Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts.
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.
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.
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.
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.
Tang L
，Zhou XD
，Wang Q
，Zhang L
，Wang Y
，Li XY
，Huang DM
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Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis.
Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects.
Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n=14) and healthy control subjects (n=8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll-like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor-κB1 and its putative inhibitor NF-κB inhibitor-like protein1, and of interleukin-1β, interleukin-6, interleukin-8, tumour necrosis factor-α, monocyte chemotactic protein-1 and regulated upon activation, normal T-cel expressed, and secreted, were assessed by real-time PCR.
Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture-positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis-negative persons.
Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis-positive donors are more responsive to an in vitro P. gingivalis challenge.
Scheres N
，Laine ML
，Sipos PM
，Bosch-Tijhof CJ
，Crielaard W
，de Vries TJ
，Everts V
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