Porphyromonas gingivalis LPS Priming Enhances TLR2‐Dependent Chemokine Responses in Human Periodontal Ligament Fibroblasts

ABSTRACT Objectives Periodontal ligament fibroblasts (PDLFs) contribute to periodontal inflammation by sensing bacterial components through Toll‐like receptors (TLRs). This study investigated the distinct inflammatory and regulatory responses of PDLFs to TLR2 stimulation and ultrapure Porphyromonas gingivalis lipopolysaccharide ( P. g . LPS), used as a TLR4 ligand, and examined whether prior exposure to P. g . LPS modulates subsequent TLR2‐mediated signaling. Materials and Methods Human PDLFs were stimulated with the TLR2 ligand Pam3CSK4 or ultrapure P. g . LPS. Chemokine gene expression was quantified by real‐time polymerase chain reaction, and IL‐8 and CCL5 concentrations in culture supernatants were measured by enzyme‐linked immunosorbent assay. Activation of the mitogen‐activated protein kinase and nuclear factor‐κB pathways and TLR2 and TLR4 protein expression were examined by western blotting. Cells were pre‐stimulated with P. g . LPS and subsequently exposed to Pam3CSK4, either with or without washing to remove the residual stimulus. TLR‐related molecules were also evaluated at the mRNA level. Results Pam3CSK4 consistently induced IL8 and CCL5 mRNA expression and activated the mitogen‐activated protein kinase and nuclear factor‐κB pathways, whereas P. g . LPS alone induced only minimal inflammatory signaling. Pre‐stimulation with P. g . LPS enhanced subsequent TLR2‐dependent responses in a manner dependent on stimulus persistence. ELISA analyses showed that IL‐8 and CCL5 secretion patterns were generally consistent with their corresponding mRNA expression profiles. Western blotting confirmed the expression of TLR2 and TLR4 proteins in PDLFs. Conclusions These findings indicate that P. g . LPS primes PDLFs for enhanced TLR2‐dependent inflammatory responses, particularly under conditions of persistent P. g . LPS exposure.