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Different Roles of Odontoblasts and Fibroblasts in Immunity

M.-J. Staquet^1,*,, S.H. Durand^1,2,, E. Colomb³, A. Roméas¹, C. Vincent³, F. Bleicher¹, S. Lebecque⁴ and J.-C. Farges^1,2

¹ "Odontoblasts and Regeneration of Dental Tissues" Group, Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Institut Fédératif de Recherches Biosciences Gerland Lyon Sud, Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon, INSERM ERI16, Faculté d’Odontologie, 11 rue Guillaume Paradin, F-69372 Lyon Cedex 08, France;
² Hospices Civils de Lyon, Service de Consultations et de Traitements Dentaires, Lyon, France;
³ Université Lyon 1, EA3732, Centre Hospitalier E. Herriot, Lyon, France; and
⁴ Université Lyon 1, UMR5201, Centre Hospitalier Lyon Sud, Lyon, France

View larger version (21K): [in this window] [in a new window]   Figure 1. LTA, poly(I:C), and LPS treatment induced TLR expression changes in human odontoblast-like cells and fibroblasts in vitro. (A) Real-time PCR analysis of TLR2, TLR3, and TLR4 genes. Results were normalized to cyclophilin A gene and expressed as fold-change values relative to control unstimulated cells. Both odontoblast-like cells and fibroblasts expressed TLR2, TLR3, and TLR4. Stimulation of cells for 8 hrs with specific agonists (LTA for TLR2, dsRNA for TLR3, and LPS for TLR4) differentially regulated TLR genes. LTA (1 µg/mL) significantly augmented TLR2 expression in odontoblast-like cells, but not in fibroblasts, and failed to modify TLR3 and TLR4 in either cell type. Poly(I:C) (25 µg/mL) increased TLR2, TLR3, and TLR4 in both odontoblast-like cells and fibroblasts. LPS (1 µg/mL) increased TLR3 in both odontoblast-like cells and fibroblasts, and TLR4 in fibroblasts. Data represent the mean ± SD obtained from 3 independent experiments. *p 0.05; **p 0.01; ***p 0.001 vs. control cells. Od: odontoblast-like cells. Fib: fibroblasts. (B) Flow cytometry analysis of TLR2, TLR3, and TLR4 proteins. Isotype controls are shown as open histograms. Up-regulation was detected for all TLRs whose gene expression was increased, except for TLR4 and TLR3 in odontoblast-like cells stimulated by poly(I:C) and LPS, respectively, and for TLR2 in fibroblasts stimulated by poly(I:C) that remained unchanged. Histograms shown are representative of 3 independent experiments.

View larger version (39K): [in this window] [in a new window]   Figure 2. Odontoblast-like cell and fibroblast LTA-, poly(I:C)-, and LPS-regulated chemokine gene expression. Gene arrays showed that odontoblast-like cells expressed the chemokine genes (also listed in the Table) CCL2 (position 6G on the membrane), CCL26 (7F), CXCL4 (5C), CXCL12 (10A), and CXCL14 (9B), and fibroblasts expressed CCL2, CCL26, CXCL2 (4C), CXCL12, and CXCL14. Other genes spotted on the membrane were never detected or not systematically found in all 3 tested samples. Upon stimulation (8 hrs) with LTA (1 µg/mL), hybridization signals were increased in both cell types for CCL2 and CCL7 (8E), whereas CXCL2 and CXCL10 (8G) were raised in odontoblast-like cells only. Poly(I:C) (25 µg/mL) increased expression of 11 and 13 chemokine genes in odontoblast-like cells and fibroblasts, respectively. LPS (1 µg/mL) up-regulated CCL2 and CXCL10 in odontoblast-like cells and CCL2, CCL7, CCL26, CXCL10, and CXCL11 (8H) in fibroblasts. Cyclophilin A tetraspots (position 14A-D) were used for normalization. The 3 pUC18 tetraspots used for background subtraction are in the position 13A-C. Autoradiograms shown are representative of 3 independent experiments.

View larger version (30K): [in this window] [in a new window]   Figure 3. LTA, poly(I:C), and LPS treatment for 8 hrs up-regulated DC-attracting CCL2, CCL7, and CCL26 in odontoblast-like cells and fibroblasts and stimulated immature DC migration. (A) Real-time PCR analysis of CCL2, CCL7, and CCL26 expression. Results were normalized to cyclophilin A gene and expressed as fold-change values relative to control cells. Statistical analysis confirmed significant up-regulation of CCL2 in odontoblast-like cells and fibroblasts by each of the 3 agonists, up-regulation of CCL7 in odontoblast-like cells stimulated by LTA and poly(I:C), and in fibroblasts stimulated by each of the 3 agonists, and up-regulation of CCL26 in fibroblasts stimulated by poly(I:C) and LPS. Data represent the mean ± SD obtained from 3 independent experiments. *p 0.05; **p 0.01 vs. control cells. Od: odontoblast-like cells. Fib: fibroblasts. (B) Antibody array analysis of CCL2, CCL7, and CCL26 release from odontoblast-like cells and pulp fibroblasts. Each anti-chemokine antibody is present on the array membrane in duplicate. After background subtraction (Neg), values were adjusted based on the intensity of control spots on the membranes (Pos). CCL2 was up-regulated in odontoblast-like cells and fibroblasts by each of the 3 agonists. CCL7 and CCL26 were not detected on the array membranes (not shown). Data represent the mean ± SD obtained from 3 independent experiments. Spots shown are representative of these experiments. *p 0.05; **p 0.01 vs. control cells. (C) Odontoblast-like cell and fibroblast supernatants treated for 8 hrs with LTA (1 µg/mL), poly(I:C) (25 µg/mL), or LPS (1 µg/mL) were tested for their ability to enhance immature DC migration in a transwell chamber migration assay. When supernatants from unstimulated odontoblast-like cells and fibroblasts were used, a mean number of 32% ± 5.0 and 27.4% ± 10.7 immature DCs migrated, respectively. The number of migratory immature DCs increased to 66.9% ± 18.6, 79.4% ± 22.6, and 77.6% ± 16.8 when odontoblast-like cells were stimulated with LTA, poly(I:C), and LPS, respectively. The number of migratory immature DCs increased to 45.7% ± 13.1, 42.8% ± 12.3, and 35.6% ± 9.6 when fibroblasts were stimulated with LTA, poly(I:C), and LPS, respectively. Statistical analysis revealed that odontoblast-like cells were more potent attractants than fibroblasts when both cell types were stimulated by the same TLR agonist. Results are expressed as the number of migrated cells in percentage of the input cell number, and are the mean ± SD of duplicates from 3 independent experiments. *p 0.05 vs. control cells. Od: odontoblast-like cells. Fib: fibroblasts.

Journal of Dental Research, Vol. 87, No. 3, 256-261 (2008)
DOI: 10.1177/154405910808700304


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