Roles of TLR2, TLR4, NOd2, and NOd1 in Pulp Fibroblasts

Pulp fibroblasts express various pro-inflammatory mediators leading to marked infiltration of inflammatory cells in the progression of pulpitis. We hypothesized that pulp fibroblasts play roles in the recognition of invaded caries-related bacteria and the subsequent innate immune responses. We found clear expressions of TLR2, NOD1, and NOD2 and a faint expression of TLR4 in human dental pulp fibroblasts (HDPF) by RT-PCR and flow cytometry. We also observed that various pro-inflammatory mediators, including cytokines, chemokines, adhesion molecules, prostaglandin E-2 and its key enzyme COX-2, not iNOS or caspase-1, were markedly up-regulated by stimulation with these TLR and NOD agonists. More over, the NOD2 agonist acted synergistically with the TLR2, not the TLR4, agonist to stimulate the production of pro-inflammatory mediators in HDPF. These findings indicate that TLR2, TLR4, NOD2, and NOD1 in HDPF are functional receptors, and NOD2 is a modulator of signals transmitted through TLR2 in pulpal immune responses, leading to progressive pulpitis. Abbreviations: HDPF, human dental pulp fibroblasts; IL, interleukin; MCP-1, monocyte-chemoattractant protein-1; IP-10, interferon-gamma-inducible protein-10; LPS, lipopolysaccharide; ICAM-1, intercellular adhesion molecule-1; PG, prostaglandin; iNOS, inducible nitric oxide synthase; PRR, pattern recognition receptor; PAMP, pattern-associated molecular pattern; TLR, Toll-like receptor; NOD, nucleotide-binding oligomerization domain; MDP, muramyldipeptide; NF-kappa B, nuclear factor-kappa B; RT-PCR, reverse-transcription polymerase chain-reaction; DMEM, Dulbecco's modified Eagle's medium; iE-DAP, gamma-D-glutamyl-meso-diaminopimelic acid; iE-Lys, gamma-D-glutamyl-lysine; MDP-LL, N-acetylmuramyl-L-alanyl-L-isoglutamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; VCAM-1, vascular cell adhesion molecule-1; FITC, fluorescein isothiocyanate; COX-2, cyclooxygenase-2.