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Infection and Immunity, April 2006, p. 2462-2467, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2462-2467.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Treponema denticola Surface Protease Dentilisin Degrades Interleukin-1ß (IL-1ß), IL-6, and Tumor Necrosis Factor Alpha

Meguru Miyamoto, Kazuyuki Ishihara,* and Katsuji Okuda

Department of Microbiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan

Received 17 September 2005/ Returned for modification 11 November 2005/ Accepted 23 January 2006


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ABSTRACT
 
Dentilisin is a major surface protease and virulence factor of the bacterium Treponema denticola. In this study, we found that T. denticola reduced inflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha, in peripheral blood mononuclear cells through degradation by dentilisin.


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TEXT
 
Chronic periodontitis is an infectious disease characterized by the accumulation of inflammatory cells in extravascular gingival connective tissue, which finally causes the breakdown of periodontal tissue and tooth loss (21, 28, 35, 36, 38). Treponema denticola has been identified as a major pathogen in human periodontal disease (1, 18, 20, 25). Previous studies have revealed that a number of inflammatory cytokines are synthesized in response to periodontopathic bacteria and their products, thus inducing and maintaining an inflammatory response in the periodontium (3, 29). Inflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-{alpha}), are closely associated with the development of periodontal lesions (9, 16, 23, 30-32, 34, 40). It has been shown that T. denticola induces the production of various cytokines, including IL-1ß, IL-6, IL-8, and TNF-{alpha} from various cell types (2, 6, 15, 26, 33). On the other hand, it has also been suggested that T. denticola hydrolyzes IL-8 (2, 26). This microorganism possesses a surface protease, dentilisin, which hydrolyzes host proteins and is believed to be a major pathogen of this microorganism (12, 13, 37). Proteases from periodontopathic bacteria such as Porphyromonas gingivalis have been reported to modulate immunoresponses by hydrolyzing inflammatory mediators (3, 5). The aim of this study was to verify the ability of dentilisin to degrade inflammatory cytokines, including IL-1ß, IL-6, and TNF-{alpha}.

In this study, we used T. denticola ATCC 35405 and dentilisin-deficient mutant K1 (12). T. denticola was propagated in TYGVS medium (27) and incubated at 37°C for 4 days under anaerobic conditions as described previously (10).

In order to estimate the effect of T. denticola on the production of inflammatory cytokines, we determined the amount of cytokines produced in human peripheral blood mononuclear cells (PBMCs) after exposure to T. denticola. Peripheral blood was obtained from three healthy adult volunteers by venipuncture after informed consents were obtained. The PBMCs were separated by density gradient centrifugation using a Lymphoprep tube (Axis-Shield, Oslo, Norway) according to the manufacturer's instructions. The cells obtained were washed with phosphate-buffered saline (PBS [pH 7.4]; Nissui, Tokyo, Japan) and resuspended in RPMI 1640 medium containing 2 mM L-glutamine (Nissui) and 10% fetal bovine serum (Bio-Whittaker, Maryland). The PBMCs (5 x 108 cells/ml) were then seeded in 96-well microtiter plates at 200 µl/well, and a 20-µl T. denticola cell suspension was then added at 1 x 107 cells/well. Cells were incubated at 37°C for 24 h in humidified air containing 5% CO2. After incubation, the amounts of cytokines in the supernatants were measured with enzyme-linked immunosorbent assay (ELISA) systems (IL-6 and TNF-{alpha}; ENDOGEN, Rockford, IL; IL-1ß, R & D systems, Minneapolis, MN) according to the manufacturer's instructions. Statistical significance was determined with an analysis of variance (ANOVA) followed by the Student-Newman-Keuls test to obtain multiple comparisons of cytokine levels and cytokine mRNA expression.

Figure 1 shows the amounts of IL-1ß, IL-6, and TNF-{alpha} produced by the PBMCs after exposure to T. denticola ATCC 35405 and K1. All the targeted cytokines in the PBMC supernatants showed significantly larger amounts after exposure to K1 cells than after exposure to ATCC 35405 (P < 0.01). The inactivation of a gene sometimes induces pleiotropic effects. In T. denticola K1, this has been shown to result in the ability to organize oligomeric proteins being affected (11). To investigate proteolytic activity by agents other than dentilisin, endo-acting proline-specific oligopeptidase (22) and trypsin-like peptidase (8) activities in both T. denticola ATCC 35405 and K1 were evaluated by using synthetic substrata, Z-Gly-Pro-4-nitroanilide (Sigma-Aldrich, Milwaukee, MI) and N{alpha}-benzoyl-DL-arginin 4-nitroanilide (Sigma), respectively. The activities of both enzymes were found to be at almost the same level. These results indicated that these other peptidase activities were not affected by inactivation of dentilisin. This suggests that dentilisin is involved in the reduction of inflammatory cytokines.


Figure 1
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  		FIG. 1. Amounts of IL-1ß, IL-6, and TNF-{alpha} in supernatant PBMCs after exposure to cells of T. denticola ATCC 35405 and dentilisin-deficient mutant K1. Each assay was performed more than three times with triplicate wells, and data are expressed as means and standard deviations (error bars). *, P was <0.01 relative to wild-type T. denticola and mutant K1 at the same concentration. Statistical analysis was performed with an ANOVA followed by the Student-Newman-Keuls test to obtain multiple comparisons.

To evaluate the degradation of cytokines by dentilisin, T. denticola and each cytokine were incubated at 37°C in PBS at a ratio of 1 x 105 cells of T. denticola/pg cytokines for 12 h. The remaining IL-1ß, IL-6, and TNF-{alpha} levels (average percent ± standard deviation) after incubation with T. denticola ATCC 35405 were 0.48 ± 1.35, 69.2 ± 5.45, and 25.6 ± 0.35, respectively. The reduction rates of IL-1ß, IL-6, and TNF-{alpha} after incubation with T. denticola K1 were 10.2 ± 3.12, 125.8 ± 4.56, and 91.7 ± 2.79, respectively. These results also indicated degradation of IL-1 and TNF-{alpha} by dentilisin. IL-6, however, showed a small decrease after exposure to dentilisin relative to the other cytokines. It is possible that the fragments containing intact epitopes detected by the ELISA kit were only the remains left over after degradation by dentilisin.

In order to evaluate the effect of dentilisin on the induction of cytokines, we used real-time reverse transcription (RT)-PCR to determine the synthesis of cytokine mRNA in PBMCs. The PBMCs (5 x 108 cells/ml) were seeded in 24-well microtiter plates at 800 µl (4 x 108 cells/well) as described above and were incubated with an 80-µl cell suspension of intact T. denticola ATCC 35405 or K1 (4 x 107 cells/well) for 6 h. Total RNAs from the PBMCs were extracted by using RNeasy (QIAGEN, Valencia, CA), and cDNA was synthesized from the total RNAs with Omniscript (QIAGEN) using an oligo(dT) primer. For real-time PCR, we used primers and a TaqMan probe for IL-1ß (Hs00174097_m1), IL-6 (Hs00174131_m1), TNF-{alpha} (Hs00174128_m1), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Hs99999905_m1) obtained from Applied Biosystems (Foster City, CA). Quantitative real-time RT-PCR was performed using an Applied Biosystems Prism 7700 sequence detection system (Applied Biosystems) according to the manufacturer's instructions. Samples were normalized using GAPDH. Triplicates were performed for each experimental point. Results are shown as a fold modulation over the control (19).

The results of the RT-PCR revealed that both strains of T. denticola induced high mRNA levels of inflammatory cytokines (Fig. 2), with T. denticola ATCC 35405 inducing almost the same levels of expression of IL-1ß and IL-6 as did K1. The protein levels of both cytokines showed a significantly larger reduction after incubation with ATCC 35405 than with K1 (P < 0.01). TNF-{alpha} induction by ATCC 35405 was somewhat lower than that with K1. However, the protein level of TNF-{alpha} induced by ATCC 35405 was significantly lower than that induced by K1 (P < 0.05). This discrepancy between the transcript and protein levels of these inflammatory cytokines suggests their degradation by dentilisin.


Figure 2
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  		FIG. 2. Real-time RT-PCR of inflammatory cytokine mRNA in T. denticola-stimulated PBMCs. IL-1ß, IL-6, and TNF-{alpha} expressions were normalized with GAPDH (internal control) expression. Results are representative of three independent experiments. Error bars indicate standard deviations. Statistical analysis was performed with an ANOVA followed by the Student-Newman-Keuls test to obtain multiple comparisons.

Immunoblot analysis confirmed the hydrolysis of IL-1ß, IL-6, and TNF-{alpha} by dentilisin. The purification of dentilisin from T. denticola ATCC 35405 was performed as described previously (12). Recombinant human IL-1ß, IL-6, and TNF-{alpha} (50 ng/ml; Techne Corporation, Minneapolis, MN) were incubated with dentilisin (50, 5, and 0.5 ng/µl) at 37°C for 3 h. Reaction mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (17) and then electrically transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking, the membrane was incubated overnight with mouse anti-human IL-1ß, IL-6, or TNF-{alpha} immunoglobulin G monoclonal antibodies (Techne Corporation) diluted at 1:2,000 at room temperature. Membranes were washed with PBS containing 0.05% Tween-20 and incubated with peroxidase-conjugated goat anti-mouse immunoglobulin G antibodies (Techne Corporation) diluted at 1:3,000 for 60 min. After washing, blots were developed for visualization by using a TMB membrane peroxidase substrate (KPL, Gaithersburg, MD).

Figure 3 shows that purified dentilisin was capable of degrading the three recombinant human cytokines and that the intensity of the cytokine bands was reduced in a dose-dependent manner. The IL-6 band disappeared when IL-6 was treated with a 5-ng/µl dilution of purified dentilisin. Intact T. denticola ATCC 35405 cells also degraded these cytokines, but those of K1 did not (data not shown). These results were confirmed using polyclonal antibody against these cytokines. SDS-PAGE using silver staining also revealed no degraded fragments of IL-1 or TNF-{alpha} (Fig. 4). Weak bands of degraded IL-6 fragments were observed, however. This formation of weak bands agreed with the small decrease in only IL-6 relative to other cytokines, as shown by ELISA, after exposure to dentilisin. On the other hand, weak bands smaller than those for IL-6 and TNF-{alpha} were observed after degradation by bovine pancreatic chymotrypsin (data not shown). This suggests that dentilisin has different recognition sites than chymotrypsin. These results indicated that dentilisin was able to degrade IL-1ß, IL-6, and TNF-{alpha}. Gingipain from P. gingivalis produces biologically active cytokine fragments (24). In investigating the cytokine degradation process by dentilisin to clarify the generation of biologically active or inactive cytokine fragments, we found no fragment formation. In the results of an earlier study, no fragments were detected after the degradation of fibronectin or {alpha}1-antitrypsin (12). This protease does cleave the prolyl-phenylalanyl and proryl-leucyl bonds, however. Its substrate specificity, though, remains to be fully elucidated. It is possible that the organization of multiple-size fragments resulted from multirecognition sites, preventing the formation of fragments in SDS-PAGE, or generated fragments that were out of the range of detectable size for the SDS-PAGE gel.


Figure 3
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  		FIG. 3. Immunoblot analysis of proteolytic degradation of human IL-1ß, IL-6, and TNF-{alpha} after incubation with purified dentilisin. After 3 h of incubation, samples were subjected to SDS-PAGE and immunoblot analysis. Lane 1, human recombinant cytokine alone; lane 2, cytokine incubated with a 0.5-ng/µl dilution of dentilisin; lane 3, cytokine incubated with a 5-ng/µl dilution of dentilisin; lane 4, cytokine incubated with a 50-ng/µl dilution of dentilisin.


Figure 4
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  		FIG. 4. SDS-PAGE of proteolytic degradation of human IL-1ß, IL-6, and TNF-{alpha} after incubation with purified dentilisin. Cytokines were incubated with 0.5 ng/µl of dentilisin. After incubation, samples were subjected to SDS-PAGE. Top panel (IL-1): lane 1, IL-1ß alone; lane 2, 1 h of incubation; lane 3, 2 h of incubation; lane 4, 3 h of incubation; lane 5, 6 h of incubation; lane 6, dentilisin alone. Middle panel (IL-6): lane 1, IL-6 alone; lane 2, 1 h of incubation; lane 3, 3 h of incubation; lane 4, 6 h of incubation; lane 5, dentilisin alone. Bottom panel (TNF-{alpha}): lane 1, TNF-{alpha} alone; lane 2, 1 h of incubation; lane 3, 6 h of incubation; lane 4; 12 h of incubation; lane 5, 24 h of incubation; lane 6, dentilisin alone.

This study revealed that T. denticola ATCC 35405 reduced inflammatory cytokines produced from PBMCs. The mRNA expression of these cytokines by T. denticola in the present report agrees with that found in previous reports (2, 6, 26, 33). Asai et al. (2) have demonstrated that T. denticola decreased the exogenous IL-8 produced by human gingival epithelial cells. In this study, the results showed that dentilisin degraded IL-1ß, TNF-{alpha}, and IL-6. In our preliminary results using the lactate dehydrogenase assay, intact T. denticola cells showed less than 5% cytotoxicity to human PBMCs (data not shown). The mRNA of proinflammatory cytokines was apparently induced, suggesting that T. denticola induces proinflammatory cytokines and degrades them by dentilisin. Beausejour et al. (4) reported that T. denticola activated IL-1ß by proteolytic activity. Our results from the immunoblot analysis indicate that IL-1ß was finally digested completely. The reduction in TNF-{alpha} may be partly attributed to the degradation by dentilisin and partly to the degradation of cell surface molecules that are associated with TNF-{alpha} production. Further analysis is required to elucidate the role of this protease in the production of TNF-{alpha}.

The perturbation of the cytokine network existing in healthy gingival tissue by periodontopathogenic bacteria is thought to play a major role in initiating inflammatory periodontal disease (6). Cytokine down-regulation has been shown to be caused by the suppression of cytokine synthesis and/or cytokine degradation (39). This bimodal effect against the host immune response may be associated with the profile of lesions infected by T. denticola. Ebersole et al. (7) reported that the abscess lesions that resulted in T. denticola-challenged mice were smaller than those seen in other periodontal bacterial challenges, such as those seen with P. gingivalis or Actinobacillus actinomycetemcomintans, but that lesions resulting from T. denticola lasted longer than those resulting from exposure to other periodontopathogens. Kesavalu et al. (14) reported that neither heat-killed nor formalin-killed T. denticola was capable of inducing abscesses with characteristics similar to those of abscesses induced by viable bacterial infection. We have demonstrated that the dentilisin-deficient mutant induced significantly smaller lesions in the murine lesion model than did the wild-type strain and that the lesions induced by the dentilisin-deficient mutant were shorter-lasting than those induced by the wild type (11). It is possible that the induction of proinflammatory cytokines and their degradation by dentilisin may be associated with long-lasting infection. Further analysis is required to clarify the relationship between the abscess-forming activity and the degradation of proinflammatory cytokines in an abscess-forming model and to elucidate the degradation of cytokines in the gingival crevicular fluid of periodontitis patients. Taken together, these results indicate that T. denticola exerts a pathogenic effect on periodontal tissue via local dysregulation of inflammatory cytokines by dentilisin.


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ACKNOWLEDGMENTS
 
This study was supported by grants to Kazuyuki Ishihara (16591837) and Katsuji Okuda (14370608) from the Ministry of Education, Science, Sport, Culture and Technology (MEXT) of Japan, grant 5A01 from the Oral Health Science Center of Tokyo Dental College, and a matching-funds subsidy from the High-Tech Research Center Project for Private Universities of MEXT (2000-2005).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan. Phone: 81-043-270-3742. Fax: 81-043-270-3744. E-mail: ishihara{at}tdc.ac.jp. Back

Editor: J. N. Weiser


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Infection and Immunity, April 2006, p. 2462-2467, Vol. 74, No. 4
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.4.2462-2467.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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