Neutrophil counts in periodontal tissue in a ligature-induced periodontitis model and neutrophil chemotaxis. (A) Neutrophil chemotaxis induced by fMLP in Mmd2A117V/+, Mmd2A117V/A117V, Mmd2R127P/+, Mmd2R127P/R127P, and Mmd2−/− mice was decreased compared with that in Mmd2+/+ and Mmd2V100L/V100L mice. (Mmd2+/+, n = 5; Mmd2V100L/V100L, n = 4; Mmd2A117V/+, n = 5; Mmd2A117V/A117V, n = 5; Mmd2R127P/+, n = 4; Mmd2R127P/R127P, n = 5; Mmd2−/−, n = 5). Mmd2+/+ versus Mmd2V100L/V100L; P = 0.6758, Mmd2+/+ versus Mmd2A117V/+; P = 0.0502, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0015, Mmd2+/+ versus Mmd2R127P/+; P = 0.0015, Mmd2+/+ versus Mmd2R127P/R127P; P < 0.0001, Mmd2+/+ versus Mmd2−/−; P < 0.0001. (B) No statistical difference in neutrophil chemotaxis induced by IL-8. The results are expressed as the mean ± SD. (Mmd2+/+, n = 5; Mmd2V100L/V100L, n = 4; Mmd2A117V/+, n = 5; Mmd2A117V/A117V, n = 5; Mmd2R127P/+, n = 4; Mmd2R127P/R127P, n = 5; Mmd2−/−, n = 5). Mmd2+/+ versus Mmd2V100L/V100L; P = 0.9669, Mmd2+/+ versus Mmd2A117V/+; P = 0.9997, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.9913, Mmd2+/+ versus Mmd2R127P/+; P > 0.9999, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.9994, Mmd2+/+ versus Mmd2−/−; P = 0.9440. (C–K) Neutrophil numbers were decreased in ligature-induced periodontitis. Immunofluorescence for Ly6G (C, F, and I) and MPO (D, G, and J) in gingival tissues at 24 h after ligation. Red = Ly6G-positive cells, green = MPO, L (white dot line) = ligature, B = alveolar bone, DP = dental pulp, D = dentin. Nuclei were visualized by DAPI (blue). n = 5. (C)Mmd2+/+ versus Mmd2A117V/+; P = 0.0185, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0048. (D)Mmd2+/+ versus Mmd2A117V/+; P = 0.0004, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0018. (E) Relative fluorescence intensity: Mmd2+/+ versus Mmd2A117V/+; P = 0.0790, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0043, bacterial DNA in gingival tissues: Mmd2+/+ versus Mmd2A117V/+; P = 0.0036, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0108. (F)Mmd2+/+ versus Mmd2R127P/+; P = 0.0339, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0042. (G)Mmd2+/+ versus Mmd2R127P/+; P = 0.0018, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0002. (H) Relative fluorescence intensity: Mmd2+/+ versus Mmd2R127P/+; P = 0.0371, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0049, bacterial DNA in gingival tissues: Mmd2+/+ versus Mmd2R127P/+; P = 0.0090, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0185. (I)Mmd2+/+ versus Mmd2−/−; P = 0.0004). (J)Mmd2+/+ versus Mmd2−/−; P = 0.0002). (K) Relative fluorescence intensity: Mmd2+/+ versus Mmd2−/−; P = 0.0001, bacterial DNA in gingival tissues: Mmd2+/+ versus Mmd2−/−; P = 0.0466. (E, H, and K) Fluorescence in situ hybridization (FISH) for bacteria rRNA in gingival tissues at 24 h after ligation. Quantification for relative fluorescence intensity were analyzed by BZ-X800 Analyzer (Keyence) in a random field. Green = bacteria rRNA. n = 4–5. Scale bar = 100 μm. Relative amounts of bacteria 16S rDNA in the gingival tissues analyzed by qPCR. The average of 16S rDNA levels in the gingival tissues from Mmd2+/+ with ligatures was set as 1. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 with one-way ANOVA with Tukey–Kramer test (A–H) or two-tailed unpaired t test (I–K). n.s = not significant. Each dot represents a biologically independent mouse. All representative images from more than three independent experiments with similar results.
Figure 4.

Neutrophil counts in periodontal tissue in a ligature-induced periodontitis model and neutrophil chemotaxis. (A) Neutrophil chemotaxis induced by fMLP in Mmd2A117V/+, Mmd2A117V/A117V, Mmd2R127P/+, Mmd2R127P/R127P, and Mmd2−/− mice was decreased compared with that in Mmd2+/+ and Mmd2V100L/V100L mice. (Mmd2+/+, n = 5; Mmd2V100L/V100L, n = 4; Mmd2A117V/+, n = 5; Mmd2A117V/A117V, n = 5; Mmd2R127P/+, n = 4; Mmd2R127P/R127P, n = 5; Mmd2−/−, n = 5). Mmd2+/+ versus Mmd2V100L/V100L; P = 0.6758, Mmd2+/+ versus Mmd2A117V/+; P = 0.0502, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0015, Mmd2+/+ versus Mmd2R127P/+; P = 0.0015, Mmd2+/+ versus Mmd2R127P/R127P; P < 0.0001, Mmd2+/+ versus Mmd2−/−; P < 0.0001. (B) No statistical difference in neutrophil chemotaxis induced by IL-8. The results are expressed as the mean ± SD. (Mmd2+/+, n = 5; Mmd2V100L/V100L, n = 4; Mmd2A117V/+, n = 5; Mmd2A117V/A117V, n = 5; Mmd2R127P/+, n = 4; Mmd2R127P/R127P, n = 5; Mmd2−/−, n = 5). Mmd2+/+ versus Mmd2V100L/V100L; P = 0.9669, Mmd2+/+ versus Mmd2A117V/+; P = 0.9997, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.9913, Mmd2+/+ versus Mmd2R127P/+; P > 0.9999, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.9994, Mmd2+/+ versus Mmd2−/−; P = 0.9440. (C–K) Neutrophil numbers were decreased in ligature-induced periodontitis. Immunofluorescence for Ly6G (C, F, and I) and MPO (D, G, and J) in gingival tissues at 24 h after ligation. Red = Ly6G-positive cells, green = MPO, L (white dot line) = ligature, B = alveolar bone, DP = dental pulp, D = dentin. Nuclei were visualized by DAPI (blue). n = 5. (C)Mmd2+/+ versus Mmd2A117V/+; P = 0.0185, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0048. (D)Mmd2+/+ versus Mmd2A117V/+; P = 0.0004, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0018. (E) Relative fluorescence intensity: Mmd2+/+ versus Mmd2A117V/+; P = 0.0790, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0043, bacterial DNA in gingival tissues: Mmd2+/+ versus Mmd2A117V/+; P = 0.0036, Mmd2+/+ versus Mmd2A117V/A117V; P = 0.0108. (F)Mmd2+/+ versus Mmd2R127P/+; P = 0.0339, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0042. (G)Mmd2+/+ versus Mmd2R127P/+; P = 0.0018, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0002. (H) Relative fluorescence intensity: Mmd2+/+ versus Mmd2R127P/+; P = 0.0371, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0049, bacterial DNA in gingival tissues: Mmd2+/+ versus Mmd2R127P/+; P = 0.0090, Mmd2+/+ versus Mmd2R127P/R127P; P = 0.0185. (I)Mmd2+/+ versus Mmd2−/−; P = 0.0004). (J)Mmd2+/+ versus Mmd2−/−; P = 0.0002). (K) Relative fluorescence intensity: Mmd2+/+ versus Mmd2−/−; P = 0.0001, bacterial DNA in gingival tissues: Mmd2+/+ versus Mmd2−/−; P = 0.0466. (E, H, and K) Fluorescence in situ hybridization (FISH) for bacteria rRNA in gingival tissues at 24 h after ligation. Quantification for relative fluorescence intensity were analyzed by BZ-X800 Analyzer (Keyence) in a random field. Green = bacteria rRNA. n = 4–5. Scale bar = 100 μm. Relative amounts of bacteria 16S rDNA in the gingival tissues analyzed by qPCR. The average of 16S rDNA levels in the gingival tissues from Mmd2+/+ with ligatures was set as 1. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 with one-way ANOVA with Tukey–Kramer test (A–H) or two-tailed unpaired t test (I–K). n.s = not significant. Each dot represents a biologically independent mouse. All representative images from more than three independent experiments with similar results.

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