| Odontoblasts (ODs) line the inner aspect of the tooth and extend their cellular processes into dentin where they are the first cell to encounter carious bacteria or their products. The goal of this study is to define the role of ODs in microbial recognition and host responses by using a combination of tooth organ cultures to maintain native human ODs and primary cultures of human OD-like cells. The gene expression profile of immune components, detected in the OD layer include various proinflammatory cytokines, chemokines, complement components, C-reactive protein, growth factors, anti-inflammatory cytokines, receptors for chemokines, cytokines, complements, and receptors for bacterial components such as toll-like receptors (TLRs). Protein expression of TLR2, TLR4, and chemokine (C-C motif) receptor 6 (CCR6) was verified in human ODs in situ. The mature human ODs attaching to the dentin are capable of mounting the inflammatory responses to a TLR4-agonist (Escherichia coli LPS) via the up-regulation of IL-1beta, tumor necrosis factor alpha (TNFalpha), CCL20, hBD2, IL8, TLR2 and TLR4 but not to a TLR2 ligand (Pam3CSK4). Consistently, human OD-like cells were more responsive to Gram-negative bacteria which mainly signal through TLR4 activation, as confirmed by TLR4 RNA-silencing. Presence of TLR2 on the cell surface allowed the responses of OD-like cells to Gram-positive bacteria. All Gram-negative bacteria, Porphyromonas gingivalis, Prevotella intermedia , and Fusobacterium nucleatum, activated NF-kappaB (a common transcription factor regulating inflammation) via both TLR2 and TLR4 whereas all Gram-positive bacteria, Streptococcus mutans, Enterococcus faecalis, and Lactobacillus casei stimulated NF-kappaB through TLR2. The anti-inflammatory cytokine transforming growth factor-beta1 (TGF-beta1) previously shown to be released from the carious dentin decreased mRNA and protein expression of TLR2 and TLR4 thereby attentuating the inflammatory responses of ODs to all bacteria. In conclusion, our results demonstrate the differential role of TLRs and TGF-beta1 in dental inflammation and propose odontoblasts as a key cell in innate host defenses of the tooth. These data also suggest a principal role of TLR4 in mounting the immune responses from human ODs and subsequent pulpal inflammation. |
