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Infection and Immunity, February 2003, p. 850-856, Vol. 71, No. 2
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.2.850-856.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells

Claudia Nonnenmacher,1,2 Alexander Dalpke,1 Stefan Zimmermann,1 Lavin Flores-de-Jacoby,2 Reinier Mutters,1 and Klaus Heeg1*

Institute of Medical Microbiology and Hygiene,1 Department of Periodontology, Philipps University, Marburg, Germany2

Received 19 June 2002/ Returned for modification 4 September 2002/ Accepted 8 November 2002


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ABSTRACT
 
Although bacterial DNA (bDNA) containing unmethylated CpG motifs stimulates innate immune cells through Toll-like receptor 9 (TLR-9), its precise role in the pathophysiology of diseases is still equivocal. Here we examined the immunostimulatory effects of DNA extracted from periodontopathogenic bacteria. A major role in the etiology of periodontal diseases has been attributed to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Peptostreptococcus micros. We therefore isolated DNA from these bacteria and stimulated murine macrophages and human gingival fibroblasts (HGF) in vitro. Furthermore, HEK 293 cells transfected with human TLR-9 were also stimulated with these DNA preparations. We observed that DNA from these pathogens stimulates macrophages and gingival fibroblasts to produce tumor necrosis factor alpha and interleukin-6 in a dose-dependent manner. Methylation of the CpG motifs abolished the observed effects. Activation of HEK 293 cells expressing TLR-9 which were responsive to bDNA but not to lipopolysaccharide confirmed that immunostimulation was achieved by bDNA. In addition, the examined bDNA differed in the ability to stimulate murine macrophages, HGF, and TLR-9-transfected cells. DNA from A. actinomycetemcomitans elicited a potent cytokine response, while DNA from P. gingivalis and P. micros showed lower immunostimulatory activity. Taken together, the results strongly suggest that DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros possesses immunostimulatory properties in regard to cytokine secretion by macrophages and fibroblasts. These stimulatory effects are due to unmethylated CpG motifs within bDNA and differ between distinct periodontopathogenic bacteria strains. Hence, immunostimulation by DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros could contribute to the pathogenesis of periodontal diseases.


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INTRODUCTION
 
Periodontitis is a chronic infectious disease caused by periodontal bacteria affecting the supporting structures of teeth (6, 41), which can lead to a loss of alveolar bone and teeth. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Peptostreptococcus micros are considered major pathogenic species in destructive periodontal disease (8, 21). Many studies have suggested that lipopolysaccharide (LPS) released from the cell walls of gram-negative bacteria or lipoteichoic acid from gram-positive bacteria is associated with the development and progression of periodontal disease (14, 30). LPS and lipoteichoic acid activate cells of the innate immune system and thus contribute to inflammatory processes. Toll-like receptors (TLR) are critically involved in recognition of these bacterial compounds. Recent experiments have identified bacterial DNA (bDNA) as another microbial stimulus which can be sensed by cells of the innate immune system such as macrophages and dendritic cells (16, 19, 23), leading to activation and eventually secretion of several proinflammatory cytokines.

The immunostimulatory properties displayed by bDNA are due to the presence of hexameric DNA motifs containing a central unmethylated CG dinucleotide (CpG). In contrast to bDNA, the frequency of unmethylated CpG dinucleotides is suppressed in vertebrate DNA (13). This structural difference distinguishes bacterial from host DNA and is used by the immune system to sense infectious danger (19, 24). The stimulatory effects of bDNA can be mimicked by short synthetic oligodeoxynucleotides (ODN) that contain one or more of the unmethylated CpG motifs (16). The mechanisms of action of CpG DNA at the molecular and cellular levels are only partly understood. Although cellular uptake seems to be a prerequisite for the action of CpG DNA, it is unclear whether a special uptake receptor exists (7, 17). TLR-9 has recently been identified to be critically involved in initiation of cellular activation by CpG DNA (12). Thus, the signal events triggered by CpG DNA merge to signal cascades that are shared with other TLR. Nevertheless, the biological valence is different: CpG DNA induces a stronger interleukin-12 (IL-12) response than other TLR ligands (4). Recent evidence suggests that murine and human TLR-9 (hTLR-9) receptors vary in their preference for certain CpG DNA motifs (2). Accordingly, murine TLR-9 recognizes the classical CpG motif (5'-RRCGYY-3') while hTLR-9 recognizes preferentially repetitive CpG dinucleotides (10, 12, 39). However, both TLR are triggered by bDNA (2, 12).

Evidence that bDNA may activate cells of the immune system was first provided by Tokunaga et al. (38), who reported that the tumoricidal effects of Mycobacterium bovis were confined to mycobacterial DNA. Since then several studies have demonstrated that DNA from gram-positive and gram-negative bacteria is a potent stimulus leading to the generation of cytokines (5, 9, 19, 32-34), influencing antigen presentation and thus shaping the adaptive immune response. Based on these observations we aimed to investigate whether DNA extracted from A. actinomycetemcomitans, P. gingivalis, and P. micros has the ability to activate macrophages and gingival fibroblasts. We found that DNA preparations from these bacteria were efficient in triggering production of proinflammatory cytokines in a CpG-specific mode in murine as well as in human cells and that these responses involved the TLR-9 signaling pathway.


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MATERIALS AND METHODS
 
ODN. Phosphorothioate-modified ODN were purchased from TibMolBiol (Berlin, Germany). The sequences used in this study were those of CpG ODN 1668 (5'-TCC ATG ACG TTC CTG ATG CT-3'), CpG ODN 2006 (5'-TCG TCG TTT TGT CGT TTT GTC GTT-3'), and GpC ODN 2006K (5'-TGC TGC TTT TGT GCT TTT GTG CTT-3').

Preparation of bDNA. DNA was prepared from A. actinomycetemcomitans (Medical Culture Collection Marburg [MCCM] 200), P. gingivalis (MCCM 3199), P. micros (MCCM 3096), Streptococcus viridans (MCCM 3223), Neisseria polysaccharea (MCCM 1714), and Escherichia coli (XL1 Blue; Stratagene, Amsterdam, The Netherlands), which served as a positive control. Calf thymus DNA purchased from Sigma (Deisenhofen, Germany) was used as a eukaryotic negative control. Columbia agar with 5% sheep blood was used for the cultivation of A. actinomycetemcomitans, a microaerophilic microorganism, and Schaedler agar plates were used for recovery of P. gingivalis and P. micros, which are obligatory anaerobic gram-negative rods and gram-positive cocci, respectively. Aliquots (1 ml) of bacteria were frozen at -70°C and periodically thawed for use. bDNA was prepared by suspending the bacteria in 50 mM Tris-HCl-5 mM EDTA, pH 8.0. Proteinase K (0.2 mg/ml) and sodium dodecyl sulfate (0.5%) were added, and the mixture was incubated at 50°C overnight. DNA was purified from the lysate by repeated extraction with phenol-chloroform-isoamyl alcohol, precipitated with sodium acetate and ethanol, and then dissolved and stored at -20°C in aliquots. The DNA content was measured with a photometer.

Methylation of bDNA. bDNA preparations were treated with SssI CpG methylase (New England Biolabs, Boston, Mass.) for 18 h at 37°C at the concentration of 2 U/µg of DNA as previously described (1).

Reverse transcription (RT)-PCR. RNA from human gingival fibroblasts (HGF), HEK 293 cells, and HEK 293 cells stably transfected with hTLR-9 (106 cells) was prepared by using a High Pure RNA isolation kit (Roche Molecular Biochemicals), which included treatment with RNase-free DNase I. cDNA was obtained from 5 µg of RNA by using 200 U of Superscript II reverse transcriptase (Life Technologies, Inc.) and 0.5 µg of oligo(dT)12-18 primer (Life Technologies, Inc.). PCR amplification of cDNA was performed with Taq polymerase by using primer pairs specific for hTLR-4 (5'-CCA GAG CCG CTG GTG TAT CT and 5'-AGA AGG CGG TAC AGC TCC AC), hTLR-9 (5'-CCA CCC TGG AAG AGC TAA ACC and 5'-GCC GTC CAT GAA TAG GAA GC), and human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (5'-ACG GAT TTG GTC GTA TTG GGC and 5'-TTG ACG GTG CCA TGG AAT TTG). Settings were initially 95°C for 30 s and then 33 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s. Contamination with genomic DNA was excluded by using RNA preparations directly in the PCR (no reverse transcriptase control). The PCR-amplified products were visualized by ethidium bromide staining on 2.4% agarose gels.

Cell preparation and cell culture. Murine macrophage cell line RAW 264.7 was cultured in Click/RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 5% (vol/vol) fetal calf serum (Sigma, Steinheim, Germany), 50 µM 2-mercaptoethanol, 2 mM L-glutamine, and antibiotics (penicillin G, 100 IU/ml; streptomycin, 100 µg/ml). The cells were cultured at 37°C in a 5% CO2 incubator. Cells (1.5 x 105 cells/well) were treated with different concentrations of DNA from A. actinomycetemcomitans, P. gingivalis, P. micros, and E. coli. In some experiments, the bDNA was pretreated with CpG methylase before stimulation.

HGF were isolated by a modification of the method of Ragnarsson et al. (26). Briefly, periodontally healthy third molars were obtained and immediately placed in ice-cold phosphate-buffered saline (PBS). The crown was dipped in a 5.25% sodium hypochlorite solution for 2 min to avoid microbial and epithelial contamination and then washed twice in PBS. The teeth were next incubated in 50 ml of a collagenase-trypsin solution (0.125% trypsin and 0.1% collagenase; Sigma, Steinheim, Germany). Following a 1-h incubation at 37°C, the teeth were removed and the tube was centrifuged at 1,000 x g for 4 min. The cell pellet was resuspended and washed twice in Click/RPMI medium and plated into culture flasks. The flasks were incubated in a humid environment at 37°C. After confluency was obtained, fibroblasts were dissociated from the outgrowth by trypsinization and harvested by centrifugation. Cells from passage 4 or passage 5 were used in the present investigation.

Cell transfection. HEK 293 cells were seeded at 105 cells per well in a 12-well plate before transfection. Cells were transfected using Superfect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions with 1.5 µg of an hTLR-9 expression construct fused to green fluorescent protein (a kind gift of T. Espevik, Boston, Mass.). Cells were grown for 24 h and overlaid by soft agar containing G418 (0.8 mg/ml; Gibco-BRL). G418-resistant clones were picked, expanded, and tested for IL-8 production. The stably transfected cells were stimulated with different concentrations of bDNA for 22 h, and human IL-8 production was monitored by enzyme-linked immunosorbent assay (ELISA).

Mice. Mice of the strain C3H/HeJ were purchased from Charles River (Sulzfeld, Germany). To obtain peritoneal macrophages, mice received intraperitoneal injections with 1 ml of 4% thioglycolate. Four days later, peritoneal exudate cells were isolated by peritoneal lavage with ice-cold PBS. Cells (1.5 x 105 cells/well) were stimulated in 96-well flat-bottom tissue culture plates with different concentrations of bDNA or LPS. The 22-h culture supernatant was analyzed for tumor necrosis factor alpha (TNF-{alpha}) and IL-6 content by an ELISA.

Cytokine measurement. Cytokine (TNF-{alpha}, IL-6, and IL-8) levels were determined using commercially available ELISA kits (OptEIA; Becton Dickinson, Heidelberg, Germany). The assays were performed according to the manufacturer's protocol, and each value shown represents the mean of duplicate values.

DNA sequence analysis. A. actinomycetemcomitans, P. gingivalis, E. coli, and human genomic sequences were obtained from the GenBank sequence database, while that of the P. micros genome has not been available yet. The Gene Runner software was used to identify the presence and frequency of CpG motifs. The expected frequency of motifs was calculated by the method described by Campbell et al. (3).


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RESULTS
 
Frequency of CpG motifs in the DNA from periodontopathogenic bacteria. In order to determine if periodontopathogenic DNA contains motifs that can elicit immune responses, we searched genomic DNA from A. actinomycetemcomitans, P. gingivalis, and E. coli genes for CG content and the frequency of CpG-containing motifs (RRCGYY). The observed frequencies of CpG dinucleotides are displayed in Table 1.


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  		TABLE 1. Frequency of CG dinucleotides and CpG motifs in bDNA

According to Karlin et al. (15), the genome signature profiles consist of the array {rho}XY = fXY/fXfY where fX denotes the frequency of the mononucleotide x and fXY denotes the frequency of dinucleotide XY. Values for {rho}XY ranging from >0.78 to <1.23 are considered normal, while underrepresentation is indicated by a {rho} of <=0.78 and overrepresentation is indicated by a {rho} of >=1.23. CG is overrepresented in A. actinomycetemcomitans, whereas in P. gingivalis and E. coli CG is in the normal range. In contrast, the human genome shows a severe underrepresentation of CG dinucleotides (f = 0.24). The relative frequency of RRCGYY is overrepresented in both E. coli and A. actinomycetemcomitans but is in the normal range in P. gingivalis.

DNA from periodontopathogenic bacteria activates macrophages to produce IL-6 and TNF-{alpha}. To analyze whether DNA from periodontopathogenic bacteria is immunogenic, various batches of bDNA from gram-negative and gram-positive sources were prepared. The purity of the nucleic acid samples was measured by means of a spectrophotometer. The ratio A260/A280, shown in Table 2, represents the purity of the nucleic acids obtained by the DNA preparation method used, which ranges between 1.80 and 2.0 for pure samples.


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  		TABLE 2. Measurement of nucleic acid content of bDNA

Next, murine macrophage cell line RAW 264.7 was treated with different concentrations of DNA extracted from A. actinomycetemcomitans, P. gingivalis, and P. micros. The supernatants were removed after 22 h, and the TNF-{alpha} and IL-6 contents were measured. As shown in Fig. 1, the DNA from both gram-negative and gram-positive bacteria was capable of inducing secretion of TNF-{alpha} and IL-6, respectively. Interestingly, the bDNAs differed slightly in their ability to stimulate murine macrophages. A. actinomycetemcomitans DNA elicited a potent cytokine response, while P. gingivalis and P. micros DNA showed a lower stimulatory effect. Furthermore, bDNA stimulated the release of cytokines in a dose-dependent manner, with high stimulatory effects at a concentration of 30 µg/ml.



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  		FIG. 1. Induction of TNF-{alpha} and IL-6 by A. actinomycetemcomitans, P. gingivalis, and P. micros DNA. Murine macrophages (1.5 x 105 cells/well) were incubated with different concentrations of bDNA. The TNF-{alpha} and IL-6 levels in the supernatant were measured after 22 h by ELISA (means ± standard deviations [error bars]). (A) Titrated amounts of DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros were used, and TNF-{alpha} was measured. E. coli DNA and CpG ODN 1668 (0.3 µM) were used as controls. (B) The same experiment as in panel A was performed, but measuring for IL-6 production. The data shown represent one of three independent experiments.

Immunostimulation by bDNA from periodontopathogenic bacteria is sensitive to CpG methylation. It has been shown that the ability of bDNA to induce cytokine synthesis is associated with the presence of unmethylated CpG motifs (9, 19, 32, 43). We therefore assessed whether the periodontopathogenic DNA activity could be blocked by methylation of the bDNA. Unmethylated DNA from periodontopathogenic bacteria significantly induced TNF-{alpha} production; in contrast, methylation of DNA markedly diminished TNF-{alpha} synthesis (Fig. 2). Hence, these results strongly suggest that the stimulatory activity of bDNA is due to the presence of unmethylated CpG motifs. In parallel, reduction of the immunostimulative capacity through methylation excludes significant endotoxin contaminations, because endotoxin-induced cytokine secretion is not altered by this treatment.



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  		FIG. 2. DNA methylation inhibits cytokine secretion induced by bDNA. DNA from A. actinomycetemcomitans, P. gingivalis, P. micros, and E. coli was treated with 2 U of CpG SssI methylase/µg of DNA and S-adenosylmethionine for 18 h at 37°C. Untreated or methylated bDNA was incubated with 1.5 x 105 RAW 264.7 cells/well at a concentration of 3 µg/ml for 22 h. TNF-{alpha} in the supernatant was measured by ELISA (means ± standard deviations [error bars]). Cytokine levels were tested for differences between untreated and methylated bDNA preparations by t test.

Murine macrophage activation by bDNA is not due to LPS contamination. To further ascertain that the observed immunostimulation is due to bDNA and not an effect of contaminating LPS in the preparation, we resorted to C3H/HeJ mice known to be nonresponsive to LPS (5, 25). Peritoneal macrophages from these C3H/HeJ mice were stimulated with different concentrations of LPS and bDNA. We found that cells from C3H/HeJ mice showed a strong TNF-{alpha} and IL-6 response following challenge with A. actinomycetemcomitans and P. gingivalis DNA, which was not seen with LPS (Fig. 3). This observation suggests that bDNA from periodontopathogenic bacteria containing specific CpG motifs triggers cytokine release in macrophages. In addition, the results demonstrate that immunostimulation by bDNA is not restricted to a macrophage cell line but also occurs in primary macrophages.



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  		FIG. 3. Peritoneal macrophages from LPS-resistant C3H/HeJ mice were stimulated with titrated amounts of DNA from A. actinomycetemcomitans, P. gingivalis, and E. coli as well as with LPS (300, 100, 30, 10, and 3 ng/ml). Levels of TNF-{alpha} (A) and IL-6 (B) in culture supernatants were determined by ELISA (means ± standard deviations [error bars]). The data are representative of three different experiments.

Immunostimulation by periodontopathogenic DNA is mediated through TLR-9. HEK 293 cells were stably transfected with hTLR-9 and stimulated with CpG ODN, LPS, and bDNA. hTLR-9-transfected HEK 293 cells stimulated with CpG ODN showed an increase in IL-8 production, while no stimulation with the control GpC ODN was observed. Calf thymus DNA as a model for eukaryotic DNA also did not elicit cytokine production. The response of the cells was CpG DNA specific, because the cells did not respond to LPS (Fig. 4A). We next tested the effect of A. actinomycetemcomitans, P. gingivalis, P. micros, and E. coli DNA on the stably transfected cells. Stimulation with bDNA elicited IL-8 production in a dose-dependent manner. Moreover, the potency of A. actinomycetemcomitans and E. coli DNA to induce IL-8 was slightly higher than that of P. gingivalis and P. micros DNA, as seen in Fig. 4B. However, further data with DNA preparations from bacteria which are not directly involved in periodontitis, such as S. viridans or N. polysaccharea, also showed immunostimulation, albeit with slightly diminished cytokine secretion (data not shown).



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  		FIG. 4. HEK 293 cells (1.5 x 105 cells/well) stably transfected with hTLR-9 were stimulated with LPS (100 ng/ml), 3 µM CpG ODN, 3 µM GpC ODN, or calf thymus DNA (100 µg/ml) (A) or different concentrations of bDNA (B) as indicated. IL-8 production was measured by ELISA after 22 h (means ± standard deviations [error bars]), and results were tested for differences relative to nonstimulated cells by t test. Results are representative of three independent experiments.

TLR-9 expression in HGF. Expression of hTLR-4, hTLR-9, and GAPDH mRNA was analyzed in HGF by RT-PCR. HEK 293 cells, which do not possess TLR, and HEK 293 cells transfected with hTLR-9 were used as controls (Fig. 5). HGF as well as the hTLR-9-transfected HEK 293 cells expressed TLR-9 mRNA. Moreover, HGF also showed a low abundance of TLR-4 mRNA transcripts. Thus, it was reasonable to test whether HGF can be stimulated by bDNA.



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  		FIG. 5. Primary HGF express TLR-9 mRNA. RT-PCR for TLR-9, TLR-4, and GAPDH was performed on cDNA from primary HGF, HEK 293, and HEK 293 cells stably transfected with hTLR-9. Genomic DNA contamination was excluded by testing RNA without reverse transcription (-). Colors are inverted.

DNA from periodontopathogenic bacteria stimulates IL-6 production in primary HGF. In order to address whether bDNA from periodontopathogenic bacteria could play a role in the pathophysiology of human periodontitis, we examined the bDNA effects on HGF. While macrophages, natural killer cells, and B cells have already been shown to release cytokines after stimulation with bDNA (1, 5, 9, 32, 33, 35), another study (36) recently showed that P. gingivalis DNA was able to induce IL-6 release by HGF. We therefore investigated the ability of bDNA to induce IL-6 production in HGF. HGF were challenged with different concentrations of DNA from periodontopathogenic bacteria, and the culture supernatant was measured for IL-6 22 h after treatment. The data in Fig. 6 show that bDNA induced IL-6 in HGF which was dose dependent, similar to what was observed with murine macrophages. Thus, bDNA from A. actinomycetemcomitans, P. gingivalis, and P. micros potently stimulates production of IL-6 in gingival fibroblasts.



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  		FIG. 6. DNA from periodontopathogenic bacteria stimulates IL-6 production by HGF. Cells (2 x 105 HGF/well) were incubated in the presence of A. actinomycetemcomitans, P. gingivalis, and P. micros DNA for 22 h. IL-6 secretion in the supernatants was then determined by ELISA (left). Control stimulations were done with LPS (0.1 µg/ml) and CpG ODN 2006 (0.3 µM) (right). Results at right are means + standard deviations [error bars]. Results in both panels are representative of three independent experiments.


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DISCUSSION
 
In this study we show that DNA extracted from the periodontopathogenic bacteria A. actinomycetemcomitans, P. gingivalis, and P. micros is a potent inducer of proinflammatory cytokines not only in murine macrophages but also in HGF. These effects are mediated by TLR-9. We here show that bDNA from odontopathogenic bacteria have the potential to stimulate cells by their DNA fractions. The immunostimulatory capacity of bDNA was dependent on unmethylated CpG motifs. Moreover, LPS-nonresponsive primary macrophages responded to the bDNA preparations, indicating that bDNA might play an important role during induction and maintenance of the pathophysiological processes in periodontal disease.

Whole periodontopathogenic bacteria as well as bacterial cell wall components including LPS and many outer membrane molecules are known to invoke inflammatory and immune responses as they interact with host cells. A role of LPS in triggering the destruction of periodontal tissues, including alveolar bone, the gingiva, and periodontal ligament, in periodontal diseases is highly probable (20, 22, 27, 30, 31, 40). LPS of P. gingivalis and A. actinomycetemcomitans stimulates the differentiation and activity of osteoclastic cells by inducing inflammatory cytokines and factors such as IL-1, TNF-{alpha}, and prostaglandin E2, which finally results in bone resorption (29, 42). However, gram-positive bacteria, which do not contain LPS, also have been implicated in the pathophysiology of periodontal diseases (37). On the other hand, several groups have reported that bDNA acts on the immune system in a manner similar to LPS (for a review, see reference 11) and that DNA from E. coli, Micrococcus lysodeikticus, and M. bovis strain BCG can induce the synthesis of an array of cytokines by immune cells (9, 38). Accordingly, Takeshita et al. (36) have shown that DNA extracted from P. gingivalis stimulated IL-6 secretion in a dose-dependent manner in HGF. We now show here that DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros is also immunostimulatory and possesses cytokine-inducing effects on murine macrophages and human fibroblasts. Moreover, we were able to show that TLR-9 which recognizes CpG DNA (12) is indeed involved in immunostimulation through the respective bDNAs. Hence, bDNA represents a microbial component which can trigger the pathogenesis of periodontal disease. Nevertheless, at present a comparison of immunostimulation from pathogenic as well as nonpathogenic bacteria is still not possible. Examinations of bigger panels of different bacteria are necessary and are currently the subject of ongoing work. Besides possible differences in immunostimulatory capacities of bDNA and differences in other microbial components between pathogenic and nonpathogenic bacteria, the composition and the cell number of different bacteria surely also determine overall immunostimulation.

The stimulation of murine immune cells by bDNA has been reported to be due to the presence of unmethylated CpG dinucleotides flanked by two 5' purines and two 3' pyrimidines (CpG motifs) (16). Microbial DNA differs from vertebrate DNA because of its higher frequency of CpG motifs, which are unmethylated, in contrast to the higher degree of methylation found in vertebrate DNA. Furthermore, the content of CG and CpG motifs may also play an important role in eliciting immune responses. In our study, A. actinomycetemcomitans DNA, which has a high CG content, elicited a higher immunostimulatory response in murine macrophages and in HGF than did P. gingivalis DNA, whose response was in a normal range.

Methylation of bDNA has been shown to inhibit immunomodulatory activities (14, 28, 32). Therefore, we tested the immunomodulatory activity of periodontopathogenic DNA after treatment with CpG methylase, observing that TNF-{alpha} secretion was abolished when the DNA was methylated. In addition, the data obtained with CpG methylase rule out the contribution of a significant LPS contamination to cytokine release by bDNA. We further ascertained this conclusion by analyzing the responses triggered by bDNA in macrophages from LPS-nonresponsive C3H/HeJ mice. These results are also consistent with the findings of Sparwasser et al. (32, 33), which have documented that DNA extracted from E. coli was able to induce TNF-{alpha} secretion. Furthermore, it is now definite that the nonresponsiveness to LPS in the C3H/HeJ mouse is due to a defect of TLR-4 (25), a molecule which is clearly not involved in bDNA responses (12).

Interestingly, multiple reports have stressed that the DNA sequence requirements for immunostimulatory oligonucleotides are different in humans and mice (10, 39). A recent report has provided the first evidence that structural differences between hTLR-9 and murine TLR-9 are responsible for this unquestionable species-specific recognition of immunostimulatory DNA (2). Although the tight DNA motif requirements manifest with short synthetic oligonucleotides (17), bDNA seems to be recognized by murine and by human cells with equal efficacies (4, 9, 18). Apparently, the large size of bDNA ascertains that distinct species-specific motifs are contained in bDNA, and thus recognition by murine as well as by human cells can take place. This, however, does not exclude the possibility that during evolution, certain bacterial strains might have suppressed those specific DNA sequences which are preferentially recognized by their host's innate immune system. Therefore, we analyzed the recognition of bDNA dependent on TLR-9 of different species in 293 cells transfected with hTLR-9. The finding that immunostimulation through DNA from periodontopathogenic bacteria is mediated by TLR-9 corroborates the data presented by Bauer et al. (2), who reported that expression of hTLR-9 is correlated with CpG DNA responsiveness in primary human cells. Furthermore, based on the data presented here, it is tempting to speculate that different bacterial species may present different pathophysiological potentials. In this context it has also been reported that inhibitory sequences exist, and thus the overall stimulation through bDNA possibly is composed of positive as well as negative regulatory sequence motifs (18).

In summary, our results demonstrate that DNA from periodontopathogenic bacteria can stimulate release of inflammatory cytokines such as TNF-{alpha} and IL-6 and that the bDNA signals through TLR-9. In addition, bDNA is sensed by the immune system in the presence of unmethylated CpG sequences. Therefore, it is conceivable that bDNA may participate in microbial pathogenesis of periodontal disease by targeting the innate arm of the immune system and by triggering the release of inflammatory cytokines. Furthermore, the observed differences in immunostimulation by different bacterial strains may reflect different pathogenic potentials.


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ACKNOWLEDGMENTS
 
We thank S. Opper, N. Schelberg, and C. Trier for technical assistance. In addition we thank C. Hermann (University of Konstanz) for help with endotoxin determinations.

This work was supported by the Deutsche Forschungsgemeinschaft (HE 1452/2-1) and the Commission of the European Communities, specific RTD program Quality of Life and Management of Living Resources, QLK2-2000-336, HOSPATH.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Hygiene, Philipps University, Pilgrimstein 2, D-35037 Marburg, Germany. Phone: 49 6421 286-6453. Fax: 49 6421 286-6420. E-mail: heeg{at}post.med.uni-marburg.de. Back

Editor: J. D. Clements


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Infection and Immunity, February 2003, p. 850-856, Vol. 71, No. 2
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.2.850-856.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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