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Functional TLRs and NODs in Human Gingival Fibroblasts

Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, Sendai, 980-8575, Japan

View larger version (66K): [in this window] [in a new window]   Figure 1. Expressions of TLR1, TLR2, TLR3, TLR4, MD-2, TLR5, TLR6, TLR7, TLR8, TLR9, MyD88, NOD1, and NOD2 mRNA in human gingival fibroblasts. Fibroblasts were cultured until confluent at 37°C. After incubation, the total RNA was extracted, and the mRNA expressions of TLR1, TLR2, TLR3, TLR4, MD-2, TLR5, TLR6, TLR7, TLR8, TLR9, MyD88, NOD1, and NOD2 were analyzed with PCR, with the primers shown in (a). The clear expressions of these mRNAs were detected (b). The results presented are representative of 3 different experiments demonstrating similar results.

View larger version (45K): [in this window] [in a new window]   Figure 2. Expressions of TLR1, TLR2, TLR3, TLR4, MD-2, TLR5, TLR6, TLR7, TLR8, TLR9, MyD88, NOD1, and NOD2 in human gingival fibroblasts, detected by flow cytometry. Fibroblasts were cultured until confluent at 37°C. The cell-surface expressions of TLR1, TLR2, TLR4, MD-2, TLR5, TLR6, and intracellular TLR3, TLR7, TLR8, TLR9, MyD88, NOD1, and NOD2 were assessed by flow cytometry. Thin lines represent the isotype Ab control. The results presented are representative of 4 different experiments demonstrating similar results.

View larger version (37K): [in this window] [in a new window]   Figure 3. Expressions of TLR1, TLR2, TLR3, TLR4, MD-2, TLR5, TLR6, TLR7, TLR8, TLR9, MyD88, NOD1, and NOD2 in human gingival fibroblasts, detected by immunostaining. Fibroblasts were cultured until confluent at 37°C. After fixation, the cells were treated with anti-TLR2, anti-TLR3, anti-TLR4, anti-TLR5, anti-TLR6, anti-TLR7, anti-TLR8, anti-TLR9, anti-MyD88, anti-NOD1, and anti-NOD2 antibodies and then visualized with Alexa Fluor 488 (green). The nuclei were visualized by being stained with 4',6-diamino-2-phenylindole (blue). Scale bars: 20 µm. The results are representative of 3 different experiments demonstrating similar results.

View larger version (10K): [in this window] [in a new window]   Figure 4. IL-6, IL-8, and MCP-1 production induced by TLR and NOD ligands in human gingival fibroblasts was mediated by NF-B. (a) Fibroblasts were incubated for 24 hrs in the presence or absence of FSL-1 (1 nM), Poly I:C (10 µg/mL), lipid A (10 ng/mL), ssPolyU (10 µg/mL), CpG DNA (1 µM), FK156 (10 µg/mL), or MDP (10 µg/mL). IL-6, IL-8, and MCP-1 levels in the culture supernatants were determined with ELISA, and expressed as means ± SD. *Values marked differed significantly from those with medium alone (none). The results presented are representative of 3 different experiments demonstrating similar results. (b) Gingival fibroblasts transfected with siRNA targeting NF-B p65 for 24 hrs were stimulated with FSL-1 (1 nM), Poly I:C (10 µg/mL), lipid A (10 ng/mL), ssPolyU (10 µg/mL), CpG DNA (1 µM), FK156 (10 µg/mL), or MDP (10 µg/mL). After 24 hrs of stimulation, the IL-8 levels in the culture supernatants were determined with ELISA, and expressed as means ± SD. *,#Values marked differed significantly from those with medium alone or respective cultures stimulated with the indicated ligands, respectively. The results presented are representative of 4 different experiments demonstrating similar results.