title: Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts.
abstract: OBJECTIVE
  Human periodontal ligament fibroblasts (HPDLFs) play a crucial role in protecting against oral bacteria in periapical tissue. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) are two major forms of innate immune sensors that recognize microbial pathogens and initiate pro-inflammatory signalling. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein for TLR-mediated nuclear factor-κB (NF-κB) signalling pathway activation that induces the production of pro-inflammatory cytokines. The aim of this study was to investigate the expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. We also investigated the expression of TRAF6 and pro-inflammatory cytokines induced by the activation of TLRs and NODs.
  
  
  METHODS
  The expression of TLR2, TLR4, NOD1, and NOD2 was measured by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and immunostaining. HPDLFs were stimulated with TLR and NOD agonists. Then, the expression of TRAF6 was measured by real-time PCR and western blot. Concentrations of IL-1β, IL-6, and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Finally, by using small interfering RNA (siRNA) for TRAF6, we analysed the production of IL-1β, IL-6, and IL-8 in HPDLFs upon stimulation with TLRs and NODs agonists.
  
  
  RESULTS
  We found clear mRNA and protein expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. The expression levels of TRAF6 and pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) were markedly up-regulated upon the activation of TLRs and NODs. Furthermore, the co-activation of TLRs and NODs had synergistic effect on the production of TRAF6 and pro-inflammatory cytokines. We also found TRAF6 suppression resulted in reduced IL-1β, IL-6, and IL-8 expression upon TLR and NOD agonists challenge.
  
  
  CONCLUSIONS
  These findings indicated that TLR2, TLR4, NOD1, and NOD2 are functional receptors in HPDLFs during innate immune responses to invading bacteria, and a combination of signalling through TLRs and NODs leads to the synergistic enhancement of inflammatory reactions in HPDLFs. In addition, TLR and NOD signalling involving TRAF6 contribute to inflammatory responses in HPDLFs.
authors: State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichan University, Chengdu 610041, China.
authors: Lu
authors: L
authors: Tang
authors: Xue-dong
authors: XD
authors: Zhou
authors: Qian
authors: Q
authors: Wang
authors: Lan
authors: L
authors: Zhang
authors: Yao
authors: Y
authors: Wang
authors: Xiao-yu
authors: XY
authors: Li
authors: Ding-ming
authors: DM
authors: Huang
mesh_terms: D017209:Apoptosis / Q000276:immunology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003960:Diaminopimelic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004926:Escherichia coli; D005347:Fibroblasts / Q000276:immunology*; D020868:Gene Silencing; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology; D018836:Inflammation Mediators / Q000032:analysis; D053583:Interleukin-1beta / Q000032:analysis; D015850:Interleukin-6 / Q000032:analysis; D016209:Interleukin-8 / Q000032:analysis; D007378:Interleukins / Q000032:analysis*; D055666:Lipopeptides / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D053474:Nod1 Signaling Adaptor Protein / Q000819:agonists / Q000032:analysis*; D053473:Nod2 Signaling Adaptor Protein / Q000819:agonists / Q000032:analysis*; D010513:Periodontal Ligament / Q000166:cytology / Q000276:immunology*; D034741:RNA, Small Interfering; D048029:TNF Receptor-Associated Factor 6 / Q000032:analysis* / Q000235:genetics; D051195:Toll-Like Receptor 2 / Q000819:agonists / Q000032:analysis*; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000032:analysis*; D015854:Up-Regulation / Q000276:immunology
chemical_list: D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D007378:Interleukins; D055666:Lipopeptides; D008070:Lipopolysaccharides; C119067:NOD1 protein, human; C429018:NOD2 protein, human; D053474:Nod1 Signaling Adaptor Protein; D053473:Nod2 Signaling Adaptor Protein; C508075:Pam(3)CSK(4) peptide; D034741:RNA, Small Interfering; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D048029:TNF Receptor-Associated Factor 6; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D003960:Diaminopimelic Acid; C034331:N(2)-(gamma-D-glutamyl)-meso-2,2'-diaminopimelic acid