Functional Analysis of Pattern Recognition Receptors Expression in Pulpal Cells

Objectives: Generally, initial sensing of microbial pathogens is mediated by the pattern recognition receptors (PRRs) for pathogen-associated molecules uniquely expressed by the microbes. Among the PRRs, Toll-like receptors (TLRs) and Nucleotide-binding oligomerization domain proteins (NODs) elicit appropriated downstream signaling events leading to inflammatory responses. We hypothesized that these PRRs play roles in pathogenesis of pulpitis following dental caries infected by caries-related bacteria. The aim of this study was to investigate the expression patterns of TLRs and NODs in human pulp-derived fibroblastic cells (HPF) and the cooperative effects of NOD ligand with TLR ligand on the induction of inflammatory mediators such as cytokines and adhesion molecules.

Methods: The mRNA and protein expression levels of PRRs in primary cultured HPF were assessed by RT-PCR and flow cytometry, respectively. To determine the PRRs functional activities, the expression levels of pro-inflammatory mediators (IL-6, IL-8, MCP-1, IP-10, ICAM-1 and PGE2) in HPF after stimulation with PRR-specific ligands were analyzed by RT-PCR, ELISA and flow cytometry. To further investigate the possible interaction between NOD2 and TLR pathways, HPF were co-stimulated with MDP (NOD2 ligand) and Pam3CSK4 (TLR2 ligand) or LPS (TLR4 ligand).

Results: In primary cultured HPF, the constitutive mRNA expressions of TLR2, TLR4, NOD1 and NOD2 were found, but the protein expression level of TLR4 was lower than that of others. Upon stimulation with MDP, Pam3CSK4 or LPS, the production levels of pro-inflammatory mediators were markedly up-regulated in a time-dependent manner. Interestingly, the synergistic effects of MDP on the productions of these mediators stimulated with Pam3CSK4, not with LPS, were observed.

Conclusion: These findings demonstrated that NOD2 ligand act cooperatively with molecules sensed by TLR2 to produce pro-inflammatory mediators in HPF and suggest that the interaction between NOD2 and TLR2 pathways may represent an important modulatory mechanism of immune responses in dental pulp tissue.