The Effect of Treponema denticola immunoinhibitory protein on cytokine expression in T cells

Lee, Sang-Yup,Shon, Won-Jun,Lee, WooCheol,Baek, Seung-Ho,Bae, Kwang-Shik,Lim, SungSam 大韓齒科保存學會 2004 Restorative Dentistry & Endodontics Vol.29 No.5

Immunoinhibitory protein extracted from sonicated Treponema denticola have been shown to suppress cell cycle progression of human lymphocytes. To study in detail about the effect of this microorganism on the function of lymphocytes. we investigated the levels of Interleukin-2 (IL-2) and Interleukin-4 (IL-4) production by T lymphocytes before and after the addition of 12.5 ㎍/ml T. denticola sonicated extracts. In this study. levels of IL-2 and IL-4 produced from T cells pretreated with sonicated extracts were evaluated by using the quantitative sandwich enzyme immunoassay technique. In response to phytohemagglutinin (PHA) stimulation. T cell produced increased levels of IL-2 and IL-4. However. the expressions of both cytokines were significantly inhibited when PHA activated-T cells were pre-exposed to sonicated T. denticola extracts (p < 0.05). These findings suggest that the T. denticola sonicated extracts induced-immunosuppression in Th1 and Th2 cell functions could be a part of the pathogenic mechanism of the endodontic failure associated with this microorganism. 감염근관내 spirochetes의 존재 유무에 대한 논란이 많았으나 최근 PCR을 사용한 세균검출 실험에서 Treponema denticola 균주가 감염근관의 50%이상의 경우에서 발견됨에 따라 이 세균이 치수 및 치근단 질환의 병인과정에 관여하는 지에 대한 관심이 높아지고 있다. 이와 관련하여 Shenker 등이 T. denticola의 sonicated extract에서 순수분리된 면역억제 단백질 (immunoinhibitory protein)이 T 임파구의 proliferation을 방해함을 보고한 바 있다. 하지만 이 세균성 단백질이 T 임파구의 기능에 어떤 영향을 미치는 지에 대한 연구는 부족한 실정이다. 따라서 T. denticola의 면역억제 단백질이 처리되기 전과 후의 T 세포에서 분비되는 cytokine Interleukine-2와 Interleukine-4의 발현 정도를 비교하여 그 작용기전을 밝히는 것이 본 연구의 목적이다. Treponema denticola LL2513를 혐기성 상태에서 TYGVS 배지에 배양한 다음 PBS 세척과 lyophilize 과정을 거친 후 sonication을 시행한다. 이 과정을 거쳐 추출된 상층액이 T. denticola의 면역억제 단백질인 sonicated extract이다. 실험을 위해 건강한 혈액 공여자로부터 T 세포를 Buoyant density 방법으로 추출해 낸 다음 24-well plate에 100만개의 세포를 주입한 다음 Group 1에는 2% FBS의 medium만으로 배양하고, Group 2에는 100 μl의 PHA로만 증식 자극을 하였고, Group 3에는 PHA처리 전 sonicated extract로 T세포를 자극하였다. 72시간 동안 배양한 다음 상층액을 추출하여 ELISA assay를 사용하여 IL-2와 IL-4의 발현정도를 측정하였다. PHA로 자극받은 Group 2에서는 IL-2와 IL-4가 대조군인 Group 1에서보다 높은 수준으로 발현되었다. 하지만 12.5 ㎍/ml의 T. denticola sonicated 추출물로 전처리한 Group 3에서는 IL-2와 IL-4의 수준이 유의성있게 억제되어 발현되었다 (p < 0.05). 이러한 결과를 통하여 T. denticola에서 추출된 면역억제 단백질이 Th1과 Th2의 cytokine 분비 기능을 억제하는 것으로 확인 되었으며 이 기전이 감염 근관에서 발견되는 T. denticola의 치수 및 치근단 질환에 대한 병인기전과 관련이 있는 것으로 사료된다.

Treponema denticola Suppresses Expression of Human ${\beta}$-Defensin-2 in Gingival Epithelial Cells through Inhibition of TNF${\alpha}$ Production and TLR2 Activation

Shin, Ji-Eun,Choi, Young-Nim Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.29 No.4

We previously reported that Treponema denticola, a periodontal pathogen, suppressed the expression of human ${\beta}$-defensins (HBDs) and IL-8 in human gingival epithelial cells. To clarify the receptor(s) involved in the suppression of HBD-2, immortalized gingival epithelial (HOK-16B) cells were infected with live or heat-killed T. denticola for 24 h, and the expression of HBD-2 was examined by real-time RT-PCR. Live T. denticola, but not heat-killed bacteria, suppressed the expression of HBD-2 about 40%. Time courses of suppression revealed that T. denticola suppressed HBD-2 expression only at late time points, which was accompanied with the suppression of TNF${\alpha}$ production. Neutralization of TNF${\alpha}$ with an antibody abrogated the suppressive effect of T. denticola on HBD-2. Accordingly, heat-killed T. denticola did not suppress TNF${\alpha}$ production. Knock-down of toll-like receptor (TLR) 2 via RNA interference reversed the suppressive effect of T. denticola on the expression of HBD-3, but not on the production of TNF${\alpha}$. Collectively, T. denticola suppresses the expression of HBD-2 in gingival epithelial cells by inhibiting the TLR2 axis and TNF${\alpha}$ production, which may contribute to the pathogenesis of periodontitis by T. denticola.

Treponema denticola Suppresses Expression of Human β-Defensin-2 in Gingival Epithelial Cells through Inhibition of TNFα Production and TLR2 Activation

Ji Eun Shin,Youngnim Choi 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.4

We previously reported that Treponema denticola, a periodontal pathogen, suppressed the expression of human β-defensins (HBDs) and IL-8 in human gingival epithelial cells. To clarify the receptor(s) involved in the suppression of HBD-2, immortalized gingival epithelial (HOK-16B) cells were infected with live or heat-killed T. denticola for 24 h,and the expression of HBD-2 was examined by real-time RT-PCR. Live T. denticola, but not heat-killed bacteria,suppressed the expression of HBD-2 about 40%. Time courses of suppression revealed that T. denticola suppressed HBD-2 expression only at late time points, which was accompanied with the suppression of TNFα production. Neutralization of TNFα with an antibody abrogated the suppressive effect of T. denticola on HBD-2. Accordingly,heat-killed T. denticola did not suppress TNFα production. Knock-down of toll-like receptor (TLR) 2 via RNA interference reversed the suppressive effect of T. denticola on the expression of HBD-3, but not on the production of TNFα. Collectively, T. denticola suppresses the expression of HBD-2 in gingival epithelial cells by inhibiting the TLR2axis and TNFα production, which may contribute to the pathogenesis of periodontitis by T. denticola.

A periodontal pathogen <i>Treponema denticola</i> hijacks the <i>Fusobacterium nucleatum</i>‐driven host response

Shin, Ji Eun,Baek, Keum Jin,Choi, Yun Sik,Choi, Youngnim Nature Publishing Group 2013 Immunology and cell biology Vol.91 No.8

<P>Periodontitis is a polymicrobial disease that arises from the dysbiosis of the plaque biofilm. To study polymicrobial interactions with gingival epithelial cells, the oral commensal <I>Fusobacterium nucleatum</I> and the periodontal pathogen <I>Treponema denticola</I> were chosen due to their opposing effects on the expression of human beta‐defensins (HBDs) and interleukin (IL)‐8 in gingival epithelial cells. Immortalized gingival epithelial HOK‐16B cells were infected with either <I>F. nucleatum</I> or <I>T. denticola</I> alone or together, and the expression of HBDs and IL‐8 was investigated. Coinfection with <I>F. nucleatum</I> and <I>T. denticola</I> neutralized the stimulatory and suppressive effects on the expression of HBD‐2 and ‐3, but the suppressive effect of <I>T. denticola</I> on IL‐8 expression remained. In CHO/CD14/TLR2 reporter cells, <I>T. denticola</I> attenuated <I>F. nucleatum</I>‐induced activation of TLR2, a receptor that mediates HBD induction. Although <I>F. nucleatum</I> facilitated the invasion of <I>T. denticola</I> into host cells, <I>T. denticola</I> interfered with the fusion of internalized <I>F. nucleatum</I> with lysosomes, which may avert TLR9‐dependent IL‐8 induction. Furthermore, <I>T. denticola</I> suppressed the <I>F. nucleatum</I>‐stimulated accumulation of intracellular reactive oxygen species (ROS), a group of essential signaling molecules for the TLR2 and TLR9 pathways. The elimination of ROS using <I>N</I>‐acetyl cysteine completely blocked the inductions of HBD‐3 and IL‐8 and significantly reduced HBD‐2 induction by <I>F. nucleatum</I>, confirming the importance of ROS in the host response. In sum, <I>T. denticola</I> incapacitates the <I>F. nucleatum</I>‐induced expression of HBDs and IL‐8 in gingival epithelial cells by interrupting endo‐lysosomal maturation and ROS‐dependent TLR activation. These results may provide new insights into polymicrobial interactions in the gingival sulcus.</P>

Complete genome sequence of Prevotella denticola KCOM 1525 isolated from human periapical abscess

임윤경,박순낭,박세호,신자영,노한성,국중기,Lim, Yun Kyong,Park, Soon-Nang,Park, Se Ho,Shin, Ja Young,Roh, Hanseong,Kook, Joong-Ki The Microbiological Society of Korea 2019 미생물학회지 Vol.55 No.2

Prevotella denticola는 그람 음성, 절대 혐기성, 비운동성이면서 아포를 형성하지 않는 막대 모양의 세균이다. P. denticola는 치주질환과 관련이 있으며, 치주질환의 위험 인자 중 하나이다. P. denticola KCOM 1525 (= ChDC B698) 균주가 사람 치근단 농양에서 분리되었다. P. denticola KCOM 1525 균주의 유전체 염기서열을 완전 해독하여 보고한다. Prevotella denticola is Gram-negative, obligately anaerobic, non-motile, non-spore forming, and rod-shaped bacterium. P. denticola is associated with periodontal disease and is a risk indicator of periodontal disease. P. denticola KCOM 1525 (= ChDC B698) was isolated from human periapical abscess. Herein, we present the complete genome sequence of P. denticola KCOM 1525.

다운증후군 환자의 치주질환 원인균의 출현율

김선미,양규호,최남기,오종석,강미선 大韓小兒齒科學會 2005 大韓小兒齒科學會誌 Vol.32 No.4

다운증후군 환자는 치주질환의 진행이 빠르고 치주조직의 파괴가 심하다. 다운증후군 환자의 치주질환 원인균의 출현율을 알아보고자 7~19세 다운증후군 환자 27명과 대조군으로 나이가 비슷한 정신지체자 27명을 대상으로 치태지수와 치은염 정도를 나타내는 치은지수를 측정하고 치은연하 치태에 존재하는 P. gingivalis, T. forsythia, T. denticola, F. nucleatum, A. actinomycetemcomitans균을 중합효소연쇄반응을 이용, 검사하고 다음과 같은 결론을 얻었다. 1. 다운증후군 환자와 대조군의 치태지수와 치은지수간에 통계적 유의성은 없었다(p>0.05). 2. 다운증후군 환자에서 F. nucleatum의 출현율이 96.3%로 가장 높았으며 그 다음이 T. forsythia 74.1%, P. gingivalis는 63.0%였으며 A. actinomycetemcomitans는 55.6%, T. denticola는 40.7%로 출현하였다. 정신지체자는 다운증후군 환자와 비슷한 순서로 치주질환 원인균이 출현하였다. 다운증후군 환자가 대조군에 비해 T. denticola를 제외한 4종의 균에서 놓은 비율로 나타났으나 치주질환 원인균 출현율에서 유의한 차이를 나타내지 않았다(p>0.05). 3. 연령에 따른 연구에서 7~10세군에서 다운증후군환자의 P. gingivalis는 16.7%로 낮은 출현율을 보였으나 연령의 증가에 따라 출현율이 평균 63.0%로 높아졌다. 반면 A. actinomycetemcomitans균은 7~10세군부터 83.3%로 높은 비율을 보였다(p<0.05). 같은 연령대에서 다운증후군 환자는 대조군에 비해 치주질환 원인균의 출현율이 더 높은 경향을 보였으나 A. actinomycetemcomitans를 제외하고는 통계적인 유의차를 보이지 않았다(p>0.05). 4. P. gingivalis, T. forsythia, T. denticola가 함께 나타난 경우는 다운증후군 환자와 대조군 모두 33%를 나타내었다. red complex에 A. actinomycetemcomitans까지가 나타난 경우는 다운증후군 환자에서 22%, 대조군에서 14%로 다운증후군 환자에서 더 높았다. 이상의 결과를 요약해보면 다운증후군 환자군과 정신지체자 대조군 모두에서 치주질환 원인균이 어린 시기부터 매우 높게 출현하였으나 전체적인 두 군간 치태지수, 치은지수, 치주질환 원인균 출현율에서 유의한 차이를 나타내지 않았다. 하지만 A. actinomycetem comitans균은 7~10세군 다운증후군 환자에서 대조군에 비해 높은 비율을 보였다. It is widely known that individuals with Down's syndrome(DS) often develop early onset severe periodontal diseases. In this study. We examined the prevalence of periodontopathic bacteria in DS patients to compare controls with mental disabilities(MD). The subjects were 27 DS patients (7 to 19 years old) and 27 age-matched controls with MD. Plaque index and gingival index were measured. And 5 pathogens, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, were surveyed in subgingival plaque samples using a polymerase chain reaction. No significant difference in plaque index and gingival index were observed between the DS and control group. The prevalence in DS was 96.3% for F. nucleatum, 74.1% for T. forsythia, 63.0% for P. gingivalis, 55.6% for A. actinomycetemcomitans, 40.7% for T. denticola. No significant differences were observed in the prevalence of periodontopathic bacterias between the DS and control. Prevalence of P.g(16.7) at age 7~10 is lower than other age group in DS, but its prevalence increased with age. Prevalence of A.a(83.3%) is peak at age 7~10 in DS. These results suggest that various periodontopathic pathogens can colo nize in the very early childhood of DS and MD patients. But no significant difference was observed in the prevalence of periodontopathic bacterias between the DS and control.

Treponema denticola와 Treponema lecithinolyticum의 분쇄액이 치은섬유아세포의 Cytokine 분비 및 Matrix metalloproteinase 활성에 미치는 영향

서혜연,최봉규,최성호,조규성,김종관,채중규,Suh, Hye-Yuhn,Choi, Bong-Kyu,Choi, Seong-Ho,Cho, Kyoo-Sung,Kim, Chong-Kwan,Chai, Jung-Kiu 대한치주과학회 1999 Journal of Periodontal & Implant Science Vol.29 No.4

본 연구에서는 치주질환과 관련이 깊은 것으로 알려진 구강내 spirochetes 균중 Treponema denticola 분쇄액(TDC)과 가장 최근에 분리 배양된 Treponema lecithinolyticum 분쇄액(TLC)이 치은섬유아세포의 cytokine 분비 및 matrix metalloproteinase(MMP) 활성에 미치는 영향을 알아 보기 위하여 균의 분쇄액을 치은섬유아세포에 처리한 후 Interleukin-6(IL-6)와 $Interleukin-1{\beta}(IL-1{\beta})$의 분비 증가 여부를 ELISA를 통하여 측정하였으며, 또한 gelatinase zymography와 gelatin 분해능 측정을 통하여 교원질 분해 효소의 하나인 pro-MMP-2(progelatinase A)의 활성화 여부를 측정한 결과 다음과 같은 결론을 얻었다. 1. TDC와 TLC가 치은섬유아세포의 IL-6 분비에 미치는 영향을 살펴 본 결과, TDC 와 TLC 처치군에서 세균 분쇄액이 없는 비처치군에 비해 IL-6 분비량이 증가하였으며 유의성 있는 차이가 있었다(p<0.05). 2. TDC 와 TLC로 처리한 치은섬유아세포의 $IL-1{\beta}$ 분비는 측정 가능치(1pg/ml)이하의 분비량이 관찰되었다. 그러므로 $IL-1{\beta}$의 분비에는 영향이 없는 것으로 보인다. 3. 치은섬유아세포에서 분비되는 분자량 72 kDa의 pro-MMP-2가 TDC와 TLC에 의해 활성형으로 발현되어 zymography상에서 62kDa의 위치에 clear band로 나타났다. 4. 치은섬유아세포가 분비하는 MMP-2의 gelatin 분해능이, TDC와 TLC 처치군에서 비처치군보다 높게 나타났으며 유의 성 있는 차이가 있었다(p<0.05). 5. TDC 처치군에서는 gelatin 분해능에 있어서 세균 자체의 serin protease의 영향이 있었으나 TLC 처치군에서는 치은섬 유아세포의 MMP에 의해서만 gelatin이 분해되었다. 이상의 결과를 보아 TDC와 TLC는 치은섬유 아세포를 자극하여 IL-6 의 분비는 증가시킬 수 있으나 $IL-1{\beta}$의 분비에는 영향을 미칠 수 없으며, 치은섬유아세포에서 분비되는 pro-MMP-2를 활성형으로 발현시켜 결합조직의 파괴를 야기함으로서 치주 질환의 병인론에 기여할 수 있음을 확인하였다. This study was investigated to observe the effect of Treponema denticola cell sonicates(TDC) and Treponema lecithinolyticum cell sonicates(TLC) on cytokine secretion and matix metalloproteinase-2(MMP-2) activation of cultured human gingival fibroblast. Several experiments were performed including $IL-1{\beta}$, IL-6 ELISA for the effect on the $IL-1{\beta}$, IL-6 secretion of human gingival fibroblast. Also gelatinase zymography and gelatin dissolubility test for the activation of MMP-2 secreted by gingival fibroblast. The results were as follows. 1. The effect of TDC and TLC on IL-6 secretion of human gingival fibroblast showed statistically significant increase of IL-6 secretion in the TDC and TLC treated group compared to no treatment group(p<0.05) . 2. The amount of $IL-1{\beta}$ secretion was below the lower limit and there was no difference in the $IL-1{\beta}$ secretion of gingival fibroblast between TDC, TLC treated group and no treatment group. 3. The active form of pro MMP-2 with 72 kDa molecular weight was activated in both TDC and TLC treated group and clear band was appeared at 62kDa site on the zymography. 4. Gelatin dissolubility of MMP-2 secreted by gingival fibroblast was higher in TDC and TLC treated group compared to no treatment group(p<0.05). 5. In the TDC treated group, serine protease of T. denticola affect gelatin dissolubility. But in the TLC treated group gelatin was degraded by only MMP secreted by gingival fibroblast. Regarding to the above results, TDC and TLC have an effect on the IL-6 secretion increase of human gingival fibroblast and appears to activate pro MMP-2 which degrades collagen.

Binding of Tp92 homolog of Treponema denticola to fibronectin and epithelial cells

Hye-Kyoung Jun,Sung-Hoon Lee,Hae-Ri Lee KOREAN ACADAMY OF ORAL BIOLOGY 2008 International Journal of Oral Biology Vol.33 No.2

Treponema denticola is the best studied oral spirochete and numerous studies have shown that it is strongly associated with periodontitis and expresses several putative virulence factors. In this study, we report on a surface protein of T. denticola, Td92, which is homologous to Tp92 of Treponema pallidum, an agent of syphilis. Immunofluorescence assay and immunogold labeling with anti- Td92 Ab revealed that Td92 had surface-exposed epitopes. And Td92 was capable of binding to fibronectin and KB cells, an oral epithelial cell line. In addition, Td92 could enter the KB cells. These results indicate that Td92 is a fibronectin-binding protein which can bind to and internalize into the host cells, facilitating the virulence of T. denticola.

Td92, an outer membrane protein of <i>Treponema denticola</i>, induces osteoclastogenesis via prostaglandin E₂-mediated RANKL/osteoprotegerin regulation

Kim, M.,Jun, H.-K.,Choi, B.-K.,Cha, J.-H.,Yoo, Y.-J. Blackwell Publishing Ltd 2010 Journal of periodontal research Vol.45 No.6

<P><I>Kim M, Jun H-K, Choi B-K, Cha J-H, Yoo Y-J. Td92, an outer membrane protein of</I> Treponema denticola<I>, induces osteoclastogenesis via prostaglandin E</I><SUB><I>2</I></SUB><I>-mediated RANKL/osteoprotegerin regulation. J Periodont Res 2010; 45: 772–779. © 2010 John Wiley & Sons A/S</I></P><P>Background and Objective: </P><P>Periodontitis is a chronic inflammatory disease of the periodontium that causes significant alveolar bone loss. Osteoclasts are bone-resorbing multinucleated cells. Osteoblasts regulate osteoclast differentiation by expression of RANKL and osteoprotegerin (OPG). Td92 is a surface-exposed outer membrane protein of <I>Treponema denticola</I>, a periodontopathogen. Although it has been demonstrated that Td92 acts as a stimulator of various proinflammatory mediators, the role of Td92 in alveolar bone resorption remains unclear. Therefore, in this study, we investigated the role of Td92 in bone resorption.</P><P>Material and Methods: </P><P>Mouse bone marrow cells were co-cultured with calvariae-derived osteoblasts in the presence or absence of Td92. Osteoclast formation was assessed by TRAP staining. Expressions of RANKL, osteoprotegerin (OPG) and prostaglandin E<SUB>2</SUB> (PGE<SUB>2</SUB>) in osteoblasts were estimated by ELISA.</P><P>Results: </P><P>Td92 induced osteoclast formation in the co-cultures. In the osteoblasts, RANKL and PGE<SUB>2</SUB> expressions were up-regulated, whereas OPG expression was down-regulated by Td92. The addition of OPG inhibited Td92-induced osteoclast formation. The prostaglandin synthesis inhibitors NS398 and indomethacin were also shown to inhibit Td92-induced osteoclast formation. The effects of Td92 on the expressions of RANKL, OPG and PGE<SUB>2</SUB> in osteoblasts were blocked by NS398 or indomethacin.</P><P>Conclusion: </P><P>These results suggest that Td92 promotes osteoclast formation through the regulation of RANKL and OPG production via a PGE<SUB>2</SUB>-dependent mechanism.</P>

Evaluation of ciprofloxacin and metronidazole encapsulated biomimetic nanomatrix gel on Enterococcus faecalis and Treponema denticola

Sagar N Kaushik,Jessica Scoffield,Adinarayana Andukuri,Grant C Alexander,Taneidra Walker,김석곤,최성철,Brigitta C Brott,Paul D Eleazer,이진용,Hui Wu,Noel K Childers,Ho-Wook Jun,박재홍,Kyounga Cheon 한국생체재료학회 2015 생체재료학회지 Vol.19 No.2

Background: A triple antibiotic mixture (ciprofloxacin; CF, metronidazole; MN, and minocycline; MC) has been used for dental root canal medicaments in pulp regeneration therapy. However, tooth discolorations, cervical root fractures, and inadequate pulp-dentin formation have been reported due to the triple antibiotic regimen. Therefore, an antibiotic encapsulated biomimetic nanomatrix gel was developed to minimize the clinical limitations and maximize a natural healing process in root canal infections. In this study, minimal bacterial concentrations (MBC) of the selected antibiotics (CF and MN) were tested in 14 representative endodontic bacterial species. Then MBC of each CF and MN were separately encapsulated within the injectable self-assembled biomimetic nanomatrix gel to evaluate antibacterial level on Enterococcus faecalis and Treponema denticola. Results: Antibiotic concentrations lower than 0.2 μg/mL of CF and MN demonstrated antibacterial activity on the 14 endodontic species. Furthermore, 6 different concentrations of CF and MN separately encapsulated with the injectable self-assembled biomimetic nanomatrix gel demonstrated antibacterial activity on Enterococcus faecalis and Treponema denticola at the lowest tested concentration of 0.0625 μg/mL. Conclusions: These results suggest that each CF and MN encapsulated within the injectable self-assembled biomimetic nanomatrix gel demonstrated antibacterial effects, which could be effective for the root canal disinfection while eliminating MC. In the long term, the antibiotic encapsulated injectable self-assembled biomimetic nanomatrix gel can provide a multifunctional antibiotic delivery method with potential root regeneration. Further studies are currently underway to evaluate the effects of combined CF and MN encapsulated within the injectable self-assembled biomimetic nanomatrix gel on clinical samples.