NOD1 and NOD2 mediate sensing of periodontal pathogens

Abstract

In bacterial infection, Nucleotide-binding Oligomerization Domain (NOD) 1 and NOD2 induce innate immune responses by recognizing fragments of the bacterial component peptidoglycan (PGN). To determine the roles of these receptors in detection of periodontal pathogens, we stimulated human embryonic kidney cells expressing NOD1 or NOD2 with heat-killed Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum or their soluble PGNs (sPGNs). All bacteria and their sPGNs could stimulate activation of NF-kappaB. However, there were differences in NOD1- and NOD2-stimulatory activities among the species of bacteria. P. gingivalis showed weaker NOD1- and NOD2-stimulatory activities than did other bacteria. These differences in activities were confirmed by production of interleukin-8 from oral epithelial cells stimulated with sPGNs. These findings indicate that both NOD1 and NOD2 might be involved in the recognition of periodontal pathogens, and that the weak NOD-stimulatory property of P. gingivalis might be helpful for survival in the periodontal pocket.

Figure 1.

Activation of NOD1 and NOD2 by stimulation with periodontopathic bacteria samples. HEK293T cells were transfected for 24 hrs with pUNO/vector, pUNO/hNod1 or pUNO/hNod2 plasmid, together with NF-κB-firefly luciferase and renilla luciferase reporter plasmid. After 24 hrs, the cells were stimulated with 1 µg/mL of A-iE-DAP, or 1 µg/mL of MDP or heat-killed bacteria. Luciferase activities were measured 12 hrs after stimulation and expressed as means ± SD of triplicate experiments. The results are representative of 3 different experiments. P.g., P. gingivalis ATCC 33277; A.a., A. actinomycetemcomitans; F.n., F. nucleatum; E.c., E. coli; A.v., A. viridans.^††P < 0.01 vs. control vector; *P < 0.05, **P < 0.01 vs. (-); ^#P < 0.05, ^##P < 0.01 vs. P. g.

Figure 2.

Activations of NOD1 and NOD2 by sPGNs from periodontopathic bacteria. (A) Diptericin expression in Drosophila S2* cells by stimulation with purified sPGNs from periodontopathic bacteria. S2* cells stably transfected with diptericin-luciferase reporter plasmid were seeded into 96-well plates and differentiated into macrophage-like cells by incubation with 1 µM 20-hydroxyecdysone for 24 hrs. Then the cells were stimulated with sPGN samples, and the luciferase activities were measured 4 hrs after stimulation and expressed as means ± SD of triplicate experiments. The results are representative of 3 different experiments. HEK293T cells were transfected for 24 hrs with pUNO/hNod1 (B) or pUNO/hNod2 plasmid (C) together with NF-κB-firefly luciferase and renilla luciferase reporter plasmid. After 24 hrs, the cells were stimulated with sPGN samples. Then luciferase activities were measured 12 hrs after stimulation and expressed as means ± SD of triplicate experiments. The results are representative of 3 different experiments. P.g., P. gingivalis ATCC 33277; A.a., A. actinomycetemcomitans; F.n., F. nucleatum; E.c., E. coli; A.v., A. viridans. *P < 0.05, **P < 0.01 vs. (-); ^#P < 0.05, ^##P < 0.01 vs. P.g.

Figure 3.

Activations of NOD1 and NOD2 by strains of P. gingivalis. HEK293T cells were transfected for 24 hrs with pUNO/hNod1 (A) or pUNO/hNod2 plasmid (B) together with NF-κB-firefly luciferase and renilla luciferase reporter plasmid for 24 hrs, and the cells were stimulated with heat-killed P. gingivalis strains: W83, TDC60, TDC117, TDC275, SU63, GAI7802, and ATCC 33277. Luciferase activities were measured 12 hrs after stimulation and expressed as means ± SD of triplicate experiments. The results are representative of 3 different experiments. *P < 0.05, **P < 0.01 vs. (-).

Figure 4.

Production of IL-8 in oral epithelial cells. (A) HSC-2 cells were stimulated with A-iE-DAP or MDP in a serum-starved condition after treatment with or without IFN-γ. At 2 hrs after stimulation, the cell culture medium was supplemented with 10% FBS by the addition of FBS and further incubated for 22 hrs. The levels of IL-8 in culture medium were measured. (B) IFN−γ-primed HSC-2 cells were stimulated with sPGNs in a serum-starved condition. At 2 hrs after stimulation, the cell culture medium was supplemented with 10% FBS by the addition of FBS and further incubated for 22 hrs. The levels of IL-8 in culture medium were measured. Data are expressed as means ± SD of triplicate experiments. The results are representative of 3 different experiments. P.g., P. gingivalis ATCC 33277; A.a., A. actinomycetemcomitans; F.n., F. nucleatum; E.c., E. coli; A.v., A. viridans. *P < 0.05, **P < 0.01 vs. (-); ^#P < 0.05, ^##P < 0.01 vs. P.g.