Limits...
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

Pam2CSK4 increases NOS activity and extracellular NO production by odontoblast-like cells. (A) Analysis of intracellular NO by the measurement of nitrite concentration in cells stimulated with 10 μg/mL Pam2CSK4 for the indicated times. Production of intracellular nitrite was strongly increased in PAM2CSK4-stimulated samples, being maximal after 8 h (n = 5). (B) Determination of NO concentration in culture supernatants of odontoblast-like cells stimulated with 10 μg/mL Pam2CSK4. Nitrite progressively accumulated in the culture medium, reaching a concentration of approximately 40 μmol/L after 24 h of stimulation (n = 5). *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4477070&req=5

Figure 2: Pam2CSK4 increases NOS activity and extracellular NO production by odontoblast-like cells. (A) Analysis of intracellular NO by the measurement of nitrite concentration in cells stimulated with 10 μg/mL Pam2CSK4 for the indicated times. Production of intracellular nitrite was strongly increased in PAM2CSK4-stimulated samples, being maximal after 8 h (n = 5). (B) Determination of NO concentration in culture supernatants of odontoblast-like cells stimulated with 10 μg/mL Pam2CSK4. Nitrite progressively accumulated in the culture medium, reaching a concentration of approximately 40 μmol/L after 24 h of stimulation (n = 5). *p < 0.05.

Mentions: To determine whether NOS2 up-regulation upon Pam2CSK4 stimulation was accompanied by an increase in NOS activity and NO production, intracellular and extracellular NO concentrations were assessed by the measure of the NO degradation end-product nitrite. We observed a strong intracellular nitrite increase that was maximal after 8 h (Figure 2A). To assess NO diffusion to the extracellular compartment, we measured nitrite concentration in supernatants of Pam2CSK4-stimulated odontoblast-like cells. We found a progressive accumulation of nitrite in the culture medium reaching a concentration of approximately 40 μmol/L after 24 h, the longest stimulation time tested (Figure 2B). In unstimulated samples, the nitrite concentration remained low and constant with time, of approximately 10 μmol/L.


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)

Pam2CSK4 increases NOS activity and extracellular NO production by odontoblast-like cells. (A) Analysis of intracellular NO by the measurement of nitrite concentration in cells stimulated with 10 μg/mL Pam2CSK4 for the indicated times. Production of intracellular nitrite was strongly increased in PAM2CSK4-stimulated samples, being maximal after 8 h (n = 5). (B) Determination of NO concentration in culture supernatants of odontoblast-like cells stimulated with 10 μg/mL Pam2CSK4. Nitrite progressively accumulated in the culture medium, reaching a concentration of approximately 40 μmol/L after 24 h of stimulation (n = 5). *p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Pam2CSK4 increases NOS activity and extracellular NO production by odontoblast-like cells. (A) Analysis of intracellular NO by the measurement of nitrite concentration in cells stimulated with 10 μg/mL Pam2CSK4 for the indicated times. Production of intracellular nitrite was strongly increased in PAM2CSK4-stimulated samples, being maximal after 8 h (n = 5). (B) Determination of NO concentration in culture supernatants of odontoblast-like cells stimulated with 10 μg/mL Pam2CSK4. Nitrite progressively accumulated in the culture medium, reaching a concentration of approximately 40 μmol/L after 24 h of stimulation (n = 5). *p < 0.05.
Mentions: To determine whether NOS2 up-regulation upon Pam2CSK4 stimulation was accompanied by an increase in NOS activity and NO production, intracellular and extracellular NO concentrations were assessed by the measure of the NO degradation end-product nitrite. We observed a strong intracellular nitrite increase that was maximal after 8 h (Figure 2A). To assess NO diffusion to the extracellular compartment, we measured nitrite concentration in supernatants of Pam2CSK4-stimulated odontoblast-like cells. We found a progressive accumulation of nitrite in the culture medium reaching a concentration of approximately 40 μmol/L after 24 h, the longest stimulation time tested (Figure 2B). In unstimulated samples, the nitrite concentration remained low and constant with time, of approximately 10 μmol/L.

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