Interaction of Porphyromonas gingivalis with KB cells: comparison of different clinical isolates

The ability of different Porphyromonas gingivalis strains (15 clinical isolates and ATCC 33277) to attach to and invade KB cells, in relation to other properties such as release of interleukin (IL)-6 and IL-8, cytotoxicity, proteolytic activity and types of fimbriae genes present, was examined. A hierarchical cluster analysis based on adherence and internalization resulted in four groups. Eight of the 15 clinical isolates belonged to a cluster group whose adherence and internalization were about 10% those of the ATCC strain. A negative correlation between lysine-specific protease activity and adherence was found. In all cases the released concentrations of IL-6 and IL-8 were very low. Only one strain was found to be cytotoxic to KB cells. Principal components analysis demonstrated correlations between adherence, internalization and autoaggregation. Most strains had fimA type I and II, type I being associated with elastase-like activity. The ability of P. gingivalis to invade epithelial cells may be a key factor for maintaining periodontal disease.     

Adherence of Porphyromonas gingivalis to gingival epithelial cells: modulation of bacterial protein expression

The protein profiles of Porphyromonas gingivalis (ATCC 33277 and W83) bound to KB gingival epithelial cells were analyzed by SDS-PAGE and immunoblotting. We found that a 51-kDa component was formed in bacteria that adhered to the KB cells, whereas 26- to 29-kDa bands were less intensive, in contrast to the protein profile of free bacteria. P. gingivalis ATCC 33277 incubated with protease-treated KB cells retained the profile of free bacteria. These results demonstrate the specificity of bacterial recognition of eukaryotic membrane components.     

The host cytokine response to Porphyromonas gingivalis is modified by gingipains

Background/aims:  Clinical studies indicate that primary proinflammatory cytokines, such as interleukin-1β (IL-1β) are elevated in the gingival crevice around teeth with periodontitis but the secondary cytokines and chemokines, IL-6 and IL-8, are not. The human gingival epithelial cells (HGECs) lining the gingival sulcus respond to perturbation by microbes of dental plaque by releasing a wide range of cytokines. Porphyromonas gingivalis , a putative periodontal pathogen, possesses numerous virulence factors some of which directly impact on the host response. In the present study, we sought to determine how P. gingivalis influences the inflammatory cytokine responses.
Methods:  HGECs were challenged with P. gingivalis and other putative periodontal pathogens, and the resultant production of IL-1β, IL-6, and IL-8 was assayed by enzyme-linked immunosorbent assay (ELISA). Culture supernatants and recombinant human cytokines were challenged with live P. gingivalis wild-type and gingipain-deficient strains and the resultant cytokine profile was assessed by ELISA and Western blot.
Results:  We show here that primary HGECs challenged with live P. gingivalis result in high levels of IL-1β but not the related secondary cytokines IL-6 and IL-8. We further demonstrate that cytokine response differences are the result of the action of P. gingivalis proteases, with lysine gingipain being the most effective.
Conclusion:  We conclude that P. gingivalis , through lysine gingipain, can subvert the protective host proinflammatory response by direct cytokine degradation. Changes in the crevicular cytokine profile have consequences in periodontal disease pathogenesis that should be considered in the development of diagnostic and therapeutic modalities.     

牙龈卟啉单胞菌刺激树突状细胞激活炎症反应

[摘要] 目的 分析树突状细胞在牙龈卟啉单胞菌感染后的差异表达基因,探讨牙周炎的致病机制及为日后研究提供线索。方法 利用高通量测序技术来分析体外诱导分化的树突状细胞在牙龈卟啉单胞菌感染情况下的差异表达基因、基因集及相关功能。 结果 本实验总计发现7305个至少有两倍变化的差异表达基因。基因本体结果显示牙龈卟啉单胞菌感染后树突状细胞的生物过程、细胞成份、分子功能均发生了较大变化。基因集富集分析结果显示肿瘤坏死因子基因集、细胞因子及炎症反应基因集、白介素1及转录因子NF-kB家族基因集、干扰素信号基因集、B细胞受体信号基因集、T细胞受体信号基因集富集明显。结论 树突状细胞在牙龈卟啉单胞菌感染后会导致基因水平的变化,引起机体免疫反应及炎症反应的发生, CXCL及SOCS的水平及作用可作为日后研究的方向。     

牙龈卟啉单胞菌红细胞凝集素A黏附和入侵人牙龈上皮细胞的研究

目的:检测牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)膜表面红细胞凝集素A(hemagglutinin A,HagA)黏附和入侵人牙龈上皮细胞的功能。方法:构建P.g381hagA基因的变异菌株,连接hagA基因到pYA292质粒上,并克隆到无毒性的沙门杆菌x4072菌中,通过比较变异菌株、野生菌株和表达hagA基因的沙门杆菌对人牙龈上皮细胞的黏附和入侵功能,检测HagA在此过程中的作用。结果:P.g381hagA基因变异菌株和野生菌株在对细胞的黏附和入侵过程中没有显著差别,但沙门杆菌x4072HagA表达菌株对细胞的黏附性和入侵性比对照组分别提高了3倍和4倍。结论:HagA参与了P.g381黏附和入侵牙龈上皮细胞的过程。     

Interaction of Porphyromonas gingivalis with gingival epithelial cells maintained in culture.

Interactions between Porphyromonas gingivalis and gingival epithelial cells were investigated. Gingival epithelial cells were cultured from surgically removed gingival tissue. Electron microscopy demonstrated adherence of P. gingivalis to the cell membranes and microvilli followed by internalization of the bacteria into the epithelial cell cytoplasm. Saliva from healthy and periodontally diseased patients inhibited P. gingivalis association with the epithelial cells. Attachment to and penetration of gingival epithelial cells by P. gingivalis may be important virulence factors in periodontal disease. Salivary molecules may play a role in modulating these interactions.     

高脂血症抑制载脂蛋白E基因敲除小鼠对牙龈卟啉单胞菌炎症反应的影响

目的:研究高脂血症在ApoE基因敲除(ApoE-/-)小鼠对牙龈卟啉单胞菌(P. gingivalis)炎症反应过程中的影响及其机制。方法:利用ApoE-/-小鼠建立长时间高脂血症模型,腹腔注射活P. gingivalis以建立腹膜炎症模型,倍比稀释法分析腹腔内清除细菌能力,ELISA法检测血液中白细胞介素6(IL-6)和单核细胞趋化因子1(MCP-1)的分泌,实时定量PCR检测腹腔细胞IL-6和MCP-1的转录水平,采用SPSS13.0软件包进行统计学分析。结果:高脂血症显著降低了腹腔清除P. gingivalis的能力,ApoE-/-小鼠腹腔细胞中IL-6和MCP-1转录水平下降,释放入血液中的IL-6和MCP-1水平下降。结论:高脂血症导致宿主对P. gingivalis炎症反应不足,清除细菌能力下降,加速了牙周病的发展进程。     

Stress response of Porphyromonas gingivalis

The heat shock response of Porphyromonas gingivalis was examined by 1 - and 2-dimensional polyacrylamide gel electrophoresis after metabolic labeling with [¹⁴C]-amino acids. When P. gingivalis cells were shifted from 37° C to 42° C, elevated synthesis of 4 proteins with the apparent molecular weight of 92, 80, 74, and 62 kDa was observed, and synthesis of a 50-kDa protein decreased during heat shock. The 74- and 62-kDa proteins were identified as homologs of Escherichia coli DnaK and GroEL respectively by Western immunoblotting. On 2-dimen-sional Western blots, 2 forms of DnaK and 4 forms of GroEL were identified due to their slightly different isolelectric points. dna K and gro EL gene homologs were identified in several P. gingivalis strains and some other black-pigmented oral species. DnaK and GroEL homolog proteins were induced when P. gingivalis cells were challenged by ethanol. These proteins were not induced by oxidative stress or change in pH.     

Porphyromonas gingivalis stimulates TACE production by T cells

Introduction: Tumour necrosis factor‐α converting enzyme (TACE), also known as ADAM17, is a membrane‐bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell‐bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T‐cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. Methods: P. gingivalis 6‐day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell‐associated TACE levels were measured by enzyme‐linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real‐time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat‐inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. Results: P. gingivalis challenge resulted in a concentration‐dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. Conclusion: The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell‐bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.     

Desulfovibrio spp. survive within KB cells and modulate inflammatory responses

Desulfovibrio are sulfate‐reducing anaerobic gram‐negative rods that have been proposed as potential periodontopathogens. We investigated the capacity of Desulfovibrio to invade epithelial cells and induce cytokine secretion from these cells. Desulfovibrio strains were co‐cultured with KB cells and counts of intracellular bacteria evaluated up to 3 days after infection. Desulfovibrio desulfuricans and Desulfovibrio fairfieldensis were able to survive within epithelial cells. Intracytoplasmic location of both bacterial species was confirmed by confocal laser scanning microscopy and transmission electron microscopy. Invasion was sensitive to nocodazole, an inhibitor of microtubule polymerization, but not to cytochalasin D, a microfilament inhibitor, suggesting that microtubule rearrangements were involved in the internalization of Desulfovibrio strains by KB cells. Infection by Desulfovibrio resulted in increased production of IL‐6 and IL‐8 by KB cells. The ability of D. desulfuricans and D. fairfieldensis to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases.     

牙龈卟啉单胞菌脂多糖对泡沫细胞凋亡基因的影响

目的 了解牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)脂多糖(lipopolysaccharide,LPS)在巨噬细胞吞噬氧化低密度脂蛋白(oxidized low density lipopmtein,oxLDL)形成泡沫细胞过程中和形成后对凋亡基因的影响,以期了解Pg影响动脉粥样硬化的可能机制.方法 以oxLDL、oxLDL+Pg-LPS刺激人急性单核细胞白血病单核细胞株THP-1源性巨噬细胞,以及oxLDL诱导巨噬细胞形成的泡沫细胞.采用吖啶橙-溴化乙锭双染色观察细胞凋亡,聚合酶链反应(PCR)芯片检测11种动脉粥样硬化相关凋亡基因的变化,实时PCR检测p53、c-Myc和半胱氨酸天冬氨酸蛋白酶(caspase)-3基因的变化.结果 Pg-LPS提高了巨噬细胞吞噬oxLDL形成泡沫细胞过程中和形成后的细胞凋亡率,分别为(5.47±0.93)%、(7.50 4-0.54)%;PCR芯片检测显示泡沫细胞形成过程中Pg-LPS上调了B细胞淋巴瘤-白血病-2相关蛋白Al的转录(>2倍),泡沫细胞形成后上调了B细胞淋巴瘤-白血病-2和B细胞淋巴瘤-白血病-2相关蛋白A1的转录(>2倍);在泡沫细胞形成过程中提高了p53和caspase-3的转录水平[分别为(4.50×10-3±4.02×10-4)和(5.30×10-2±4.58×10-3)],抑制了c-Myc的转录水平(1.53×10-2±5.77×10-4);在泡沫细胞形成后降低了p53转录水平(4.23×10-3±5.85×10-4),促进了caspase-3的转录水平(6.00×10-2±6.08×10-3),P<0.05.结论 Pg-LPS影响了泡沫细胞形成过程中和形成后细胞中多种凋亡基因的转录,促进了凋亡的发生.     

Immunization to Porphyromonas gingivalis enhances the local pro-inflammatory response to subcutaneous bacterial challenge

BACKGROUND, AIMS: Human and animal studies have suggested that immunization to P. gingivalis might be beneficial for controlling periodontitis, by the induction of protective antibody response. The present study was designed to examine the effect of immunization on the local cellular, cytokine and antibody response to P. gingivalis in mice. METHODS: Subcutaneous chambers were implanted in 3 groups of mice. 2 groups were then immunized with P. gingivalis in either incomplete Freund's (IFA) or an Alum-based adjuvant. The 3rd group served as the control. At baseline, all mice were challenged with an intra-chamber injection of P. gingivalis. Chamber exudates were sampled at baseline, 1 and 7 days post-challenge, following by determination of leukocyte counts and the cytokines TNF-alpha, IFNgamma (pro-inflammatory) and IL-10 (anti-inflammatory). IgG levels to P. gingivalis were analyzed in both the exudates and serum. RESULTS: Leukocyte accumulation increased in the chambers over the study period and was more marked in the immunized groups. P. gingivalis challenge induced the expression of the tested cytokines in all groups. Levels of IFNgamma showed a significantly greater increase in the immunized groups on day 1 post-challenge. By day 7, the levels in the controls had reached those of the immunized groups. IL-10 levels were significantly higher in the control group compared to the immunized groups on day 1 and by day 7 they were reduced significantly in all groups to barely detectable levels. While there were no significant differences in TNF-alpha levels between IFA and control groups, they were significantly higher in the Alum group on day 0 and 7. Both immunization protocols induced anti-P. gingivalis IgG. The Alum group achieved the highest antibody levels, which were due to the increased expression of IgG1, a marker of a Th2-response. CONCLUSIONS: The results suggest that immunization to P. gingivalis results in enhanced expression of pro-inflammatory, tissue-destructive cytokines in the inflammatory site. The nature of the adjuvant used for immunization allows manipulation of the T-cell response, and alum was more effective in reducing the inflammatory response than IFA.     

Porphyromonas gingivalis gingipains mediate the shedding of syndecan-1 from the surface of gingival epithelial cells

Porphyromonas gingivalis gingipains are thought to be critical virulence factors in periodontitis. Increased serum levels of the soluble ectodomains of surface effectors have been reported to occur during bacterial infections. In the present study, we show that the cell surface proteoglycan syndecan-1 was highly expressed on human gingival epithelial cells. Treatments with P. gingivalis culture supernatants consistently mediated the shedding of syndecan-1 from the surface of epithelial cells. Concomitantly, the amount of soluble syndecan-1 detected in the culture medium increased significantly in a time-dependent manner. However, neither a heat-inactivated supernatant nor a supernatant from a gingipain-deficient mutant had a significant effect on syndecan-1 shedding. Such a shedding process may play an important role in the bacterial invasion of periodontal tissue and the modulation of host defences.     

Production of inflammatory cytokines by human gingival fibroblasts stimulated by cell-surface preparations of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. Inflammatory cytokines are believed to be the major pathological mediators in periodontal diseases. We therefore investigated the productions of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) in human gingival fibroblasts treated with lipopolysaccharide, polysaccharide and outer-membrane proteins from P. gingivalis ATCC 53977. Outer-membrane protein from P. gingivalis enhanced the production of IL-6 and IL-8 from the cells of periodontium in vitro as well as lipopolysaccharide did. The IL-8 production activity of polysaccharide from P. gingivalis was higher than that of other cell-surface components. The levels of IL-6 and IL-8 released from the P. gingivalis lipopolysaccharide-treated human gingival fibroblasts were lower than those of the same cells treated with lipopolysaccharides from Actinobacillus actinomycetemcomitans or Escherichia coli. Rabbit antisera against either outer-membrane protein or lipopolysaccharide inhibited the IL-6 and IL-8 production derived from human gingival fibroblasts stimulated sonicated supernatants from P. gingivalis. The present study suggests that, in addition to lipopolysaccharide, outer-membrane protein and polysaccharide of P. gingivalis are also pathological mediators in periodontal diseases.     

牙龈卟啉单胞菌膜泡诱导牙龈上皮细胞炎性反应的体外研究

目的建立牙龈卟啉单胞菌膜泡诱导牙龈上皮细胞炎性反应的体外模型，探讨牙龈卟啉单胞菌在牙周炎中的致病作用。方法用酶联免疫吸附法检测牙龈卟啉单胞菌膜泡对牙龈上皮细胞前列腺素E2（prostaglandin E2，PGE2）分泌的影响，以实时反转录聚合酶链反应法检测牙龈卟啉单胞菌膜泡对牙龈上皮细胞环氧化物酶（cyclooxygenase，COX）-2和白细胞介素（interleukin，IL）-6、IL-8基因表达的作用。结果牙龈卟啉单胞菌膜泡浓度依赖性地促进了牙龈上皮细胞PGE2的分泌，并使COX-2、IL-6、IL-8的mRNA表达水平显著上调。结论牙龈卟啉单胞菌膜泡诱导牙龈上皮细胞发生的细胞炎性反应，可能是牙周炎发生、发展的重要因素。     

Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis

Takahashi N, Honda T, Domon H, Nakajima T, Tabeta K, Yamazaki K. Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis. J Periodont Res 2010; 45: 512–519. © 2010 John Wiley & Sons A/S Background and Objective: Recent studies have revealed that negative regulatory molecules, including interleukin‐1 receptor‐associated kinase‐M (IRAK‐M), control the overactivation of Toll‐like receptor (TLR) signaling. The role of IRAK‐M in human gingival epithelial cells (HGECs), which express TLRs, remains unclear. The present study examined the role of IRAK‐M on interleukin‐8 and macrophage chemoattractant protein‐1 (MCP‐1) expression in HGECs stimulated with Porphyromonas gingivalis and TLR ligands. Material and Methods: Primary HGECs and an SV40 T‐antigen‐immortalized HGEC line (epi 4) were stimulated with live or heat‐killed P. gingivalis, P. gingivalis lipopolysaccharide or the synthetic lipopeptide PAM₃CSK₄, and subsequent expression of IRAK‐M, interleukin‐8 and MCP‐1 was evaluated at the mRNA and protein levels. The effects of IRAK‐M on interleukin‐8 and MCP‐1 expressions were evaluated by IRAK‐M‐specific RNA interference (RNAi)‐based loss‐of‐function assay. Results: All tested stimulants up‐regulated the expression of IRAK‐M in HGECs. The P. gingivalis lipopolysaccharide or PAM₃CSK₄ increased MCP‐1 expression, whereas live P. gingivalis down‐regulated the MCP‐1 expression in HGECs. However, IRAK‐M RNAi increased the expression of MCP‐1 irrespective of up‐ or down‐regulation mediated by the respective stimulants. Interleukin‐8 gene expression, up‐regulated by all tested stimulants, was further enhanced by IRAK‐M RNAi. In contrast, IRAK‐M RNAi had no effect on the interleukin‐8 protein levels, irrespective of the stimulant, indicating that post‐translational modification, not IRAK‐M, controls interleukin‐8 protein expression. Conclusion: Interleukin‐1 receptor‐associated kinase‐M appeared to have distinct regulatory roles on the interleukin‐8 and MCP‐1 produced by HGECs, further suggesting an important role for interleukin‐8 in the immune reponse to periodontopathic bacteria.     

Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis

Multiple periodontal pathogens sequentially colonize the subgingival niche during the conversion from gingivitis to destructive periodontal disease. An animal model of sequential immunization with key periodontal pathogens has been developed to determine whether T- and B-lymphocyte effector functions are skewed and fail to protect the host from pathogenic challenge. The present study was performed to evaluate the immunomodulatory effect of exposure to Fusobacterium nucleatum prior to Porphyromonas gingivalis. Group 1 (control) mice were immunized with phosphate-buffered saline, group 2 were immunized with F. nucleatum prior to P. gingivalis and group 3 were immunized with P. gingivalis alone. All the T-cell clones derived from group 2 demonstrated type 2 helper T-cell clone (Th2 subsets), whereas those from group 3 mice demonstrated Th1 subsets. Exposure of mice to F. nucleatum prior to P. gingivalis interfered with the opsonophagocytosis function of sera against P. gingivalis. In adoptive T-cell transfer experiments, in vivo protective capacity of type 2 helper T-cell clones (Th2) from group 2 was significantly lower than type 1 helper T-cell clones (Th1) from group 3 against the lethal dose infection of P. gingivalis. Western blot analysis indicated a different pattern of recognition of P. gingivalis fimbrial proteins between sera from group 2 and group 3. In conclusion, these studies suggest that exposure of a host to F. nucleatum prior to the periodontal pathogen P. gingivalis modulates the host immune responses to P. gingivalis at the humoral, cellular and molecular levels.