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Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation.

Farges JC, Bellanger A, Ducret M, Aubert-Foucher E, Richard B, Alliot-Licht B, Bleicher F, Carrouel F - Front Physiol (2015)

Bottom Line: We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones.Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME.In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, UMR5242 Centre National de la Recherche Scientifique/ENS/Université Lyon 1, Equipe Physiopathologie des Odontoblastes Lyon, France ; Faculté d'Odontologie, Université Lyon 1, Université de Lyon Lyon, France ; Hospices Civils de Lyon, Service de Consultations et Traitements Dentaires Lyon, France ; Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, Institut de Biologie et Chimie des Protéines, UMR5305 Centre National de la Recherche Scientifique/Université Lyon 1 Lyon, France.

ABSTRACT
The penetration of cariogenic oral bacteria into enamel and dentin during the caries process triggers an immune/inflammatory response in the underlying pulp tissue, the reduction of which is considered a prerequisite to dentinogenesis-based pulp regeneration. If the role of odontoblasts in dentin formation is well known, their involvement in the antibacterial response of the dental pulp to cariogenic microorganisms has yet to be elucidated. Our aim here was to determine if odontoblasts produce nitric oxide (NO) with antibacterial activity upon activation of Toll-like receptor-2 (TLR2), a cell membrane receptor involved in the recognition of cariogenic Gram-positive bacteria. Human odontoblast-like cells differentiated from dental pulp explants were stimulated with the TLR2 synthetic agonist Pam2CSK4. We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones. NOS2 was the most up-regulated gene. NOS1 and NOS3 proteins were not detected in Pam2CSK4-stimulated or control cultures. NOS2 protein synthesis, NOS activity and NO extracellular release were all augmented in stimulated samples. Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME. In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones. NOS2 protein was immunolocalized in odontoblasts situated beneath the caries lesion but not in pulp cells from healthy teeth. These results suggest that odontoblasts may participate to the antimicrobial pulp response to dentin-invading Gram-positive bacteria through NOS2-mediated NO production. They might in this manner pave the way for accurate dental pulp healing and regeneration.

No MeSH data available.


Related in: MedlinePlus

NO released from unstimulated or Pam2CSK4-stimulated odontoblast-like cells alters Streptococcus mutans viability. Analysis of Streptococcus mutans growth after contact of bacteria for 15, 30, 60, or 90 min with culture supernatants of cells challenged with 10 μg/mL Pam2CSK4 for 24 h in the presence or in the absence of L-NAME. The number of Streptococcus mutans colony-forming units was reduced when odontoblast-like cells were stimulated with Pam2CSK4 compared to unstimulated ones. Pretreatment with the NOS inhibitor L-NAME increased the number of Streptococcus mutans colony-forming units in both unstimulated and Pam2CSK4-stimulated samples (n = 6). #p < 0.05 vs. control unstimulated cells. *p < 0.05 vs. cultures without L-NAME.
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Figure 3: NO released from unstimulated or Pam2CSK4-stimulated odontoblast-like cells alters Streptococcus mutans viability. Analysis of Streptococcus mutans growth after contact of bacteria for 15, 30, 60, or 90 min with culture supernatants of cells challenged with 10 μg/mL Pam2CSK4 for 24 h in the presence or in the absence of L-NAME. The number of Streptococcus mutans colony-forming units was reduced when odontoblast-like cells were stimulated with Pam2CSK4 compared to unstimulated ones. Pretreatment with the NOS inhibitor L-NAME increased the number of Streptococcus mutans colony-forming units in both unstimulated and Pam2CSK4-stimulated samples (n = 6). #p < 0.05 vs. control unstimulated cells. *p < 0.05 vs. cultures without L-NAME.

Mentions: Next, to evaluate the NO effect on the growth of cariogenic microorganisms, odontoblast-like cells were stimulated or not with 10 μg/mL Pam2CSK4 for 24 h with or without pretreatment with the NOS inhibitor L-NAME. Culture supernatants were collected and placed into contact with Streptococcus mutans bacteria for 15, 30, 60, or 90 min. In unstimulated samples, pretreatment with L-NAME increased the number of Streptococcus mutans colony-forming units, suggesting that NO from unstimulated odontoblast-like cells limits Streptococcus mutans growth (Figure 3). We observed that the number of Streptococcus mutans colony-forming units was clearly reduced in odontoblast-like cells stimulated with Pam2CSK4 compared to unstimulated ones, indicating a stronger antibacterial effect of culture supernatants from TLR2-activated cells. The decrease in NO production owing to increasing concentrations of L-NAME in Pam2CSK4-stimulated samples led to an augmentation of the number of Streptococcus mutans colony-forming units, thus indicating that NO production by odontoblast-like cells was indeed responsible for the observed slowdown of bacterial growth.


Human odontoblast-like cells produce nitric oxide with antibacterial activity upon TLR2 activation.

Farges JC, Bellanger A, Ducret M, Aubert-Foucher E, Richard B, Alliot-Licht B, Bleicher F, Carrouel F - Front Physiol (2015)

NO released from unstimulated or Pam2CSK4-stimulated odontoblast-like cells alters Streptococcus mutans viability. Analysis of Streptococcus mutans growth after contact of bacteria for 15, 30, 60, or 90 min with culture supernatants of cells challenged with 10 μg/mL Pam2CSK4 for 24 h in the presence or in the absence of L-NAME. The number of Streptococcus mutans colony-forming units was reduced when odontoblast-like cells were stimulated with Pam2CSK4 compared to unstimulated ones. Pretreatment with the NOS inhibitor L-NAME increased the number of Streptococcus mutans colony-forming units in both unstimulated and Pam2CSK4-stimulated samples (n = 6). #p < 0.05 vs. control unstimulated cells. *p < 0.05 vs. cultures without L-NAME.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4477070&req=5

Figure 3: NO released from unstimulated or Pam2CSK4-stimulated odontoblast-like cells alters Streptococcus mutans viability. Analysis of Streptococcus mutans growth after contact of bacteria for 15, 30, 60, or 90 min with culture supernatants of cells challenged with 10 μg/mL Pam2CSK4 for 24 h in the presence or in the absence of L-NAME. The number of Streptococcus mutans colony-forming units was reduced when odontoblast-like cells were stimulated with Pam2CSK4 compared to unstimulated ones. Pretreatment with the NOS inhibitor L-NAME increased the number of Streptococcus mutans colony-forming units in both unstimulated and Pam2CSK4-stimulated samples (n = 6). #p < 0.05 vs. control unstimulated cells. *p < 0.05 vs. cultures without L-NAME.
Mentions: Next, to evaluate the NO effect on the growth of cariogenic microorganisms, odontoblast-like cells were stimulated or not with 10 μg/mL Pam2CSK4 for 24 h with or without pretreatment with the NOS inhibitor L-NAME. Culture supernatants were collected and placed into contact with Streptococcus mutans bacteria for 15, 30, 60, or 90 min. In unstimulated samples, pretreatment with L-NAME increased the number of Streptococcus mutans colony-forming units, suggesting that NO from unstimulated odontoblast-like cells limits Streptococcus mutans growth (Figure 3). We observed that the number of Streptococcus mutans colony-forming units was clearly reduced in odontoblast-like cells stimulated with Pam2CSK4 compared to unstimulated ones, indicating a stronger antibacterial effect of culture supernatants from TLR2-activated cells. The decrease in NO production owing to increasing concentrations of L-NAME in Pam2CSK4-stimulated samples led to an augmentation of the number of Streptococcus mutans colony-forming units, thus indicating that NO production by odontoblast-like cells was indeed responsible for the observed slowdown of bacterial growth.

Bottom Line: We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones.Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME.In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génomique Fonctionnelle de Lyon, UMR5242 Centre National de la Recherche Scientifique/ENS/Université Lyon 1, Equipe Physiopathologie des Odontoblastes Lyon, France ; Faculté d'Odontologie, Université Lyon 1, Université de Lyon Lyon, France ; Hospices Civils de Lyon, Service de Consultations et Traitements Dentaires Lyon, France ; Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, Institut de Biologie et Chimie des Protéines, UMR5305 Centre National de la Recherche Scientifique/Université Lyon 1 Lyon, France.

ABSTRACT
The penetration of cariogenic oral bacteria into enamel and dentin during the caries process triggers an immune/inflammatory response in the underlying pulp tissue, the reduction of which is considered a prerequisite to dentinogenesis-based pulp regeneration. If the role of odontoblasts in dentin formation is well known, their involvement in the antibacterial response of the dental pulp to cariogenic microorganisms has yet to be elucidated. Our aim here was to determine if odontoblasts produce nitric oxide (NO) with antibacterial activity upon activation of Toll-like receptor-2 (TLR2), a cell membrane receptor involved in the recognition of cariogenic Gram-positive bacteria. Human odontoblast-like cells differentiated from dental pulp explants were stimulated with the TLR2 synthetic agonist Pam2CSK4. We found that NOS1, NOS2, and NOS3 gene expression was increased in Pam2CSK4-stimulated odontoblast-like cells compared to unstimulated ones. NOS2 was the most up-regulated gene. NOS1 and NOS3 proteins were not detected in Pam2CSK4-stimulated or control cultures. NOS2 protein synthesis, NOS activity and NO extracellular release were all augmented in stimulated samples. Pam2CSK4-stimulated cell supernatants reduced Streptococcus mutans growth, an effect counteracted by the NOS inhibitor L-NAME. In vivo, the NOS2 gene was up-regulated in the inflamed pulp of carious teeth compared with healthy ones. NOS2 protein was immunolocalized in odontoblasts situated beneath the caries lesion but not in pulp cells from healthy teeth. These results suggest that odontoblasts may participate to the antimicrobial pulp response to dentin-invading Gram-positive bacteria through NOS2-mediated NO production. They might in this manner pave the way for accurate dental pulp healing and regeneration.

No MeSH data available.


Related in: MedlinePlus