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* Vaccine Research Center and
Department of Pathology, Emory University, Atlanta, GA 30329;
Department of Periodontology and Oral Biology, Boston University School of Medicine, Boston, MA 02118;
Alzheimer Research Laboratory, Departments of Neurology and Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106;
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School of Dentistry, University of Michigan, Ann Arbor, MI 48109;
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Amgen, Seattle, WA 98101; and
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Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; in contrast, helminths induce the differentiation of Th2 cells, whose cytokines (principally IL-4, IL-5, and IL-10) induce IgE and eosinophil-mediated destruction of the pathogens (1, 2, 3, 4). At the individual T cell level, considerable heterogeneity of cytokine profiles can be seen with T cell clones, raising the possibility that the canonical Th1 and Th2 global phenotypes only represent two polar extremes of all possible single cell phenotypes (5).
Although there is much knowledge about the cytokines produced early in the response, and the transcription factors that determine Th polarization in T cells (3, 4), the early "decision-making mechanisms" which result in a given type of immune response are poorly understood. Several recent observations bear on this problem. First, distinct types of dendritic cell (DC)3 subsets can differentially induce Th1 and Th2 responses (6, 7, 8). For example, in mice, CD8
+ DCs can elicit Th1 cells, while CD8− DCs can induce Th2/Th0 cells (6, 7). Second, the dose of Ag can play an important role in influencing the Th1/Th2 balance (9). Third, different microbial stimuli signal through distinct pattern recognition receptors on APCs (10, 11, 12). For example, LPS from Escherichia coli signals through Toll-like receptor (TLR)-4 (13), while TLR-2 appears to have a broad spectrum of ligands, including peptidoglycan of Staphylococcus aureus (14), lipoproteins from Mycobacterium tuberculosis (15, 16), and Sacharomyces cerevisiae zymosan (17). Fourth, different microbial stimuli differentially activate DCs to elicit distinct classes of immune responses (18, 19, 20, 21, 22, 23). For example, Toxoplasma extracts (24) and E. coli LPS stimulate IL-12(p70) production in CD8+ DCs and prime a Th1 response (25), and certain viruses induce IFN- from plasmacytoid DCs and stimulate Th1 responses (26, 27). In contrast, schistosome egg Ags (28), filarial Ags (29), cholera toxin (30), lipoxins stimulated by Toxoplasma (31), certain forms of Candida albicans (32), or highly purified preparations of Porphyromonas gingivalis LPS (25), do not stimulate IL-12(p70), and favor Th2-like responses. Interestingly, a recent report suggests that P. gingivalis LPS signals via TLR-2 in murine macrophages (33). In this context, it is unclear whether signaling through distinct TLRs can shift the balance toward the Th2 phenotype. Although, our recent work with P. gingivalis LPS suggests that signaling through TLR-2 can stimulate Th2-like responses (25), it is not clear whether this is characteristic of all TLR-2 ligands, or whether this is a unique feature of P. gingivalis LPS. If indeed, signaling via different TLRs instructs DCs to elicit distinct Th responses, then the intracellular signaling pathways, which mediate such different outcomes, are not known. In this study, we address these questions, using a synthetic TLR-2 ligand, Pam3cys-Ser-Lys4 (Pam-3-cys) (12), and highly purified preparations of E. coli LPS. Our data suggest that signaling via distinct TLR-2 conditions DCs to modulate the Th balance toward the Th2 pathway, via a mechanism involving enhanced induction of extracellular signal-regulated kinase (ERK) and the early growth transcription factor, c-Fos.
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Materials and Methods |
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C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Male B6.PL-Thy 1a (B6.PL) mice were purchased from The Jackson Laboratory or were bred at the Rodent Vivarium of the Yerkes National Primate Center (Emory University, Atlanta, GA). B6129/F1/Tac (B6129) mice were purchased from Taconic Farms (Germantown, NY). TLR-2 knockout mice (TLR-2−/−) (14), and myeloid differentiation factor 88 (MyD88) knockout (MyD88−/−) mice (34), originally generated in the laboratory of S.A., were kindly provided by Drs. R. Medzhitov (Yale University, New Haven, CT) and A. Sher (National Institutes of Health, Bethesda, MD), and bred at the Yerkes Vivarium. ERK1 knockout (ERK1−/−) (35) mice, in the C57BL/6 background, were provided by G.L., and bred at the Yerkes Vivarium. OT-2 TCR transgenic mice (strain 426-6) (36), generated by Drs. W. Heath (Walter and Elisa Hall Institute, Melbourne, Australia) and F. Carbone (Monash University, Melbourne, Australia) were obtained from Dr. J. Kapp (Emory University) and bred at the Yerkes Animal Facility. OT-1 TCR transgenic mice (37) were obtained from The Jackson Laboratory, and bred at the Yerkes Vivarium. All mice were aged 6- to 10-wk. c-fos−/− mice (38), in the B6/129 background, were provided by L.M., and sex and age-matched littermate controls were used. These mice were used at 3–4 wk of age. All animal studies were approved by the Institutional Animal Care and Use Committee (Emory University). For adoptive transfer studies, age-matched B6.PL recipients were given 2.5 x 106 of either OT-2 or OT-1 TCR transgenic T cells i.v.
Microbial stimuli
Highly purified E. coli LPS (strain 25922) and P. gingivalis LPS (A7436) were generous gifts from T.V.D. Pam-3-cys was obtained from G. Jung (Eberhard Karls Universitat Tübingen, Tübingen, Germany) and reconstituted in endotoxin-free water. All Ags were sonicated before use.
Injections
B6.PL mice reconstituted with OT-2 TCR transgenic T cells were injected i.p. with 50 µg of MHC class II-restricted OVA peptide (ISQVHAAHAEINEAGR; OVA323–339) in PBS alone, or PBS containing either 25 µg of E. coli LPS or 50 µg of Pam-3-cys. OT-1 TCR transgenic T cell-reconstituted B6.PL mice were injected in a similar fashion except with 50 µg of MHC class I-restricted OVA peptide (SIINFEKL; OVA257–264). The OVA peptides were obtained from BioSynthesis (Lewisville, TX) and from Dr. B. Evavold (Emory University, Atlanta, GA).
To investigate the effect of TLR ligands on DC in vivo, B6129 or TLR-2 knockout mice were injected with PBS containing either 25 µg of E. coli LPS or 50 µg of Pam-3-cys. Six hours later, the spleens were removed and a small portion were digested with Collagenase, Type 4 (1 mg/ml; Worthington Biochemical, Lakewood, NJ) in complete DMEM + 2% FBS for 30 min at 37°C. The RBC were lysed and the cell suspension was washed twice before analysis of cell surface expression of activation markers by flow cytometry.
Flow cytometry
All Abs used were from BD PharMingen (San Diego, CA). For analysis of activation of DC after injection of TLR ligands in vivo, RBC-lysed, collagenase-digested spleen cells were incubated at 4°C with FITC-conjugated CD11c, PE-conjugated CD11b, and either biotin-labeled CD86 or MHC class II followed by labeling with streptavidin allophycocyanin. For analysis of OT-2 cells, cell suspensions prepared from spleen cells were incubated at 4°C with allophycocyanin-conjugated CD4 and PE-conjugated Thy 1.2. OT-1 cells were analyzed in a similar fashion, but with allophycocyanin-conjugated CD8 and PE-conjugated Thy 1.2 Abs.
Purification of DC
CD11c+CD11b+ and CD11c+CD11b− DC subsets were purified from spleen cell suspensions from mice treated for 9 days with Flt-3 ligand (Immunex, Seattle, WA). Briefly, spleens from Flt-3 ligand-treated mice were dissected, cut into small fragments,and then digested with Collagenase, Type 4 (1 mg/ml) in complete DMEM + 2% FBS for 30 min at 37°C. Cells were washed twice and frozen in FBS + 10% DMSO (Sigma-Aldrich, St. Louis, MO). Thawed cells were washed twice and the CD11c+ DCs were enriched using the CD11c+ microbeads from Miltenyi Biotec (San Diego, CA). The resulting purity of CD11c+ DCs was 
95%. The enriched DCs were stained with FITC-conjugated CD11c (BD PharMingen) and PE-conjugated CD11b (BD PharMingen) and sorted into the CD11c+CD11b+ and CD11c+CD11b− DC subsets using a high speed modular flow cytometer (MoFlo; Cytomation, Fort Collins, CO)
CD40 ligand (CD40L) fibroblasts
3T3-CD40L fibroblasts (Ref.35 ; a kind gift of Dr. P. Hwu, National Cancer Institute, Bethesda, MD) were grown in complete DMEM + 10% FBS + 2 mg/ml G418 sulfate. Cells were harvested using trypsin-EDTA and irradiated at 3000 rad (2853 cGy; Gammacell 3000 Elan; MDS Nordion, Ottawa, Ontario, Canada) to prevent further replication. Cells were plated at 4 x 104 cells per well in a 48-well plate and incubated overnight at 37°C in a humidified atmosphere of 5% CO2 in air.
In vitro cultures
Total spleen cells from TLR-2 knockout or B6129 mice were depleted of RBC by RBC lysis and washed three times. Spleen cells were plated at 2 x 106 cells/ml in 48-well cultures dishes with various concentrations of E. coli LPS, Pam-3-cys or P. gingivalis LPS and incubated in a humidified atmosphere of 5% CO2 in air at 37°C for 48 h. Culture supernatants were collected and assayed for the presence of IL-6. CD11c+-enriched DC or CD11c+CD11b+ and CD11c+CD11b− DC subsets were plated with either E. coli LPS or Pam-3-cys in complete DMEM + 10% FBS onto a confluent layer of irradiated CD40L fibroblasts to induce CD40L triggering. Mitogen-activated protein (MAP) kinase inhibitor UO126 (10 µM; Calbiochem, San Diego, CA) was added before the addition of the TLR ligands to the CD11c+-enriched DC. Cultures were incubated in a humidified atmosphere of 5% CO2 in air at 37°C for 24–48 h. The culture supernatants were collected and assayed for the presence of IL-12(p70), IL-10, and IL-6.
Four days after in vivo priming with OVA peptide, OVA peptide + E. coli LPS, or OVA peptide + Pam-3-cys, RBC-depleted spleen cells were cultured in triplicate in 96-well round-bottomed plates (1 x 106 cells/well) in complete DMEM + 10% FBS together with different concentrations of OVA peptide. Proliferative responses were assessed after 72 h of culture in a humidified atmosphere of 5% CO2 in air at 37°C. Cultures were pulsed with 1 µCi of [3H]thymidine for 12 h and incorporation of the radionucleotide was measured by 
-scintillation spectroscopy. For cytokine assays, aliquots of culture supernatants were removed after 90 h, pooled, and assayed for the presence of IL-4, IL-5, IL-13, and IFN- by ELISA.
Measurement of cytokine production
IL-4, IL-5, IL-6, IL-12(p70), and IFN- were quantitated by ELISA sets from BD PharMingen, and IL-13 was measured by an ELISA kit from R&D Systems (Minneapolis, MN).
Western blotting
CD11c+ DC (1 x 106) were stimulated for 20 min or 2 h with either E. coli LPS (10 µg/ml) or Pam-3-cys (100 µg/ml), in the presence of an anti-CD40 Ab (BD PharMingen), and lysed with cell extraction buffer (Immunoassay kit; BioSource International, Camarillo, CA). Total protein (80–100 µg) was resolved on 10% SDS-PAGE gels and transferred to Immuno Blot polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). Blotting was performed with phospho-p38 MAP kinase Ab, phospho-p44/42 MAP kinase Ab, p38 kinase Ab, or p44/42 kinase Ab (Cell Signaling Technology, Beverly, MA). Bands were visualized with secondary HRP-conjugated Ab (Cell Signaling Technology) and the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
Intracellular c-Fos staining
CD11c+ DC (1 x 106) were stimulated for 20 min or 2 h with anti-mouse CD40 mAb (5 µg/ml) and either E. coli LPS (10 µg/ml) or Pam-3-cys (100 µg/ml). Cells were fixed in 2% paraformaldehyde and cell membranes were permeabilized with 90% methanol. After washing, cells were incubated with PE-conjugated c-FOS (Santa Cruz Biotechnology, Santa Cruz, CA) and washed extensively before detecting intracellular c-FOS by flow cytometry.
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Results |
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Our recent data suggest that E. coli LPS, but not P. gingivalis LPS, induces IL-12(p70) from the CD11c+CD11b−CD8+ splenic DC subset. Recent work from Vogel and coworkers (33) suggests that this P. gingivalis LPS signals through TLR-2 in splenic macrophages. Given, the striking differences in immune responses stimulated by E. coli LPS vs P. gingivalis LPS (25), it was important to confirm that our preparation of P. gingivalis LPS did indeed signal through TLR-2. Therefore, we cultured spleen cells from wild-type and TLR-2−/− mice with E. coli LPS, P. gingivalis LPS, and Pam-3-cys for 48 h. Then the supernatants were assayed for IL-6. As shown in Fig. 1, induction of cytokines by P. gingivalis LPS, but not E. coli LPS, is severely impaired in TLR-2−/− mice. Consistent with previous reports by Akira et al. (12), cytokine induction by the synthetic molecule Pam-3-cys is impaired in TLR-2−/− mice.
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The induction of high levels of IL-10, a cytokine which induces Th2 or T regulatory cells, by Pam-3-cys was intriguing. It has been suggested that signaling via any of the TLRs results in Th1 cytokines (15, 16, 41), and mice deficient in the TLR adaptor protein MyD88 have been reported to have a selective impairment in Th1 responses (42, 43). Thus, it was important to determine whether IL-10 induction by Pam-3-cys was truly dependent on TLR-2 and MyD88, or whether this was mediated via some other accessory receptor. Splenic DCs were isolated from wild-type, TLR-2, and MyD88-deficient mice, and cultured with the different stimuli in the presence of the CD40L-expressing fibroblasts. As shown in Fig. 2, b and c, IL-10 induction by Pam-3-cys is impaired in TLR-2-deficient DCs, as well as in MyD88-deficient DCs. Thus, Pam-3-cys induces IL-10, largely through a mechanism dependent on both TLR-2 and MyD88. Contrary to previous reports, this suggests that TLR-2 and MyD88 can also induce certain Th2 cytokines such as IL-10.
E. coli LPS and Pam-3-cys activate splenic CD11c+CD11b− and CD11c+CD11b+ DC subsets in vivo
We next determined whether TLR-4 and TLR-2 ligands could activate splenic DC subsets in vivo, by injecting the ligands i.p. into wild-type or TLR-2-deficient mice, and examining the microenvironmental localization of DCs, and their expression of costimulatory molecules, 4 or 6 h after injection. As shown in Fig. 3, E. coli LPS and Pam-3-cys induce equivalent up-regulation of CD86 and MHC class II(I-Ab) on both CD11c+CD11b+ and CD11c+CD11b− DCs in wild-type mice. In TLR-2-deficient mice, the induction of CD86 and I-Ab by Pam-3-cys was severely impaired, but the effects of E. coli LPS were unaffected. Therefore, the synthetic molecule Pam-3-cys appears to activate DCs in vivo, via TLR-2.
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Given the significant differences in the relative levels of IL-12(p70) and IL-10 induced by TLR-4 and TLR-2 ligands, we wondered whether they stimulated different types of CD4+ Th immune responses in vivo. We addressed this question, using OVA-specific, MHC class II-restricted (I-Ab), TCR transgenic mice (OT-2 mice) (36). In these mice, the CD4+ OVA-specific T cells express V2 and V5, and recognize the amino acid 323–339 peptide fragment (hereafter denoted as OVA323–339) from OVA. TCR transgenic T cells were adoptively transferred into Thy-1 congenic B6.PL.Thy-1a (B6.PL) mice, such that they constituted a small, but detectable proportion of all T cells (44, 45, 46). In this system, the fate of OVA-specific, transgenic T cells was followed using the Thy-1.2 Ab, which stains the transferred cells, but not the host cells. Cells with the phenotype Thy-1.2+CD4+V2+V5+ are considered OVA-specific CD4+ T cells. In some of the experiments, we simply used Thy-1.2 in combination with CD4, to detect the OVA-specific T cells.
The reconstituted mice were injected with 50 µg of OVA323–339 peptide alone, or OVA323–339 + E. coli LPS, or OVA323–339 + Pam-3-cys i.p. Injection of OVA323–339 alone did not induce any significant clonal expansion of the CD4+Thy-1.2+ cells in the spleens of mice (Fig. 4a). However, both E. coli LPS and Pam-3-cys significantly enhanced the clonal expansion of CD4+ Thy-1.2+ cells. Previous work has shown that productive T cell immunity is elicited only when the Ag is injected with an adjuvant, and that injections of soluble Ags only result in a transient and abortive clonal expansion, in which Ag-specific T cells cannot be efficiently restimulated in vitro, with protein or peptide (44, 45, 46). Thus, we examined the in vitro proliferative capacity of OVA-specific T cells from the various cohorts of mice, by culturing single cell suspensions of the spleen with varying concentrations of OVA. As shown in Fig. 4b, mice that received OVA323–339 + E. coli LPS or OVA323–339 + Pam-3-cys had greatly enhanced responses, compared with those that received OVA323–339 peptide alone.
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, IL-4, IL-5, and IL-13. There were significant differences between mice injected with OVA323–339 peptide alone, or OVA323–339 + E. coli LPS, or OVA323–339 + Pam-3-cys (Fig. 4c). In cultures from mice injected with OVA257–264 peptide alone, there was little, if any, IFN-, IL-4, IL-5, or IL-13. In contrast, and consistent with previous reports (25, 46), in cultures from mice injected with OVA323–339 + E. coli LPS, there were high levels of IFN-, and low levels of IL-4 (8 pg/ml) and IL-5 (30 pg/ml). In addition, there was a significant level of IL-13. Considering that the sensitivity of the cytokine ELISA is 8 pg/ml (dotted red line, Fig. 4c), the levels of IL-4 and IL-5 induced by E. coli LPS are either below or barely above the threshold of detection, and thus E. coli LPS biases the response toward the Th1 pathway. This Th1 induction by E. coli LPS was dependent on IL-12(p70), because its neutralization, in vivo, with an Ab, largely impaired IFN- production (data not shown). However, the induction of significant levels of IL-13, as observed previously (25), suggests that the response induced does not fit the "canonical Th1 profile." In striking contrast to this response, in cultures from mice injected with OVA323–339 + Pam-3-cys, there were much lower levels of IFN-, significantly higher levels of IL-5 (70 pg/ml) and IL-4 (30 pg/ml), and similar levels of IL-13 as that induced by E. coli LPS. Although the absolute levels of cytokines induced varied from experiment to experiment, in every experiment Pam-3-cys induced a much greater Th2 bias than LPS. Thus, Pam-3-cys induces a response in which there is a preferential bias toward the Th2 pathway (Fig. 2), consistent with its effective induction in DCs of IL-10, a Th2-inducing cytokine (40). This response is unlikely to represent a T regulatory response, because Pam-3-cys was able to induce significant clonal expansion and in vitro proliferation (Figs. 4 and 5). Thus, although neither stimulus induces canonical Th1 or Th2 responses, they exert strikingly different influences on modulating the Th1/Th2 balance. This is further illustrated by the ratios of Th1:Th2 cytokines induced by the E. coli LPS vs Pam-3-cys (IFN-:IL-4, 975 vs 40; IFN-:IL-5, 162 vs 18; IFN-:IL-13, 4.5 vs 1.2) (data not shown).
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The propensities of E. coli LPS and Pam-3-cys to stimulate different classes of Th responses in vivo prompted us to consider whether these stimuli could induce distinct types of CD8+ T cell responses in vivo. We investigated this, using OT-1 mice (H-2Kb-restricted, OVA-specific TCR transgenic mice) (37). A total of 2.5 x 106 spleen cells from OT-1 mice were transferred into B6.PL (Thy1.2) mice. Cohorts of host mice were injected with either OVA257–264 + E. coli LPS or OVA257–264 + Pam-3-cys. Clonal expansion of OVA-specific CD8+ T cells (CD8+Thy-1.2+) was assessed by flow cytometry (Fig. 5a). Both E. coli LPS + OVA323–339 and Pam-3-cys + OVA257–264 enhanced the clonal expansion of OVA-specific CD8+ T cells.
We then examined the in vitro proliferative capacity of the OVA-specific CD8+ T cells from the various cohorts of mice, by culturing single cell suspensions of the spleen with varying concentrations of OVA257–264. As shown in Fig. 5b, mice that received an injection of either E. coli LPS + OVA257–264, or Pam-3-cys + OVA257–264 had greatly enhanced responses, compared with those that received OVA257–264 alone.
Cytokine production by Ag-specific T cells was measured by assaying the culture supernatants from the cultures described above for IFN-, IL-4, IL-5, and IL-13 (Fig. 5c). There were significant differences between mice injected with OVA257–264 peptide alone, or OVA257–264 + E. coli LPS, or OVA257–264 + Pam-3-cys. In cultures from mice injected with OVA323–339 peptide alone, there was little, if any, IFN-, IL-4, IL-5, or IL-13. In contrast, and consistent with previous reports (44, 45), in cultures from mice injected with OVA257–264 + E. coli LPS, there were high levels of IFN-, and much lower levels of IL-4, IL-5, and IL-13. Thus, E. coli LPS appears to skew the T cytotoxic (Tc) balance toward the Tc1 pathway. However, compared with the cultures from the mice injected with E. coli LPS, in cultures from mice injected with OVA257–264 + Pam-3-cys, there were lower levels of IFN-, but higher levels of IL-4, IL-5, and IL-13. Therefore, Pam-3-cys appears to shift the balance toward the Tc2 pathway. This is further illustrated by the ratios of Tc1:Tc2 cytokines induced by the E. coli LPS vs Pam-3-cys (IFN-:IL-4, 260 vs 28; IFN-:IL-5, 685 vs 21; IFN-:IL-13, 22 vs 4) (data not shown).
Pam-3-cys induces enhanced phosphorylation of ERK1/2, which suppress IL-12(p70) and promote IL-10 in DCs
To gain insights into the potential intracellular signaling mechanisms which may mediate the different DC responses, we focused on the MAP-kinase signaling pathway, one of the most ancient signal transduction pathways in mammalian cells (47, 48, 49). MAP kinases consist of three major groups: p38 MAP kinases, the ERKs (ERK1 and 2), and the c-Jun NH2-terminal kinases 1 and 2. Previous reports indicate a critical role for MAP kinases in regulating Th1/Th2 balance in T cells (47), and emerging evidence suggests a role for these proteins in regulating cytokine production from APCs (50, 51). Therefore, we sought to determine the phosphorylation of p38 and ERK1/2 in DCs stimulated with various stimuli. As shown in Fig. 6, and consistent with previous studies (52, 53), both E. coli LPS and Pam-3-cys induced the phosphorylation of p38, but DCs cultured with anti-CD40 Ab alone also induced significant p38 phosphorylation, which is consistent with previous reports (54, 55). Blocking the activity of p38 with a synthetic inhibitor resulted in a profound inhibition of IL-12(p70) and IL-6, and to a lesser extent IL-10 (data not shown).
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Pam-3-cys enhances DC expression of the immediate early gene product c-Fos, which regulates the production of IL-12(p70) and IL-10
How does the enhanced ERK1/2 signaling induced by Pam-3-cys result in suppression of IL-12(p70), and enhanced IL-10? Recent work (56), suggests that enhanced ERK signaling results in the phosphorylation and stabilization of the immediate early gene product c-Fos, in a fibroblast cell line. In fact, ERK phosphorylation appears to be essential for enhanced c-Fos expression (56). Thus, we determined expression of c-Fos, in DCs stimulated with the different stimuli, using an Ab directed against c-Fos. As shown in Fig. 7a, the level of expression of c-Fos (as assessed by the mean fluorescence intensity of staining) in DCs stimulated by Pam-3-cys is greater than in DCs stimulated with E. coli LPS, or in unstimulated DCs. Furthermore, c-Fos expression was maintained, even at 45 min poststimulation, in DCs stimulated with Pam-3-cys, but not with E. coli LPS. The mean fluorescence intensities of the levels of c-Fos expression, at 20 min for DCs cultured alone, or with LPS, or Pam-3-cys are 14.8, 8.5, and 17.1, respectively. At 45 min, the corresponding values are 10.1, 10.8, and 19.1, respectively (data not shown). Similar results were obtained with the Western blotting technique to detect c-Fos (data not shown). Therefore, stimulation of DCs by Pam-3-cys, which induce enhanced ERK1/2 signaling, results in increased c-Fos expression. It should be noted that even unstimulated DCs, or DCs stimulated with E. coli LPS, do express levels of c-Fos that are greater relative to the isotype control. However, the levels induced by Pam-3-cys appear to be greater than in unstimulated DCs, or after LPS stimulation.
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secretion, whereas only LPS was capable of inducing IL-1 and NO secretion (52), although the functional significance of this difference in IL-1 and TNF production is not clear. Second, a study by Re and Strominger (53) suggests that activating human monocyte-derived DCs with different TLR agonists induces distinct cytokines, but the consequences of these different cytokines on adaptive immunity, or the signaling mechanisms which elicit the production of the different cytokines are not known. Third, our data suggests that highly purified P. gingivalis LPS, a TLR-2 ligand (33), fails to induce IL-12(p70) in murine DCs, but rather induces Th2 responses (25). This has recently also been shown to be the case with human monocyte-derived DCs (58). Fourth, it has recently been shown that triggering TLR-4, but not TLR-2, signaling mediates IFN--induced STAT1-dependent gene expression in macrophages (59). Finally, a recent report suggests that triggering human monocyte-derived DCs or plasmacytoid DCs through TLR-7 results in differential induction of IL-12 and IFN- (60). Despite this emerging picture, knowledge about the signaling mechanisms which mediate such diverse functional responses is sparse. Furthermore, the consequences that signaling through different TLRs may have on the Th1/Th2 balance is not known. In this regard, our present observations offer some novel perspectives (Fig. 8). First, the data suggest that activation of innate immune cells via TLRs do not always result in polarized Th1 responses (15, 16, 41, 61), but can also bias the response toward the Th2 pathway. Although the Th1/Th2 paradigm has yielded insights of profound importance, emerging evidence suggests this framework may be an oversimplification of the complexity that underlies an immune response. For example, individual T cells display a rather complex spectrum of cytokine profiles, in which the canonical Th1 and Th2 cells represent only the very extreme ends of an axis (5). In this context, it must be emphasized that our data suggests that neither stimulus induces a typical Th1 or Th2 response. Rather, each stimulus appears to modulate the response toward opposite ends of the Th1/Th2 spectrum (Fig. 8). The notion that activating DCs via TLR-2 may bias toward Th2 responses is further supported by our recent data that other TLR-2 ligands such as peptidoglycan and zymosan, also induce abundant levels of IL-10, and little IL-12(p70), in DCs and favor a Th2 bias (data not shown). However, because these ligands also signal through certain other receptors (NOD1 in the case of peptidoglycan (62, 63), and dectin in the case of zymosan (64)), the interpretation of these results must be approached with caution.
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It is interesting to consider the proximal signaling molecules that might mediate the distinct DC responses induced by TLR-4 and TLR-2 ligands. To date, TLR-2 is known to associate with two adaptor proteins, MyD88 and TIRAP (12, 68, 69), while TLR-4 is known to associate with two additional proteins, TRIF (70, 71) and TRAM (72). The latter two are believed to mediate STAT 1 phosphorylation, and type 1 IFN production (70, 71), and given their absence in TLR-2 signaling, they are not likely to mediate the Th2 bias observed with TLR-2 ligands. MyD88 is currently believed to mediate Th1 responses, as MyD88−/− mice display a selective impairment in Th1 responses; however, it remains to be seen whether the Th2-biased responses induced by Pam-3-cys are also impaired in these mice. Indeed, our data (Fig. 2c), suggests that the induction of the Th2-inducing cytokine, IL-10 by Pam-3-cys, is largely dependent on MyD88. Furthermore, a potential role for TIRAP in the Th2 bias induced by Pam-3-cys remains to be determined. Thus, the precise roles played by each of these adaptor proteins, their influence on one another, and their possible segregation in different subsets of immunocytes, including dendritic cells, needs to be investigated.
Finally, from an evolutionary viewpoint, because a given bacterium contains ligands that engage multiple TLRs, the notion that different TLRs induce distinct Th responses may be teleologically untenable. However, it should be noted that many pathogens that are conventionally defined as "Th2 inducing" also contain Th1-inducing stimuli. For example, schistosomes are thought to induce strong Th2 responses, but there is evidence that they also stimulate Th1 responses (73). Conversely, many bacteria which contain pathogen-associated molecular patterns that stimulate Th1 responses also express Th2-inducing stimuli. For example, Vibrio cholera contains CpG DNA and other pathogen-associated molecular patterns that promote Th1 responses, but it also expresses the cholera toxin subunit B which is a strong Th2 inducer (30). Extending this argument even further, even different forms of the same pathogen differentially induce Th1 and Th2 responses (e.g., C. albicans; Ref.32). Thus, there are numerous examples of pathogens which are endowed with stimuli that induce both Th1 and Th2 responses. The evolutionary struggle between the immune system and pathogens is an abiding one; in many instances, the state of host-pathogen interactions is not necessarily something that is optimally suited to either the host or the pathogen, but rather is an uneasy truce between the host and the pathogen. In this context, it is instructive to view the global immune response against a given pathogen as a complex summation of all the input signals delivered from the pathogen to the host immune system. However, the predominance of a particular agonist in an infection might dictate the type of Th response.
In summary, the present data offer some novel perspectives on the mechanisms which underlie the complex decision-making processes which determine the striking diversity of immune responses generated against different microbes. These data are largely consistent with our recent published work with human monocyte-derived DCs, which suggests that Pam-3-cys and the classic Th2 adjuvant schistosome egg Ag both suppress IL-12(p70) production from DCs via a mechanism involving ERK and c-Fos, and bias toward Th2 responses (74). However, one interesting difference between the human study (73) and the present study concerns the induction of IL-10; while both E. coli LPS and Pam-3-cys could both efficiently induce abundant IL-10 in human DCs (74), only Pam-3-cys could do so with murine DCs (Fig. 2a). The reason for this difference is at present unclear, but it is noteworthy that while in humans IL-10 is predominantly considered a "regulatory cytokine (22)," in mice it can also induce Th2 responses (40).
Furthermore, the data underscore novel therapeutic opportunities that will be gained by modulating critical parameters (e.g., TLRs, MAP kinases, transcription factors) in the immune therapy of cancer, allergy, autoimmunity, and transplantation.
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2 Address correspondence and reprint requests to Dr. Bali Pulendran, Vaccine Research Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329. E-mail address:bpulend{at}rmy.emory.edu
3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; Pam-3-cys, Pam3cys-Ser-Lys4; ERK, extracellular signal-regulated kinase; MyD88, myeloid differentiation factor 88; CD40L, CD40 ligand; MAP, mitogen-activated protein; Tc, T cytotoxic.
Received for publication November 17, 2003. Accepted for publication February 10, 2004.
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