulate specific host immune response functions in accordance with the specialization of host cell types in the generation of immune responses. While B. bronchiseptica may suppress specific MAPK pathways in macrophages to suppress macrophage activation, it may interact with the same pathways in dendritic cells in other specific ways to modulate their interactions with T cells. The identification of Bordetella type III-secreted effectors and how they interact with specific members of the MAPK pathways in different cell types will allow the elucidation of the mechanisms of cell type-specific modulation of signaling pathways by the Bordetella type III secretion system.
ACKNOWLEDGMENTS
We thank the members of the Yuk laboratory for critical reading of the manuscript and encouraging discussions. We also thank Nigel Fraser and his laboratory for sharing technical expertise in real-time reverse transcription-PCR analysis.
This work was supported by grants from the NIH (AI049346) and Philip Morris Research Management Group.
REFERENCES
1. Anderson, C. F., and D. M. Mosser. 2002. A novel phenotype for activated macrophages: the type II activated macrophage. J. Leukoc. Biol. 72:101-106.
2. Boyd, A. P., N. Grosdent, S. Ttemeyer, C. Geuijen, S. Bleves, M. Iriarte, I. Lambermont, J.-N. Octave, and G. R. Cornelis. 2000. Yersinia enterocolitica can deliver Yop proteins into a wide range of cell types: development of a delivery system for heterologous proteins. Eur. J. Cell Biol. 79:659-671.
3. Chang, L., and M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature 410:37-40.
4. Cornelis, G. R. 2002. The Yersinia Ysc-Yop ‘type III’ weaponry. Nat. Rev. Mol. Cell. Biol. 3:742-752.
5. Cornelis, G. R., and F. Van Gijsegem. 2000. Assembly and function of type III secretory systems. Annu. Rev. Microbiol. 54:735-774.
6. Cotter, P. A., and J. F. Miller. 1994. BvgAS-mediated signal transduction: analysis of phase-locked regulatory mutants of Bordetella bronchiseptica in a rabbit model. Infect. Immun. 62:3381-3390.
7. Cotter, P. A., and J. F. Miller. 1997. A mutation in the Bordetella bronchiseptica bvgs gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens. Mol. Microbiol. 24:671-685.
8. Cotter, P. A., M. H. Yuk, S. Mattoo, B. J. Akerley, J. Boschwitz, D. A. Relman, and J. F. Miller. 1998. Filamentous hemagglutinin of Bordetella bronchiseptica is required for efficient establishment of tracheal colonization. Infect. Immun. 66:5921-5929.
9. Dong, C., Davis, R. J., and R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55-72.
10. Fleisher, T. A., S. E. Dorman, J. A. Anderson, M. Vail, M. R. Brown, and S. M. Holland. 1999. Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin. Immunol. 90:425-430.
11. Frevel, M. A. E., Bakheet, T., Silva, A. M., Hissong, J. G., Khabar, K. S. A., and B. R. G. Williams. 2003. p38 mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-rich element-containing transcripts. Mol. Cell. Biol. 23:425-436.
12. Galán, J. E., and A. Collmer. 1999. Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284:1322-1328.
13. Germann, T., F. Mattner, A. Partenheimer, E. Schmitt, A. B. Reske-Kunz, H.-G. Fischer, and E. Rüde. 1992. Different accessory function of TH1 cells of bone marrow derived macrophages cultured in granulocyte macrophage colony stimulating factor or macrophage colony stimulating factor. Int. Immunol. 4:755-764.
14. Giulietti, A., L. Overbergh, D. Valckx, B. Decallonne, R. Bouillon, and C. Mathieu. 2001. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386-401.
15. Guo, X., Gerl, R. E., and J. W. Schraders. 2003. Defining the involvement of p38 MAPK in the production of anti- and proinflammatory cytokines using an SB 203580-resistant form of the kinase. J. Biol. Chem. 278:22237-22242.
16. Guo, Z., M. Zhang, H. An, W. Chen, S. Liu, J. Guo, Y. Yu, and X. Cao. 2003. Fas ligation induces IL-1-dependent maturation and IL-1-independent survival of dendritic cells: different roles of ERK and NF-B signaling pathways. Blood 102:4441-4447.
17. Harvill, E. T., P. A. Cotter, M. H. Yuk, and J. F. Miller. 1999. Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity. Infect. Immun. 67:1493-1500.
18. Hoffmeyer, A., Grosse-Wilde, A., Flory, E., Neufeld, B., Kunz, M., Rapp, U. R., and S. Ludwig. 1999. Different mitogen-activated protein kinase signaling pathways cooperate to regulate tumor necrosis factor gene expression in T lymphocytes. J. Biol. Chem. 274:4319-4327.
19. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.
20. Jendrossek, V., H. Grassme, I. Mueller, F. Lang, and E. Gulbins. 2001. Pseudomonas aeruginosa-induced apoptosis involves mitochondria and stress-activated protein kinases. Infect. Immun. 69:2675-2683.
21. Jia, J., M. Alaoui-El-Azher, M. Chow, T. C. Chambers, H. Baker, and S. Jin. 2003. c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-induced apoptosis. Infect. Immun. 71:3361-3370.
22. Juris, S. J., F. Shao, and J. E. Dixon. 2002. Yersinia effectors target mammalian signalling pathways. Cell. Microbiol. 4:201-211.
23. Kuwae, A., M. Ohishi, M. Watanabe, M., Nagai, and A. Abe. 2003. BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells. Cell. Microbiol. 5:973-983.
24. Lin, S. L., T. X. Le, and D. S. Cowen. 2003. SptP, a Salmonella typhimurium type III-secreted protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Ras activation. Cell. Microbiol. 5:267-275.
25. Lutz, M. B., N. Kukutsch, A. L. J. Ogilvie, S. Rner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77-92.
26. Lutz, M. B., and G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity Trends Immunol. 9:445-449.
27. Martinez de Tejada, G., J. F. Miller, and P. A. Cotter. 1996. Comparative analysis of the virulence control systems of Bordetella pertussis and Bordetella bronchiseptica. Mol. Microbiol. 22:895-908.
28. Mathur, R. K., Awasthi, A., Wadhone, P., Ramanamurthy, B., and B. Saha. 2004. Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. Nat. Med. 10:540-544.
29. Mattoo, S., J. F. Miller, and P. A. Cotter. 2000. Role of Bordetella bronchiseptica fimbriae in tracheal colonization and development of a humoral immune response. Infect. Immun. 68:2024-2033.
30. Mosser, D. M. 2003. The many faces of macrophage activation. J. Leukoc. Biol. 73:209-212.
31. Murli, S., R. O. Watson, and J. E. Galán. 2001. Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of salmonella with host cells. Cell. Microbiol. 3:795-810.
32. Mynott, T. L., B. Crossett, and S. R. Prathalingam. 2002. Proteolytic inhibition of Salmonella enterica serovar typhimurium-induced activation of the mitogen-activated protein kinases ERK and JNK in cultured human intestinal cells. Infect. Immun. 70:86-95.
33. Nogawa, H., Kuwae, A., Matsuzawa, T., and A. Abe. 2004. The type III secreted protein BopD in Boretella bronchiseptica is complexed with BopB for pore formation on the host plasma membrane. J. Bacteriol. 186:3806-3813.
34. Parton, R. 1999. Review of the biology of Bordetella pertussis. Biologicals 27:71-76.
35. Rosenberger, C. M., and B. B. Finlay. 2003. Phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens. Nat. Rev. Mol. Cell. Biol. 4:385-396.
36. Ross, P. J., Lavelle, E. C., Mills, K. H., and A. P. Boyd. 2004. Adenylate cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells. Infect. Immun. 72:1568-1579.
37. Rutherford, M. S., and L. B. Schook. 1992. Differential immunocompetence of macrophages derived using macrophage or granulocyte-macrophage colony-stimulating factor. J. Leukoc. Biol. 51:69-76.
38. Sansonetti, P. 2001. Phagocytosis of bacterial pathogens: implications in the host response. Semin. Immunol. 13:381-390.
39. Skinner, J. A., Reissinger, A., Shen, H., and M. H. Yuk. 2004. Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state. J. Immunol. 173:1934-1940.
40. Tato, C. M., and C. A. Hunter. 2002. Host-pathogen interactions: subversion and utilization of the NF-B pathway during infection. Infect. Immun. 70:3311-3317.
41. Valledor, A., Comalada, M., Xaust, J., and A. Celada. 2000. The differential time-course of extracellular-regulated kinase activity correlates with the macrophage response toward proliferation or activation. J. Biol. Chem. 10:7403-7409.
42. van der Zee, A., F. Mooi, J. Van Embden, and J. Musser. 1997. Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences. J. Bacteriol. 179:6609-6617.
43. Viboud, G. I., S. Shu Kin So, M. B. Ryndak, and J. B. Bliska. 2003. Proinflammatory signalling stimulated by the type III translocation factor YopB is counteracted by multiple effectors in epithelial cells infected with Yersinia pseudotuberculosis. Mol. Microbiol. 47:1305-1315.
44. Walker, K. E., and A. A. Weiss. 1994. Characterization of the dermonecrotic toxin in members of the genus Bordetella. Infect. Immun. 62:3817-3828.
45. Wells, A. D., Gudmundsdottir, H., and L. A. Turka. 1997. Following the fate of individual T cells throughout activation and clonal expansion. J. Clin. Investig. 100:3173-3183.
46. Yuk, M. H., E. T. Harvill, and J. F. Miller. 1998. The BvgAS virulence control system regulates type III secretion in Bordetella bronchiseptica. Mol. Microbiol. 28:945-959.
47. Yuk, M. H., E. T. Harvill, P. A. Cotter, and J. F. Miller. 2000. Modulation of host immune responses, induction of apoptosis and inhibition of NF-B activation by the Bordetella type III secretion system. Mol. Microbiol. 35:991-1004.
48. Zhou, D., M. S. Mooseker, and J. E. Galán. 1999. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283:2092-2095.(Annette Reissinger, Jason)