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

Abstract

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.

Keywords: Gingival fibroblast; Inflammation; Lipopolysaccharide; Salivary pellet; Toll-like receptor.

Fig. 1

To observe chemokine expression, gingival fibroblasts were exposed to a series of dilutions of salivary pellet resuspended in serum-free medium or pellet supernatants for 6 hours. The numbers (1, 2, 4, 8) indicate the number of washing steps. A panel of chemokines was subjected to RT-qPCR assay. Data were normalized to expression levels of control cultures with serum-free medium alone. Circles represent the mean ± standard deviation of three experiments with two cell donors. Not indicated are p-values > 0.1

Fig. 2

To analyze chemokine protein release, gingival fibroblasts were exposed to a series of dilutions of salivary pellet resuspended in serum-free medium or pellet supernatants for 6 hours. The numbers (1, 2, 4, 8) indicate the number of washing steps. Protein release for CXCL8 and CXCL1 was measured by immunoassays. Data were normalized to expression levels of control cultures with serum-free medium alone. Circles represent the mean ± standard deviation of three experiments with two cell donors. Not indicated are p-values > 0.1

Fig. 3

Gingival fibroblasts were exposed to different dilutions of resuspended salivary pellet or pellet supernatant for 6 hours to measure viability. Viability was determined via the conversion rate of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan crystals and optical density was measured with a photometer. Data were normalized to untreated control. Circles represent the mean ± standard deviation of three experiments with two cell donors. Not indicated are p-values > 0.1

Fig. 4

Prior phase contrast and live-dead staining microscopy gingival fibroblasts were exposed to a series of dilutions of salivary pellet and pellet supernatant, 1, 2, 4, 8 fold, respectively, for 6 h. Visual inspection using phase contrast microscopy revealed a regular fibroblastic morphology. Live-dead staining showed that most of the cells were colored green, indicating that they were vital (10 fold magnification)

Fig. 5

To reveal any impact on proliferation, gingival fibroblasts were exposed to a series of dilutions of salivary pellet resuspended in serum-free medium or pellet supernatants for 24 h. Proliferation was measured according to the bromdesoxyuridin (BrdU) labeling and detection protocol. Data were normalized to expression levels of control cultures with serum-free medium alone. Circles represent the mean ± standard deviation of three experiments with two cell donors. Not indicated are p-values > 0.1

Fig. 6

To reveal the involvement of TLR4 signaling endotoxin, removal agents were used and signal pathways were blocked. Gingival fibroblasts were exposed to salivary pellet that had been washed four times and filtered saliva for 6 hours. a Endotoxin removal resins reduced endotoxin-induced chemokine expression. Signal pathways were blocked with the TLR4 inhibitor TAK-242 (25 μM). b Salivary pellet that had been washed four times and filtered saliva increased phospho-p65 signaling in gingival fibroblasts, and TAK-242 reduced this process. Tumor necrosis factor (TNF)-α (10 ng/mL), served as positive, and serum-free medium as negative control. Data were normalized to positive expression levels of salivary pellet that had been washed four times and filtered saliva. Circles represent the mean ± standard deviation of five experiments. Not indicated are p-values > 0.1