Published online before print October 26, 2007
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Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
1 Correspondence: Department of Viral Diseases and Immunology, National Public Health Institute, Mannerheimintie 166, FI-00300 Helsinki, Finland. E-mail: ville.veckman{at}ktl.fi
ABSTRACT
Human peripheral blood contains two major dendritic cell (DC) populations, namely CD11c–CD123+ plasmacytoid DCs (PDCs) and CD11c+CD123– myeloid DCs (MDCs). Although the activation of these DC types by various TLR ligands has been relatively well-characterized, less is known about the ability of whole live bacteria to induce PDC and MDC activation. In the present report, we have compared the activation of human PDCs and MDCs in response to major human bacterial pathogen Streptococcus pyogenes (group A streptococci) and influenza A virus. S. pyogenes stimulation resulted in the maturation of both DC types, as evidenced by enhanced expression of costimulatory molecules and production of proinflammatory cytokines and chemokines. Furthermore, S. pyogenes-stimulated PDCs and MDCs activated naïve CD4+ T cells and enhanced their Th1 cytokine production. Influenza A virus infection induced rapid PDC activation, whereas MDCs were extremely sensitive to influenza A virus-induced cell death. The most significant differences between DC types were seen in the production of IL-10 and IL-12, which were only produced by S. pyogenes-stimulated MDCs. Although S. pyogenes was able to induce PDC activation, only influenza A virus infection resulted in detectable IFN-
production. Our results show that depending on the infecting microbe, the functions of PDCs and MDCs may be partially overlapping, suggesting a considerable flexibility of the human DC system.
Key Words: cytokines • bacteria • virus • innate immunity
INTRODUCTION
Dendritic cells (DCs) are APCs that act as a bridge between innate and adaptive immunity. DCs reside in peripheral tissues in an immature state, where they are on alert for invading pathogens or other danger signals. A contact with nonself structures such as microbes or their structural components activates DC maturation, which is characterized by enhanced expression of costimulatory molecules, production of cytokines and chemokines, and a change in the cell surface chemokine receptor expression pattern. Moreover, maturation renders DCs capable for efficient antigen presentation to T cells [1 ].
Human blood contains a small number of terminally differentiated DCs, which typically constitute 0.5–2% of PBMCs. Circulating human DCs are HLA-DR+ lineage– and can be classified into CD11c+CD123– myeloid DCs (MDCs) and CD11c–CD123+ plasmacytoid DCs (PDCs). There is, however, accumulating evidence that human CD11c+ MDCs are heterogeneous and consist of several different subpopulations. The existence of at least blood DC antigen (BDCA)-1+, BDCA-3+, and CD16+ MDCs in human blood has been reported. Although the relationship between different human MDC populations is currently not well characterized, a recent microarray analysis of MDC subpopulations revealed that based on gene expression pattern, BDCA1+ and BDCA3+ populations group together, and CD16+ MDCs show a more unique gene expression pattern [2 ]. It has also been demonstrated that different MDC types show a distinct T cell stimulatory capacity in allogenic mixed lymphocyte reaction (MLR) [3 ].
The phenotypic and functional differences between human PDCs and MDCs have been relatively well-characterized. A hallmark for virus-infected PDCs is the production of large amounts of IFN- accompanied with the production of other proinflammatory cytokines. In contrast, MDCs produce proinflammatory cytokines together with IL-10 and IL-12 in response to microbial stimulation. The key difference between PDCs and MDCs is their differential expression of TLRs. PDCs express mainly TLR7 and TLR9 and thus, respond to ssRNA and unmethylated CpG-rich DNA, respectively. However, low levels of TLR1, TLR6, and TLR10 mRNAs have also been detected in PDCs [4
5
]. MDCs show a broader TLR expression pattern, and these cells express all TLRs, except TLR7 and TLR9 [4
5
]. The activation of human PDCs and MDCs in response to purified TLR ligands has been analyzed extensively. However, far less is known about the ability of live bacteria to induce the activation of different human blood DC subtypes, especially that of PDCs. Although TLR ligands provide a clean model system for analyzing DC responses, it is unlikely that PDCs or MDCs encounter only a single microbial structure in vivo. In contrast, whole bacteria contain components that interact with multiple TLRs and other pattern recognition receptors (some of which may still be unknown). Bacteria-induced activation of PDCs has not been systematically analyzed, although some earlier reports have shown that Gram-positive Staphylococcus aureus bacteria and Escherichia coli-derived DNA may induce IFN production in PDCs [6
7
].
We have demonstrated previously that an important human-specific pathogen, Gram-positive Streptococcus pyogenes, activates an inflammatory cytokine response from several leukocyte types including PBMCs, monocytes, macrophages, and monocyte-derived DCs [8 9 10 11 12 ]. In the present study, we have analyzed the ability of S. pyogenes and influenza A virus to activate primary human blood-derived PDCs and BDCA-1+ MDCs. In this report, we show that S. pyogenes induces the maturation and production of proinflammatory cytokines and chemokines in PDCs and MDCs. In addition, S. pyogenes-stimulated PDCs and MDCs were able to stimulate naïve CD4+ T cell proliferation and drive their polarization toward a Th1 phenotype. Certain important differences between DC types and between bacterial and viral infection were observed. It is most notable that IL-10 and IL-12 were only produced by S. pyogenes-stimulated MDCs, and efficient induction of IFNs was only seen in influenza A virus-infected DCs. In conclusion, our results demonstrate that depending on the infecting microbe, the functions of human PDCs and MDCs may be partially overlapping, which suggests a considerable flexibility of the human DC system. Moreover, our results show that PDCs, which have been analyzed mostly in the context of viral infections, can also respond efficiently to certain bacterial pathogens.
MATERIALS AND METHODS
Isolation of DCs and CD4+ T cells
DCs were isolated from freshly collected, leukocyte-rich buffy coats obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). PBMCs were isolated by a density gradient centrifugation over Ficoll-Paque density gradient (GE Healthcare, UK), as described previously [11
]. PDCs were isolated by positive selection using BDCA-4-conjugated magnetic beads (Miltenyi Biotec, Gladbach, Germany). To enhance cell purity, the labeled PDCs were separated twice through MACS LS columns. Otherwise, the protocol was carried out according to the manufacturer’s instructions. The purity of isolated CD123+ BDCA-2+ double-positive cells was constantly over 95%. MDCs were isolated from PBMCs by using the BDCA-1 DC isolation kit, according to the manufacturer’s instructions (Miltenyi Biotec). Briefly, CD19+ cells were first removed by positive selection, after which BDCA-1+ DCs were positively selected. BDCA-1+ cells were run twice through the LS column to increase their purity. CD11c and CD123 staining from two representative donors is shown in Figure 1 A
. Naïve CD4+ T cells were isolated from cord blood mononuclear cells by negative selection using the CD4+ T cell isolation kit II (Miltenyi Biotec). The purity of CD4+ cells was higher than 90%.
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Figure 1. (A) Purity of human DC populations. PDCs and MDCs were purified with BDCA-4- or BDCA-1-conjugated magnetic beads, respectively, as described in Materials and Methods. After isolation, cells were fixed and stained with anti-CD11c or anti-CD123 antibodies (black histograms) and respective isotype controls (solid line). Results from two representative blood donors are shown. (B) Microbe-induced TNF- production from human PDCs. Results are from two independent experiments. Freshly isolated PDCs were left unstimulated or stimulated with live or UV-inactivated influenza A virus [IA; multiplicity of infection (MOI), 5], S. pyogenes (S.p; MOI, 5), LPS (100 ng/ml), Salmonella enterica serovar typhimurium (S.t; MOI, 5), or Lactobacillus rhamnosus (L.r; MOI 5). After 24 h, cell culture supernatants were collected, and TNF- production was measured.
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Flow cytometry
The purity of DCs was verified by staining paraformaldehyde-fixed cells with anti-CD123, anti-BDCA-2, or anti-CD11c antibodies (Miltenyi Biotech and BD Biosciences, Sunnyvale, CA, USA). Cell surface expression of CD40, CD86, and HLA class II was analyzed by using FITC-conjugated antibodies and respective isotype controls (Caltag Laboratories, Burlingame, CA, USA). DC viability was measured with Dead cell discriminator reagent (Caltag Laboratories). The expression of cell surface proteins and DC viability was analyzed with FACScan flow cytometer and CellQuest software (BD Biosciences).
Cytokine measurements
Cytokine production was measured from microbe-stimulated DC culture supernatants. The production of proinflammatory and Th1/Th2 cytokines (including CXCL8 chemokine) was measured with FlowCytomix human Th1/Th2 Plex kit II (Bender Medsystems, Vienna, Austria). IFN- levels were measured with a human IFN- ELISA kit obtained from PBL Biomedical Laboratories (New Brunswick, NJ, USA). The antibody pairs and standards for measuring CCL2, CCL3, and CXCL10 chemokine levels were purchased from BD Biosciences.
DC-T cell coculture
In coculture experiments, PDCs and MDCs were left untreated or were infected with influenza A virus or stimulated with S. pyogenes for 24 h. DCs were washed twice with RPMI-1640 medium and incubated with allogenic cord blood-derived, naïve CD4+ cells at a DC:T cell ratio of 1:5. Cells were cocultured in 96-well round-bottom plates in RPMI-1640 medium supplemented with 10% FCS for 5 days. Fresh medium was added at Day 3. To measure the T cell cytokine profile in response to restimulation, cocultured cells were washed with RPMI medium and stimulated with PMA (50 ng/ml) and ionomycin (2 µg/ml) for 20 h (Alexis Biochemicals, Lausen, Switzerland). Cell culture supernatants were collected, and cytokine levels were analyzed as described above.
Statistical analysis
Statistical significance was determined by paired, two-tailed Student’s t-test. The statistically significant differences between different groups are indicated in each figure (*, P > 0.05; **, P > 0.01).
RESULTS
Human PDCs and MDCs were purified from the PBMC population by immunomagnetic beads. The purity of different DC types was confirmed by staining PDCs and MDCs with anti-CD11c and anti-CD123 antibodies. PDCs were constantly CD123+CD11c–, and MDCs were CD123–CD11c+. Cell populations were free of monocytes, T cells, or B cells (data not shown). Representative CD11c and CD123 stainings from two different blood donors are shown in Figure 1A .
In the first set of experiments, the ability of whole bacteria to activate PDCs was analyzed. PDCs were stimulated with Gram-positive S. pyogenes, nonpathogenic L. rhamnosus, or Gram-negative S. typhimurium at 5:1 bacteria:DC ratios for 24 h. As a positive and a negative control, PDCs were also infected with influenza A virus and stimulated with LPS, respectively. In these experiments, TNF- production was used as the readout. Influenza A virus infection and S. pyogenes stimulation resulted in a significant TNF- production from PDCs (Fig. 1B)
. In contrast, when PDCs were stimulated with live L. rhamnosus, S. typhimurium, or purified LPS, low or no TNF- production was detected, as compared with unstimulated PDCs. It is interesting that UV inactivation of influenza A virus or S. pyogenes had no effect on TNF- production in PDCs (Fig. 1B)
.
Effect of microbe stimulation on DC viability
Next, we broadened the analysis to cover PDCs and MDCs. We first analyzed whether S. pyogenes or influenza A virus was able to maintain DC viability in the absence or presence of cytokines that regulate DC development and survival. PDCs and MDCs were cultured with or without IL-3 and IL-4 + GM-CSF, respectively, and stimulated with S. pyogenes or influenza A virus for 24 h. DC viability was assayed by propidium iodide (PI) staining and flow cytometry.
IL-3 was absolutely necessary for PDC viability, as in the absence of IL-3, nearly 60% of unstimulated PDCs were PI-positive after 24 h of culture (Fig. 2 , upper panel). In the absence of IL-3, microbe stimulation enhanced PDC survival, and the number of PI-positive cells was reduced to 50% and 40% for S. pyogenes and influenza A virus-infected PDCs, respectively (P<0.05). When PDCs were cultured and stimulated in the presence of IL-3, the amount of PI-positive cells was reduced markedly compared with cells cultured in the absence of IL-3. Moreover, no significant difference between unstimulated and microbe-stimulated PDCs was observed when IL-3 was present in the culture medium. In contrast to PDCs, MDCs did not require additional cytokines for maintaining their viability (Fig. 2 , lower panel). No statistically significant difference was observed in the viability of unstimulated or S. pyogenes-stimulated MDCs, whether the cells were cultured in the presence or absence of IL-4 + GM-CSF. However, MDCs were found to be sensitive to influenza A virus infection, as nearly 60% of cells were PI-positive at 24 h post-influenza A virus infection. Culturing MDCs in the presence of IL-4 + GM-CSF had no protective effect on influenza A virus-induced cell death. When MDCs were infected with lower MOIs of influenza A virus, no cytokine response was observed, and the number of virus-infected MDCs was reduced drastically (data not shown). To be able to compare the responses of unstimulated and stimulated cells reliably, both DC types were cultured in the presence of cytokines in the following experiments.
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Figure 2. DC viability in response to S. pyogenes stimulation or influenza A virus infection. PDCs (upper panel) were cultured in the presence or absence of IL-3 and left unstimulated or stimulated with S. pyogenes (MOI, 5) or infected with influenza A virus (MOI, 5). MDCs (lower panel) were cultured in the presence or absence of IL-4 and GM-CSF and stimulated with microbes as described for PDCs. DC viability was analyzed by PI staining after cell isolation (0 h) and after 24 h culture. Results from one experiment (three different blood donors) out of two experiments performed are shown. The statistical significance of microbe-induced enhancement of PDC viability was analyzed by using Student’s t-test (*, P<0.05).
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Figure 3. S. pyogenes and influenza A virus-induced maturation of PDCs (left panel) and MDCs (right panel). DCs were stimulated with S. pyogenes (MOI, 5) or infected with influenza A virus (MOI, 5) for 24 h or 48 h, and cell surface expression of CD40, CD86, and HLA class II was analyzed by flow cytometry. Results are shown as fold induction compared with unstimulated cells (24 h). Statistical significance of the differences between groups was tested by Student’s t-test (**, P<0.01; *, P<0.05). Results from one experiment (four different blood donors) out of three experiments performed are shown.
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S. pyogenes and influenza A virus-induced cytokine and chemokine response
Next, we analyzed the cytokine production profile of S. pyogenes-stimulated and influenza A virus-infected DCs. PDCs produced nearly similar amounts of TNF- in response to S. pyogenes stimulation and influenza A virus infection (Fig. 4
). In contrast, MDCs produced high levels of TNF-, only in response to S. pyogenes stimulation, and S. pyogenes-induced TNF- production levels were 
15-fold higher compared with those induced by influenza A virus. The production of IL-6 was better induced by S. pyogenes as compared with influenza A virus in PDCs and MDCs. The most significant differences between PDCs and MDCs were seen in the production of IL-10 and IL-12. These cytokines were produced by S. pyogenes-stimulated MDCs, and PDCs were devoid of their production almost completely. Moreover, influenza A virus failed to induce the production of these cytokines in either of the DC types. A clear difference between S. pyogenes and influenza A virus was seen in their ability to induce IFN- production. This cytokine was only detected in influenza A virus-infected PDC and MDC cell culture supernatants. The levels of all analyzed cytokines reached their maximum at 24 h after stimulation, except for IFN-, whose production continued to increase up to 48 h in PDCs and MDCs (Fig. 4)
.
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Figure 4. DC cytokine response to S. pyogenes and influenza A virus. PDCs (left panel) and MDCs (right panel) were left unstimulated or stimulated with S. pyogenes (MOI, 5) or influenza A virus (MOI, 5) for 24 h or 48 h. Cell culture supernatants were collected, and cytokine production was analyzed by cytometric bead assay (TNF-, IL-6, IL-10, and IL-12) or ELISA (IFN-). Values (note the differences in scale) are in ng/ml. Results are the means from three independent experiments (nine different blood donors). Error bars indicate SD of the means. Statistical significance of the differences between groups was tested by Student’s t-test (**, P<0.01; *, P<0.05).
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Figure 5. Microbe-induced inflammatory chemokine production in PDCs and MDCs, which were stimulated with S. pyogenes (MOI, 5) or infected with influenza A virus (MOI, 5) for 24 h, and chemokine production was analyzed by ELISA. Results are the means from two independent experiments (seven different blood donors). Error bars indicate SD of the means. Statistical significance of the differences between groups was tested by Student’s t-test (**, P<0.01; *, P<0.05).
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Coculture of CD4+ T cells with S. pyogenes or influenza A virus-stimulated PDCs resulted in the production of IFN-
, IL-2, and IL-10 upon restimulation (Fig. 6
). Higher cytokine levels were seen for S. pyogenes-stimulated PDCs compared with influenza A virus-infected PDCs. T cells cultured with unstimulated PDCs showed low or no cytokine response upon restimulation. S. pyogenes-stimulated MDCs induced CD4+ cells to produce IFN-. In contrast to PDCs, IL-10 production was low in MDC-T cell cocultures. Influenza A virus-infected MDCs showed a similar or even lower ability to induce T cell cytokine response as unstimulated MDCs. This is likely a result of influenza A virus-induced MDC death (see Fig. 2
), which leads to impaired antigen presentation and T cell response. It is interesting that coculture of MDCs with naïve CD4+ T cells resulted in high IL-2 production upon restimulation in microbe-stimulated and unstimulated MDC cocultures. In our experimental setting, no significant IL-4 or IL-5 production was detected (Fig. 6
, and data not shown).
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Figure 6. PDC- and MDC-induced T cell cytokine profile. PDCs and MDCs were isolated from the same individuals (two different donors), and the cells were stimulated with S. pyogenes (MOI, 5) or infected with influenza A virus (MOI, 5). At 24 h after stimulation, DCs were washed and cocultured with cord blood-derived, purified CD4+ T cells for 5 days (three different cord blood donors). After coculture, cells were washed and stimulated with PMA and ionomycin for 20 h. Cell culture supernatants were analyzed by flow cytometric bead assay. Cytokine concentrations are in ng/ml, and horizontal lines indicate the medians. Each symbol represents one DC-T cell donor pair (n=6 in this experiment). Results from one of two experiments performed are shown. The statistical significance was analyzed by using Student’s t-test (*, P<0.05).
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In this report, we show that Gram-positive bacterium S. pyogenes is able to activate human PDCs and MDCs. Both DC types produced proinflammatory cytokines and chemokines and showed enhanced expression of costimulatory molecules following stimulation with live S. pyogenes. Moreover, S. pyogenes-stimulated PDCs and MDCs promoted the activation and Th1 polarization of naïve cord blood-derived CD4+ T cells. Bacteria-induced activation of human blood-derived primary DCs, especially PDC activation, has not been analyzed previously in detail. There are only a few studies that show that bacterial DNA and heat-killed Gram-positive S. aureus bacteria induce the production of IFN in PDCs [6
7
]. In the initial experiments, we observed that bacteria-induced PDC activation is significantly dependent on the type of bacteria. Although S. pyogenes induced a clearly detectable TNF- production in PDCs, another Gram-positive bacteria, L. rhamnosus, Gram-negative S. typhimurium, or purified E. coli LPS, failed to induce significant TNF- production in PDCs (Fig. 1)
. As PDCs do not express TLR4 [4
15
16
], the inability of LPS to induce TNF- production in PDCs also demonstrates that our PDC cultures are pure and devoid of myeloid cell contamination.
S. pyogenes-stimulated PDCs produced proinflammatory cytokines TNF- and IL-6 in nearly similar or even higher levels as compared with influenza A virus-infected PDCs (Fig. 4)
. A major difference between S. pyogenes and influenza A virus was seen in the production of IFN-, which was induced exclusively by influenza A virus in PDCs and MDCs. Previous studies using the mouse model system have shown that TLR-induced IFN response in PDCs is mediated by the MyD88-IFN regulatory factor 7 (IRF7) pathway [17
18
]. Thus, our results suggest that S. pyogenes is unable to induce IRF7 activation in PDCs. However, as S. pyogenes-stimulated PDCs produced proinflammatory cytokines TNF- and IL-6, it is likely that S. pyogenes is able to activate other signaling cascades, such as NF-
B and MAPK pathways in PDCs. Unfortunately, the small number of blood-derived DCs prevents an extensive biochemical characterization of the activation of these signaling pathways. Despite the lack of IFN- production, S. pyogenes stimulation resulted in the maturation of PDCs and MDCs, which suggests that IFN- is not essential for microbe-induced maturation of human DCs. In line with this hypothesis, Gibson et al. [19] and Ito et al. [20] have demonstrated that IFN- acts as a survival, but not as a maturation factor, for human PDCs. Although IFN- alone enhances the expression of costimulatory molecules and promotes a T cell stimulatory capacity of human MDCs (monocyte-derived and CD11c+) [20
21
22
23
], it has been shown by IFN- neutralization assays that secreted IFN- has only a minor role in LPS or virus-induced maturation of human MDCs [24
25
26
]. In conclusion, our results, together with previous observations, suggest that direct microbe-induced signals are sufficient to induce full maturation of human DCs. Secreted IFN- is likely to contribute to the fine-tuning of DC activation by enhancing the expression of IFN-inducible genes such as the CXCL10 chemokine (Fig. 5)
. Moreover, DC-derived IFN- may enhance the local antimicrobial response significantly in a paracrine manner, as IFN- controls the expression of several microbe-recognition receptors, transcription factors, and antimicrobial compounds. Thus, DC-derived IFN- may locally prime uninfected cells for more efficient microbe-recognition and immune activation.
Although PDCs and MDCs produced proinflammatory cytokines in response to S. pyogenes stimulation, certain differences between the two DC types were observed. When the cytokine production profile of MDCs and PDCs was compared, we noticed that IL-10 and IL-12 were only produced by S. pyogenes-stimulated MDCs. Although some earlier studies suggest that human PDCs produce IL-12 [15 16 27 ], more recent reports have demonstrated that unlike mouse PDCs, human PDCs do not produce IL-10 or IL-12 [5 28 29 30 ]. The differential expression of IL-10 and IL-12 between PDCs and MDCs also demonstrates further that S. pyogenes-induced PDC activation is not mediated by myeloid cell contamination in our experimental setting. MDC-derived IL-12 may play a critical role in controlling S. pyogenes infection. In this study, we observed that S. pyogenes-stimulated MDCs enhanced Th1 polarization of naïve T cells, and IL-12 most likely has an important role in this process. In addition, previous studies using the mouse skin infection model have shown that IL-12 and NK cell activation induced by IL-12 is crucial for the control of S. pyogenes infection [31 32 ]. Although S. pyogenes has traditionally been considered as an extracellular pathogen, recent evidence suggests that this bacterium can also invade epithelial cells and reside viable inside phagocytic cells [33 34 35 ]. Therefore, S. pyogenes-induced IL-12 production by DCs and the resulting NK activation and Th1 adaptive immune response may be critical in controlling the intracellular phases of streptococcal infections.
The functionality of S. pyogenes and influenza A virus-infected primary DCs was analyzed by characterizing the ability of microbe-stimulated PDCs and MDCs to direct T cell polarization. PDCs stimulated with influenza A virus or S. pyogenes induced naïve T cells to produce IFN- or IFN- and IL-10, respectively (Fig. 6)
. The production of Th2 cytokines IL-4 and IL-5 was low and not influenced by microbe stimulation (Fig. 6
, and data not shown). The T cell response induced by bacteria-stimulated PDCs has not been analyzed previously. However, a similar Th1 response has been described previously for PDCs infected with influenza A, Sendai, or Herpes simplex virus [29
36
37
38
]. S. pyogenes-stimulated MDCs also directed T cells to produce IFN-. However, in contrast to S. pyogenes-PDCs, S. pyogenes-MDCs did not induce T cells to produce IL-10 upon restimulation. This suggests that S. pyogenes-MDCs preferentially drive Th1 polarization, and S. pyogenes-PDCs may also enhance the generation of IL-10-producing, tolerogenic type 1 regulatory T cells. In our experimental setting, we observed that influenza A virus-infected MDCs were inefficient in driving T cell polarization. This most likely reflects the high susceptibility of MDCs to influenza A virus-induced cell death (Fig. 2)
. In conclusion, our results show that S. pyogenes and influenza A virus render PDCs capable of inducing T cell polarization and to secrete Th1 cytokines. Furthermore, as MDCs were found to be highly susceptible to influenza A virus-induced cell death, PDCs may play a critical role in generating adaptive immune responses against influenza A virus.
Although our results clearly demonstrate that S. pyogenes activates PDCs and MDCs, the receptor(s) responsible for these bacteria-induced responses remain elusive. One of the candidate S. pyogenes receptors in PDCs is TLR9, which recognizes unmethylated CpG-rich DNA found in bacteria and DNA viruses. However, when S. pyogenes-stimulated PDCs were treated with inhibitory CpG oligonucleotide or chloroquine, no reduction in TNF- production was detected (data not shown). These data suggest that TLR9 is not involved in S. pyogenes-induced PDC activation, possibly as a result of inaccessibility of S. pyogenes DNA to TLR9. Intracellular NOD proteins could also serve as candidate receptors for S. pyogenes. The most studied NOD receptors are NOD1 and NOD2, which recognize bacterial peptidoglycan structures and are able to activate NF-B and MAPK pathways [39
40
41
]. In a transfection model, it has been shown that Gram-positive Streptococcus pneumoniae induces NF-B activation via NOD2 [42
]. Although we observed low NOD2 mRNA expression in PDCs (data not shown), the contribution of NOD receptors in PDC signaling and especially in S. pyogenes-induced responses is currently not known. PDCs also express several scavenger Fc and lectin-type receptors on their cell surface, which may contribute to pathogen recognition, internalization, and cell activation. The CD36 scavenger receptor is expressed in a variety of cells including PDCs [36
], and CD36 engagement has been shown to activate MAPKs and cytokine production in macrophages [43
]. Recently, CD36 was shown to be important for a Gram-positive bacteria-induced cytokine response in macrophages and act in synergy or cooperation with TLRs [44
]. Further studies are, however, needed to reveal whether any of the above-mentioned candidate receptors are involved in S. pyogenes-induced PDC activation.
In the present study, we have demonstrated that human PDCs and MDCs, isolated directly from blood, show a partially overlapping and partially specific cytokine production pattern in response to virus infection or bacterial stimulation. It is most important that we show that an important human Gram-positive, bacterial pathogen S. pyogenes activates PDCs and MDCs rendering them able to stimulate T cells toward Th1 polarization. This report and our previous observations show that S. pyogenes infection leads to efficient activation of several different leukocyte types, such as monocytes, macrophages, MDCs, and PDCs [8 9 10 11 12 ]. The powerful leukocyte activation by S. pyogenes most likely results in efficient control of streptococcal infections in vivo. However, excessive activation of the immune system may also lead to direct tissue damage or to the development autoimmune symptoms, which have been shown to take place after untreated streptococcal infections [45 46 ]. In conclusion, our results demonstrate that different human pathogenic microbes can induce a greatly variable cytokine response in different human DC subtypes. Moreover, "virus-specific" PDCs may show a much greater flexibility in their microbe recognition as previously anticipated, as they are also responsive to certain bacterial pathogens.
ACKNOWLEDGEMENTS
This work was supported by the Academy of Finland (Microbes and Man Program) and the Sigrid Juselius Foundation. Mari Aaltonen, Johanna Lahtinen, and Hanna Valtonen are acknowledged for their expert technical assistance.
Received July 12, 2007; revised September 12, 2007; accepted October 5, 2007.
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  T. G. Loof, O. Goldmann, and E. Medina Immune Recognition of Streptococcus pyogenes by Dendritic Cells Infect. Immun., June 1, 2008; 76(6): 2785 - 2792. [Abstract] [Full Text] [PDF]
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