Salivary pellets induce a pro-inflammatory response involving the TLR4–NF-kB pathway in gingival fibroblasts

BACKGROUND Whole saliva provokes a substantial pro-inflammatory response in gingival fibroblasts. This raises the question whether the salivary pellet, which is used for diagnostic purposes, also has a pro-inflammatory capacity and, if yes, what the underlying mechanisms at the molecular level are. METHODS We examined the ability of extensively washed salivary pellets to provoke the expression of chemokines in gingival fibroblasts by real-time polymerase chain reaction and immunoassays. Protein composition was determined with proteomic analysis. Endotoxins were analyzed by a Limulus assay and removed by affinity chromatography. The inhibitors TAK-242 and BAY11-7082 were used to determine the involvement of the TLR4 and NF-kB signaling, respectively. Western blot was performed to detect phosphorylated p65. RESULTS The experiments show that salivary pellets and the corresponding washing solution contain pro-inflammatory activity without impairing cell viability. Proteomic analysis revealed proteins with a binding capacity for lipopolysaccharides, and the Limulus assay indicated the presence of endotoxin in the salivary pellets. Blocking TLR4 with TAK-242 and depletion of endotoxins both lowered the capacity of salivary pellets to increase chemokine expression and phosphorylation of p65. BAY11-7082 suppressed chemokine expression in response to the salivary pellets. Autoclaving salivary pellets also reduced their pro-inflammatory activity. CONCLUSIONS The data support the molecular mechanism of a TLR4-NF-kB-dependent pro-inflammatory response of the gingival fibroblasts exposed to preparations of washed salivary pellets. Together, the data indicate that the salivary pellet is rich in endotoxin but it is mainly a heat labile fraction that accounts for the chemokine expression in the bioassay.