Synergism between TLRs and NOD1/2 in oral epithelial cells

Oral epithelium is the first barrier against oral bacteria in periodontal tissue. Oral epithelial cells constitutively express Toll-like receptors (TLRs) and NOD1/2, functional receptors which induce the production of antibacterial factors such as peptidoglycan recognition proteins (PGRPs) and beta-defensin 2, but not pro-inflammatory cytokines such as interleukin (IL)-8. In this study, we hypothesized that innate immune responses in the oral epithelium are enhanced in inflamed tissue. We found that NOD1 and NOD2 agonists, in combination with TLR agonists, synergistically induced production of PGRPs and of beta-defensin 2 in human oral epithelial cells via NF-kappaB. In contrast, co-stimulation with NOD1/2 and TLR ligands had no effect on the production of pro-inflammatory cytokines (IL-6, IL-8, and monocyte chemoattractant protein-1). These findings indicate that, in innate immune responses to invading microbes, a combination of signaling through TLRs and NODs leads to the synergistic activation of antibacterial responses in the oral epithelium.     

TRAF6在粪肠球菌感染人成骨样细胞炎症反应中的作用

目的 探讨肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)在粪肠球菌引发的人成骨样细胞MG63炎症反应中的作用.方法 采用siRNA瞬时干扰沉默MG63细胞的TRAF6基因,用粪肠球菌灭活全菌及其脂磷壁酸(lipoteichoic acid,LTA)刺激MG63细胞不同时间,定量PCR检测细胞中Toll样受体2(toll-like receptor 2,TLR2)和TRAF6的表达量;ELISA法检测MG63细胞产生的致炎因子白细胞介素1β(interleukin 1β,IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达量.结果 粪肠球菌及其LTA感染MG63细胞,TLR2受体和TRAF6的基因水平都有不同程度的上升(P<0.05).IL-1β和TNF-α的表达量都显著增高(P<0.05).用siRNA抑制MG63细胞TRAF6表达后,促炎因子IL-1β和TNF-α的表达量显著下降(P<0.05).结论 MG63细胞主要通过TLR2受体来识别粪肠球菌及其毒性成分.粪肠球菌引起的根尖周感染中,其主要毒力因子是其细胞壁上的LTA.     

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation

Background and Objective:  While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-κB (NF-κB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins.
Material and Methods:  Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-κB were measured using enzyme-linked immunosorbent assay.
Results:  The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-κB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-κB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts.
Conclusion:  These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.     

TLR4,NOD2和NLRP3在牙髓炎中的表达

目的:分析牙髓炎中TLR4、NOD2和NLRP3的表达特征。方法:收集健康、龋病及牙髓炎的患牙共47颗分为3个组,采用免疫组织化学染色和单色免疫荧光染色方法检测每组牙髓组织中TLR4、NOD2、NLRP3的表达。结果:健康组中TLR4、NOD2和NLRP3仅在成牙本质细胞中少量表达。TLR4、NOD2和NLRP3在牙髓炎组及龋病组表达均明显高于正常组,大量表达于排列紊乱的成牙本质细胞和多种炎症细胞中,而在炎性病灶周边的牙髓成纤维细胞中三者也有表达。结论:本研究表明TLR4、NOD2和NLRP3在炎性牙髓组织中表达同时增高,提示三者均与牙髓炎的进展相关。     

Functional TLRs and NODs in human gingival fibroblasts

Since human gingival fibroblasts are the major cells in periodontal tissues, we hypothesized that gingival fibroblasts are endowed with receptors for bacterial components, which induce innate immune responses against invading bacteria. We found clear mRNA expression of Toll-like receptors (TLR)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, MD-2, MyD88, NOD1, and NOD2 in gingival fibroblasts. Gingival fibroblasts constitutively expressed these molecules. Upon stimulation with chemically synthesized ligands mimicking microbial products for these receptors, the production of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1, was markedly up-regulated. Furthermore, the production of pro-inflammatory cytokines induced by TLR and NOD ligands was significantly inhibited by an RNA interference assay targeted to NF-kappaB. These findings indicate that these innate immunity-related molecules in gingival fibroblasts are functional receptors involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis.     

TLR expression profile of human gingival margin-derived stem progenitor cells

Background

Gingival margin-derived stem/progenitor cells (G-MSCs) show remarkable periodontal regenerative potential in vivo. During regeneration, G-MSCs may interact with their inflammatory environment via toll-like-receptors (TLRs). The present study aimed to depict the G-MSCs TLRs expression profile.

Material and Methods

Cells were isolated from free gingival margins, STRO-1-immunomagnetically sorted and seeded to obtain single colony forming units (CFUs). G-MSCs were characterized for CD14, CD34, CD45, CD73, CD90, CD105, CD146 and STRO-1 expression, and for multilineage differentiation potential. Following G-MSCs’ incubation in basic or inflammatory medium (IL-1β, IFN-γ, IFN-α, TNF-α) a TLR expression profile was generated.

Results

G-MSCs showed all stem/progenitor cells’ characteristics. In basic medium G-MSCs expressed TLRs 1, 2, 3, 4, 5, 6, 7, and 10. The inflammatory medium significantly up-regulated TLRs 1, 2, 4, 5, 7 and 10 and diminished TLR 6 (p≤0.05, Wilcoxon-Signed-Ranks-Test).

Conclusions

The current study describes for the first time the distinctive TLRs expression profile of G-MSCs under uninflamed and inflamed conditions. Key words:Stem cells, TLR, gingiva, polymerase chain reaction, FACS.     

TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens

Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone‐marrow‐derived macrophages (BMDM) from wild‐type (WT) and Toll‐like receptor (TLR)‐specific and MyD88 knockouts (KOs), we demonstrated that heat‐killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1‐specific KOs and NOD2‐expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855).     

Toll-like receptors, NOD1, and NOD2 in oral epithelial cells

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR)4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of beta-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.     

白藜芦醇对高糖环境下牙龈上皮细胞TLR4表达及炎症因子分泌的影响

目的:研究白藜芦醇对高糖环境下牙龈上皮细胞TLR4表达及炎症因子分泌的影响,探讨白藜芦醇对伴糖尿病牙周炎患者的治疗作用及相关分子机制.方法:体外培养牙龈上皮细胞,按照作用方式不同分为正常对照组、高糖组和高糖+白藜芦醇组.荧光定量PCR检测TLR4表达情况;取第3代牙龈上皮细胞,高糖环境下采用或不采用白藜芦醇处理24 h,随后用100 ng/mL LPS处理2 h,ELISA检测IL-1β 、IL-6、IL-8和TNF-α分泌;Western印迹法检测TLR4信号通路下游分子NF-κB p65、p38MAPK和STAT3磷酸化.采用SPSS 17.0软件包对数据进行统计学分析.结果:白藜芦醇可以逆转高糖环境下牙龈上皮细胞TLR4水平的升高,同时抑制高糖环境下LPS诱导的IL-1β、IL-6、IL-8和TNF-α表达.Western印迹结果显示,白藜芦醇也可抑制TLR4信号通路下游分子NF-κB p65、p38MAPK和STAT3磷酸化.结论:白藜芦醇通过负向调控TLR4信号通路减轻高糖环境下牙龈上皮细胞炎症因子的分泌.     

内毒素对人牙周膜细胞Toll样受体2和Toll样受体4表达的影响

目的:观察内毒素(LPS)刺激后人牙周膜细胞Toll样受体2和Toll样受体4的表达,探讨牙周膜细胞作为免疫活性细胞在牙周炎局部免疫反应中的作用。方法:组织块法原代培养人牙周膜细胞,经来源鉴定后,取生长良好的第4代细胞用LPS进行刺激,分别于刺激0、4、8、12、24 h,运用免疫细胞化学染色法检测TLR2和TLR4在牙周膜细胞中的表达;利用多功能真彩色细胞分析管理系统进行图像分析,对TLR2和TLR4进行免疫组化评分(IHS)。结果:无LPS刺激的对照组(刺激O h)TLR2和TLR4的表达均为弱阳性,加入LPS后,染色增强,且随着刺激时间的延长而增加(P<0.05),LPS刺激后各时间点TLP2和TLR4的IHS分值与对照组相比差异均有统计学意义(P<0.05)。结论:内毒素的刺激可以使牙周膜细胞TLR2和TLR4表达增多,提示牙周膜细胞参与牙周局部免疫反应。     

Toll样受体2和4在脂多糖诱导人牙周膜成纤维细胞表达细胞核因子-κB受体活化因子配基中的作用

目的 观察在脂多糖（LPS）刺激下,人牙周膜成纤维细胞（HPDLFs）中Toll样受体2（TLR2）和Toll样受体4（TLR4）表达水平的抑制对其表达细胞核因子-κB受体活化因子配基（RANKL）的影响。方法 选用100 ng·mL-1、1 μg·mL-1、10 μg·mL-1大肠杆菌LPS分别刺激HPDLFs,刺激6、12、24、48 h后,采用酶联免疫吸附试验（ELISA）检测HPDLFs表达RANKL的水平。分别运用不同滴度的anti-TLR2+anti-TLR4、anti-TLR2、anti-TLR4抗体预处理HPDLFs,观察
1 μg·mL-1 LPS刺激下,其RANKL表达水平的变化。结果 LPS刺激HPDLFs 6 h后,即可检测到RANKL的表达,24 h达到顶峰,然后逐渐下降;各LPS质量浓度组的规律基本一致。分别用anti-TLR2+anti-TLR4、anti-TLR2、anti-TLR4抗体预处理HPDLFs,在1 μg·mL-1 LPS刺激下,其产生RANKL的水平明显下降（P<0.05）;3组中,RANKL的表达水平有明显差异（P<0.05）,其中anti-TLR2+anti-TLR4抗体处理组RANKL表达量最少,anti-TLR4抗体处理组次之,anti-TLR2
抗体处理组RANKL的表达量最高。结论TLR2、TLR4均参与了LPS诱导HPDLFs表达RANKL的过程;与anti-TLR2抗体相比,anti-TLR4抗体能更有效地抑制LPS刺激后HPDLFs表达RANKL的能力。     

Modulation of cell proliferation,survival and gene expression by RAGE and TLR signaling in cells of the innate and adaptive immune response: role of p38 MAPK and NF-KB

Objective

The aim of this study was to evaluate a possible synergism between AGE-RAGE and TLR4 signaling and the role of p38 MAPK and NF-kB signaling pathways on the modulation of the expression of inflammatory cytokines and proliferation of cells from the innate and adaptive immune response.

Material and Methods

T lymphocyte (JM) and monocyte (U937) cell lines were stimulated with LPS and AGE-BSA independently and associated, both in the presence and absence of p38 MAPK and NF-kB inhibitors. Proliferation was assessed by direct counting and viability was assessed by a biochemical assay of mitochondrial function. Cytokine gene expression for RAGe, CCL3, CCR5, IL-6 and TNF-α was studied by RT-PCR and RT-qPCR.

Results

RAGE mRNA expression was detected in both cell lines. LPS and AGE-BSA did not influence cell proliferation and viability of either cell line up to 72 hours. LPS and LPS associated with AGE induced expression of IL-6 and TNF-α in monocytes and T cells, respectively.

Conclusions

There is no synergistic effect between RAGE and TLR signaling on the expression of IL-6, TNF-α , RAGE, CCR5 and CCL3 by monocytes and lymphocytes. Activation of RAGE associated or not with TLR signaling also had no effect on cell proliferation and survival of these cell types.     

Roles of dental pulp fibroblasts in the recognition of bacterium-related factors and subsequent development of pulpitis

As caries-related bacteria invade deeply into dentin and come into close proximity to the pulp, inflammatory cells (such as lymphocytes, macrophages and neutrophils) infiltrate into the bacterium-invaded area and consequently pulpitis develops. Many types of cytokines and adhesion molecules are responsible for the initiation and progression of pulpitis. Dental pulp fibroblasts, a major cell type in the dental pulp, also have capacity to produce pro-inflammatory cytokines and express adhesion molecules in response to pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide. The innate immune system senses microbial infection using pattern recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD), for PAMPs. In this review, we summarize the roles of dental pulp fibroblasts in the recognition of invaded bacterium-related factors via TLR and NOD pathways, and the subsequent pulpal immune responses, leading to progressive pulpitis.     

LPS对人牙周膜细胞Toll样受体4及下游炎症因子表达的影响

目的：观察革兰氏阴性菌外膜成分脂多糖（LPS）对牙周膜细胞（PDLCs）Toll样受体4（TLR4）及下游炎症因子表达的影响。方法：用浓度为10μg／mL的LPS对牙周膜细胞进行刺激,Western Blot检测牙周膜细胞上TLR4受体表达,同时ELISA检测培养基上清中炎症因子TNF－α、IL－1β的含量。结果：加入LPS后,牙周膜细胞上TLR4蛋白表达增强,炎症因子TNF－α、IL－1β含量增多,且随LPS的刺激时间增加而呈上升趋势。结论：LPS促进PDLCs的TLR4表达,并导致炎症因子分泌增加,TLR4信号通路在牙周炎发病过程中具有重要作用。     

IL-1β作用于人牙周膜成纤维细胞后TRAF6蛋白表达的研究

目的通过研究IL-1β干预下,TRAF6在人牙周膜成纤维细胞中的蛋白表达,为细胞因子网络对细胞功能调控作用的研究提供新资料。方法原代培养人牙周膜成纤维细胞,并进行免疫学鉴定。取5～8代细胞以IL-1β梯度浓度干预,采用免疫组织化学方法检测TRAF6在人牙周膜成纤维细胞中的表达。结果TRAF6在正常的人牙周膜成纤维细胞中呈阴性表达,IL-1β干预后,TRAF6呈阳性表达,并且随IL-1β干预的浓度的升高而增强。结论IL-1β作用后,TRAF6在牙周膜成纤维细胞出现表达,且与IL-1β作用浓度相关。     

Expression of HMGB1 and HMGN2 in gingival tissues, GCF and PICF of periodontitis patients and peri-implantitis

Background and objective

High mobility group chromosomal protein B1 (HMGB1) and N2 (HMGN2), two members of high mobility group (HMG) family, play important role in inflammation. The purpose of this study was to investigate the expression of HMGB1 and HMGN2 in periodontistis.

Materials and methods

The expression of HMGB1 and HMGN2 mRNA in gingival tissues and gingival crevicular fluid (GCF) in chronic periodontitis (CP), generalised aggressive periodontitis (G-AgP) patients and healthy subjects was detected by real-time PCR. The protein level of HMGB1 and HMGN2 in peri-implant crevicular fluid (PICF), peri-implant crevicular fluid of peri-implantitis (PI-PICF) and normal patients was determined by Western blotting. Furthermore, IL-1β, IL-6, IL-8, TNF-α and HMGB1 levels in GCF, PI-PICF and healthy-PICF samples from different groups were determined by ELISA.

Results

HMGN2 expression was increased in inflamed gingival tissues and GCF from CP and G-ApG groups compared to control group. HMGB1 expression was the highest in the gingival tissues and GCF from CP patients and was accompanied by increased concentrations of IL-1β, IL-6, IL-8 proinflammaory cytokines.

Conclusion

To our knowledge, this is the first study reporting that the expression of HMGB1 and HMGN2 was increased in the gingival tissues and GCF in CP and G-AgP and the PICF in PICF. Our data suggest that HMGB1 may be a potential target for the therapy of periodontitis and PI.     

Immunocompetent cells and cytokine expression in the rat periodontal ligament at the initial stage of orthodontic tooth movement

Objective

The aim of the present study is to investigate the involvement of immunocompetent cells, pro-inflammatory cytokines and HSP, to evaluate a change of periodontal ligament during the initial stage of orthodontic tooth movement.

Design

In the present study, we investigated the distributional density of immunocompetent cells, the localisation of cytokines, and the expression levels of their mRNA in the periodontal ligament during the initial stage of orthodontic tooth movement, using immunohistochemistry and real-time PCR.

Results

Orthodontic tooth movement led to significant recruitment of OX6⁺ cells and ED1⁺ cells in the rat PDL. Double-immunofluorescence staining showed that some ED1⁺ cells expressed pro-inflammatory factors of IL-1β and TNF-α in the PDL during orthodontic tooth movement. Real-time PCR analysis revealed that the expression levels of IL-1β (Il1b) and TNF-α (Tnf) mRNA gradually increased following its decrease after 1 h of orthodontic tooth movement. These findings suggest that ED1⁺ cells are involved in the expression of TNF-α and IL-1β and the subsequent regulation of bone resorption on pressure side. HSP27 (Hspb1) mRNA levels were significantly increased as compared with the control at 1 h of the initial stage of treatment.

Conclusion

ED1⁺ cells involved in the expression of TNF-α and IL-1β may play an important role in the initial reaction of the PDL and in the induction of the osteoclastic bone resorption during orthodontic tooth movement.     

Pressure induces interleukin-6 expression via the P2Y6 receptor in human dental pulp cells

Background and objective

An increase in intrapulpal pressure occurs during inflammation and restorative procedures; however, the role of the pressure on human dental pulp cell (HDPC) is not yet clarified. In this study, the effect of pressure on interleukin-6 (IL-6) expression of HDPCs was examined.

Design

HDPCs were applied with pressure (0.7–1.4 g/cm²). The level of IL-6 mRNA and protein release was determined by RT-PCR and ELISA, respectively. The signalling pathways were investigated using inhibitors, antagonists, and small interfering RNA.

Results

The results showed that pressure up-regulated IL-6 mRNA expression and protein release in a time- and dose-dependent manner. The implication of P2Y receptor was exhibited by a significant inhibition of pressure-induced IL-6 expression by suramin, an antagonist for the non-specific purinergic receptor family. Using loss of function experiments, we showed MRS2578 (a specific P2Y6 antagonist), as well as P2Y6 small interfering RNA, abolished pressure-induced IL-6, whilst MRS2179 (a specific P2Y1 antagonist) and NF449 (a P2X1, P2X3, P2Y1, and P2Y2 antagonist) had no effect. Finally, we demonstrated that either the conditioned medium collected from pressurised dental pulp cells or addition of UDP, a selective agonist of P2Y6, up-regulated IL-6 expression in HDPCs.

Conclusions

These results indicate that pressure could induce IL-6 expression through the P2Y6 receptor in HDPCs, leading to a new insight of the role of pressure on cytokine release during pulpal inflammatory process.