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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

NOS2 transcript and protein are up-regulated in inflamed pulps from decayed teeth compared to healthy ones. (A) Analysis of NOS2 gene expression in healthy and inflamed pulps with real-time RT-PCR (n = 3). *p < 0.05. (B) Immunohistochemical localization of NOS2 protein in healthy and bacteria-challenged inflamed pulps. No NOS2 staining was found in healthy sample, but odontoblasts and subodontoblast cells were stained in inflamed pulp beneath the caries lesion. D, dentin; Od, odontoblast layer; P, pulp. Data shown are representative of results obtained from independent experiments performed with two healthy and two carious teeth.
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Figure 4: NOS2 transcript and protein are up-regulated in inflamed pulps from decayed teeth compared to healthy ones. (A) Analysis of NOS2 gene expression in healthy and inflamed pulps with real-time RT-PCR (n = 3). *p < 0.05. (B) Immunohistochemical localization of NOS2 protein in healthy and bacteria-challenged inflamed pulps. No NOS2 staining was found in healthy sample, but odontoblasts and subodontoblast cells were stained in inflamed pulp beneath the caries lesion. D, dentin; Od, odontoblast layer; P, pulp. Data shown are representative of results obtained from independent experiments performed with two healthy and two carious teeth.

Mentions: To assess the in vivo relevance of these findings and determine whether NOS2 is expressed in odontoblasts, NOS2 transcript and protein were examined in healthy dental pulps and inflamed samples from carious teeth. We found that the NOS2 gene was strongly up-regulated in inflamed pulps compared to healthy ones (Figure 4A). NOS2 protein was clearly detected by immunostaining in odontoblasts and subodontoblast cells in the inflamed area, but not in cells in the non-inflamed area far from the lesion (not shown) or in healthy pulps (Figure 4B). Staining controls performed by using the mouse immunoglobulin G1 isotype were negative.


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)

NOS2 transcript and protein are up-regulated in inflamed pulps from decayed teeth compared to healthy ones. (A) Analysis of NOS2 gene expression in healthy and inflamed pulps with real-time RT-PCR (n = 3). *p < 0.05. (B) Immunohistochemical localization of NOS2 protein in healthy and bacteria-challenged inflamed pulps. No NOS2 staining was found in healthy sample, but odontoblasts and subodontoblast cells were stained in inflamed pulp beneath the caries lesion. D, dentin; Od, odontoblast layer; P, pulp. Data shown are representative of results obtained from independent experiments performed with two healthy and two carious teeth.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: NOS2 transcript and protein are up-regulated in inflamed pulps from decayed teeth compared to healthy ones. (A) Analysis of NOS2 gene expression in healthy and inflamed pulps with real-time RT-PCR (n = 3). *p < 0.05. (B) Immunohistochemical localization of NOS2 protein in healthy and bacteria-challenged inflamed pulps. No NOS2 staining was found in healthy sample, but odontoblasts and subodontoblast cells were stained in inflamed pulp beneath the caries lesion. D, dentin; Od, odontoblast layer; P, pulp. Data shown are representative of results obtained from independent experiments performed with two healthy and two carious teeth.
Mentions: To assess the in vivo relevance of these findings and determine whether NOS2 is expressed in odontoblasts, NOS2 transcript and protein were examined in healthy dental pulps and inflamed samples from carious teeth. We found that the NOS2 gene was strongly up-regulated in inflamed pulps compared to healthy ones (Figure 4A). NOS2 protein was clearly detected by immunostaining in odontoblasts and subodontoblast cells in the inflamed area, but not in cells in the non-inflamed area far from the lesion (not shown) or in healthy pulps (Figure 4B). Staining controls performed by using the mouse immunoglobulin G1 isotype were negative.

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