Role of MyD88‐dependent and MyD88‐independent signaling in Porphyromonas gingivalis‐elicited macrophage foam cell formation

Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as Porphyromonas gingivalis and atherosclerosis and formation of macrophage foam cells. Toll‐like receptors and molecules governing their intracellular signaling pathways such as MyD88 play roles in atherosclerosis, as well as host response to P. gingivalis. The aim of this study was to define roles of MyD88 and TRIF during macrophage foam cell formation in response to P. gingivalis. In the presence of human low‐density lipoprotein (LDL) mouse bone‐marrow‐derived macrophages (BMφ) cultured with P. gingivalis responded with significant reduction in tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). The BMφ stained strongly with oil red O, regardless of whether bacterial challenge occurred concurrent with or before LDL treatment. Heat‐killed P. gingivalis stimulated foam cell formation in a similar way to live bacteria. The BMφ from MyD88‐knockout and Lps2 mice revealed a significant role for MyD88, and a minor role for TRIF in P. gingivalis‐elicited foam cell formation. Porphyromonas gingivalis‐elicited TNF‐α and IL‐6 were affected by MyD88 ablation and to a lesser extent by TRIF status. These data indicate that LDL affects the TNF‐α and IL‐6 response of macrophages to P. gingivalis challenge and that MyD88 and TRIF play important roles in P. gingivalis‐elicited foam cell formation.     

Inhibition of Porphyromonas gingivalis‐induced periodontal bone loss by CXCR4 antagonist treatment

Microbial pathogens have evolved mechanisms to proactively manipulate innate immunity, thereby improving their fitness in mammalian hosts. We have previously shown that Porphyromonas gingivalis exploits CXC‐chemokine receptor‐4 (CXCR4) to instigate a subversive crosstalk with Toll‐like receptor 2 that inhibits leukocyte killing of this periodontal pathogen. However, whether CXCR4 plays a role in periodontal disease pathogenesis has not been previously addressed. Here, we hypothesized that CXCR4 is required for P. gingivalis virulence in the periodontium and that treatment with AMD3100, a potent CXCR4 antagonist, would inhibit P. gingivalis‐induced periodontitis. Indeed, mice given AMD3100 via osmotic minipumps became resistant to induction of periodontal bone loss following oral inoculation with P. gingivalis. AMD3100 appeared to act in an antimicrobial manner, because mice treated with AMD3100 were protected against P. gingivalis colonization and the associated elevation of the total microbiota counts in the periodontal tissue. Moreover, even when administered 2 weeks after infection, AMD3100 halted the progression of P. gingivalis‐induced periodontal bone loss. Therefore, AMD3100 can act in both preventive and therapeutic ways and CXCR4 antagonism could be a promising novel approach to treat human periodontitis.     

大鼠舌鳞癌诱变过程中MMP-3、TIMP-1的表达变化及相关性研究

目的:研究基质金属蛋白酶-3(MMP-3)、基质金属蛋白酶组织抑制因子-1(TIMP-1)在大鼠舌鳞癌诱变过程中转录水平表达变化及两者之间的相关性。方法:60只SD大鼠随机分为3组,每组20只,正常对照组,给予正常饮食;另2组每天喂养0.002%4-硝基喹啉-1-氧化物(4NQO),于16周和24周处死;16周组可见上皮异常增生,而24周组已经为舌鳞癌,分别选取8个标本作检测。各组利用定量逆转录聚合酶链反应(qRT-PCR)检测MMP-3、TIMP-1在不同病变时期舌组织中转录水平的表达变化。结果:MMP-3及TIMP-1的mRNA于正常对照组相对表达量较少,上皮异常增生组相对表达量较正常组显著增加,舌鳞癌组的相对表达量最高。MMP-3与TIMP-1的mRNA表达之间成显著相关性。结论:MMP-3与TIMP-1随癌变的发生表达显著上调,两者间平衡失调可导致舌鳞癌的发生及发展。     

Interferon‐Gamma and Fas Are Involved in Porphyromonas gingivalis–Induced Apoptosis of Human Extravillous Trophoblast‐Derived HTR8/SVneo Cells via Extracellular Signal‐Regulated Kinase 1/2 Pathway

Background: A number of studies recently revealed a link between periodontal disease and preterm birth (PTB). PTB can be induced by dental infection with Porphyromonas gingivalis (Pg), a periodontopathic bacterium. This study aims to investigate responses of human extravillous trophoblast‐derived HTR8/SVneo cells to Pg infection. Methods: Cell apoptosis, cell viability, protein expression, and cytokine production in HTR8 cells were measured via: 1) flow cytometry, 2) CCK‐8 assay, 3) western blot, and 4) enzyme‐linked immunosorbent assay methods, respectively. Results: Pg decreased cell viability and increased cell apoptosis, active caspase‐3 and Fas expression, and interferon‐gamma (IFN‐γ) secretion in HTR8 cells. Extracellular signal‐regulated kinase (ERK) 1/2 inhibitor U0126 and FasL neutralizing antibody NOK1 that blocks FasL/Fas interaction both significantly suppressed Pg‐induced apoptosis. U0126 also inhibited IFN‐γ secretion and Fas expression close to control levels. Moreover, treatment with recombinant IFN‐γ also significantly decreased number of viable HTR8 cells and increased Fas expression, suggesting IFN‐γ may play an important role in Pg‐induced apoptosis of HTR8 cells, at least partially through regulation of Fas expression. Conclusions: To the best of the authors’ knowledge, this is the first study to demonstrate Pg induces IFN‐γ secretion, Fas expression, and apoptosis in human extravillous trophoblast‐derived HTR8/SVneo cells in an ERK1/2‐dependent manner, and IFN‐γ (explored by recombinant IFN‐γ) and Fas are involved in Pg‐induced apoptosis. The finding that Pg infection abnormally regulates inflammation and apoptosis of human trophoblasts may give new insights into the possible link of PTB with maternal periodontal disease and periodontal pathogens.     

Response of periodontitis and healthy patients in a Porphyromonas gingivalis‐stimulated whole‐blood model

Aim: To investigate the inflammatory responses of periodontitis patients and healthy patients in a whole‐blood model stimulated with Porphyromonas gingivalis. Methods: Whole blood collected from 17 periodontitis patients and six healthy patients was stimulated with Porphyromonas gingivalis cells. The secretion of cytokines and matrix metalloproteinases was quantified by enzyme‐linked immunosorbent assay. An analysis of covariance with the ancova model was used to evaluate the significance of differences in secreted host molecules by whole blood from the periodontitis and healthy groups. Results: Porphyromonas gingivalis induced the secretion of interleukin‐1β, interleukin‐6, interleukin‐8, tumor necrosis factor‐α, monocyte chemoattractant protein‐1, interferon inducible protein‐10 by whole blood from patients in the periodontitis and healthy groups. Matrix metalloproteinase‐8 and ‐9 levels secreted by whole blood also increased following stimulation. No significant differences (P < 0.05) in the amounts of secreted host molecules were observed between periodontitis and healthy patients. Conclusion: This study suggests that Porphyromonas gingivalis can provoke an inflammatory response and promote the progression of periodontitis by inducing the secretion of high levels of cytokines and matrix metalloproteinases by a mixed leukocyte population. However, the whole‐blood model did not reveal any significant differences in the inflammatory response between periodontitis patients (n = 17) and periodontally‐healthy patients (n = 6).     

基质金属蛋白酶及其抑制剂与口腔鳞癌颈淋巴结转移的关系

目的:探讨基质金属蛋白酶-2,9(MMP-2,9)和基质金属蛋白酶抑制剂-1,2(TIMP-1,2)在口腔鳞癌中的表达形式以及其活性与颈淋巴结转移的关系。方法:应用原位杂交法(in situ hybridization,ISH)检测30例口腔鳞癌组织,口腔鳞癌转移细胞系GNM和人舌鳞癌细胞系TSCCa MMP-2,9和TIMP-1,2的表达;应用胶质酶谱法(zomography)分析上述标本MMP-2,9的活性以及应用体外侵袭实验检测两细胞系侵袭力的差异。结果:MMP-2,9和TIMP-1,2 mRNA 在肿瘤细胞和间质细胞均有表达;有转移鳞癌组织MMP-2,9,以及TIMP-1,2阳性率均高于无转移患者(P<0105), GNMMMP-2,9阳性率高于TSCCa;MMP-2,9活性在转移组明显高于未转移组(P<0105);GNM条件培养上清液中 MMP-2,9活性以及体外侵袭力均高于TSCCa。结论:MMP-2,9在口腔鳞癌转移中有重要作用,TIMP-1,2表达上升可能是肿瘤细胞与间质相互作用的结果。     

血管内皮生长因子对口腔鳞癌细胞系基质金属蛋白酶-2和-9活性的影响

目的　探讨血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)和口腔鳞癌侵袭转移的关系。方法　采用蛋白酶谱分析法测量不同浓度VEGF作用于口腔鳞癌TSCCa细胞系及颈淋巴转移癌GNM细胞系后基质金属蛋白酶 2 (matrixmetal loproteinase 2 ,MMP 2 )和MMP 9的活性 ,同时用BoydenChamber观察VEGF诱导口腔鳞癌TSCCa细胞转移的作用。结果　不同浓度的VEGF (1、5、10ng/ml)作用于GNM细胞 2h后 ,MMP 2和MMP 9的活性与对照组相比 ,差异无显著性 (P >0 0 5 ) ;而不同浓度的VEGF作用于TSCCa细胞 2h后 ,MMP 2和MMP 9的活性与对照组相比 ,差异有显著性 (P <0 0 5或 0 0 1) ,MMP 2和MMP 9的活性与VEGF有剂量依赖关系 ,用VEGF 1、5、10ng/ml培养口腔鳞癌TSCCa细胞系 2h ,BoydenChamber小室下室浸润的口腔鳞癌细胞数分别为 (6 6 7± 1 78)× 10 4/ml、(17 17± 2 38)× 10 4/ml、(2 2 33± 2 5 4 )× 10 4/ml ,分别高于对照组 (2 4 8±1 0 2 )× 10 4/ml (P <0 0 5或 0 0 1)。结论　VEGF可促进口腔鳞癌细胞侵袭转移。     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

In Vivo Inhibition of Porphyromonas gingivalis Growth and Prevention of Periodontitis With Quorum‐Sensing Inhibitors

Background: Autoinducer (AI)‐2 has an important role in biofilm formation in the oral environment. Mature biofilms formed as a result of the cell‐to‐cell communication make it difficult to overcome periodontitis with the use of antibiotics. Previous in vitro studies suggest that quorum‐sensing inhibitors (QSIs) interfere with AI‐2. This study compares the QSI effects resulting from an oral inoculation of Porphyromonas gingivalis in an experimental animal model. Methods: Forty‐five male mice were divided into three groups (n = 15 each): 1) infection; 2) QSI; and 3) control. Infection and QSI groups received oral inoculation of P. gingivalis, whereas treatment with QSIs (furane compound and d ‐ribose) was only performed in the QSIs group. The control group was a negative control not receiving manipulation. After 42 days, mice were sacrificed, and the distance from the alveolar bone crest (ABC) to the cemento‐enamel junction (CEJ) was measured by microcomputed tomography. P. gingivalis DNA was quantified in the soft and hard tissues around the molar teeth by real‐time polymerase chain reaction. Results: Distance from ABC to CEJ was significantly increased in the P. gingivalis infection group compared with the control group (P = 0.02) and significantly decreased in the QSI group compared with the infection group (P = 0.02). The QSI group contained 31.64% of the bacterial DNA count of the infection group. Conclusion: Use of QSIs in the mice infection model showed a reduction of bone breakdown and a decrease in the number of bacteria in vivo, suggesting that QSIs can be a new approach to prevention and treatment of periodontitis.     

Iron‐ and hemin‐dependent gene expression of Porphyromonas gingivalis

Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron‐dependent regulation is not well known in anaerobic bacteria. Here, we investigated iron‐ and hemin‐dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole‐genome microarrays of P. gingivalis genes were used to compare the levels of gene expression under iron‐replete and iron‐depleted conditions as well as under hemin‐replete and hemin‐depleted conditions. Under iron‐depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron‐depleted conditions to those from hemin‐depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron‐dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron‐regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron‐stressed bacterium than the iron‐replete P. gingivalis cells. The major iron‐regulated proteins encoded by PG1019–20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole‐genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis.