Microbe-Dependent Exacerbated Alveolar Bone Destruction in Heterozygous Cherubism Mice

Abstract

Cherubism (OMIM#118400) is a craniofacial disorder characterized by destructive jaw expansion. Gain-of-function mutations in SH3-domain binding protein 2 (SH3BP2) are responsible for this rare disorder. We have previously shown that homozygous knock-in (KI) mice (Sh3bp2 KI/KI ) recapitulate human cherubism by developing inflammatory lesions in the jaw. However, it remains unknown why heterozygous KI mice (Sh3bp2 KI/+ ) do not recapitulate the excessive jawbone destruction in human cherubism, even though all mutations are heterozygous in humans. We hypothesized that Sh3bp2 KI/+ mice need to be challenged for developing exacerbated jawbone destruction and that bacterial stimulation in the oral cavity may be involved in the mechanism. In this study, we applied a ligature-induced periodontitis model to Sh3bp2 KI/+ mice to induce inflammatory alveolar bone destruction. Ligature placement induced alveolar bone resorption with gingival inflammation. Quantification of alveolar bone volume revealed that Sh3bp2 KI/+ mice developed more severe bone loss (male: 43.0% ± 10.6%, female: 42.6% ± 10.4%) compared with Sh3bp2 +/+ mice (male: 25.8% ± 4.0%, female: 30.9% ± 6.5%). Measurement of bone loss by the cement-enamel junction-alveolar bone crest distance showed no difference between Sh3bp2 KI/+ and Sh3bp2 +/+ mice. The number of osteoclasts on the alveolar bone surface was higher in male Sh3bp2 KI/+ mice, but not in females, compared with Sh3bp2 +/+ mice. In contrast, inflammatory cytokine levels in gingiva were comparable between Sh3bp2 KI/+ and Sh3bp2 +/+ mice with ligatures. Genetic deletion of the spleen tyrosine kinase in myeloid cells and antibiotic treatment suppressed alveolar bone loss in Sh3bp2 KI/+ mice, suggesting that increased osteoclast differentiation and function mediated by SYK and accumulation of oral bacteria are responsible for the increased alveolar bone loss in Sh3bp2 KI/+ mice with ligature-induced periodontitis. High amounts of oral bacterial load caused by insufficient oral hygiene could be a trigger for the initiation of jawbone destruction in human cherubism. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Keywords: CHERUBISM; ORAL MICROBES; OSTEOCLASTS; PERIODONTITIS; SH3BP2.

© 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Figures

Figure 1

The gain‐of‐function mutation of SH3BP2 exacerbates alveolar bone loss in ligature‐induced periodontitis. (A) H&E staining of gingival tissue in males between the first and second molars. Scale bar = 100 μm. Arrowheads indicate inflammatory infiltrates surrounding the ligated silk sutures. (B) Tartrate‐resistant acid phosphatase (TRAP) staining of tissues in males between two buccal roots. Arrowheads indicate multinucleated cells that are not located on the alveolar bone surface. Scale bar = 50 μm. (C) qPCR analysis with RNA isolated from the gingiva. Average levels in unligated WT mice were set as 1. (D) 3D μCT images surrounding the maxillary second molar (top), 2D coronal plane μCT images in the middle of the maxillary second molar (middle), and the region of interest (ROI) used for bone volume measurement (bottom). Arrowheads indicate the areas of alveolar bone loss. P = Palatal side; B = buccal side. (E) Alveolar bone volume between two buccal roots underneath the maxillary second molar. (F) Percentage of bone loss against the contralateral unligated side. ANOVA with Tukey–Kramer post hoc test. Mean ± SD. *p < 0.05. NS = not significant.

Figure 2

Gain‐of‐function of SH3BP2 increases differentiation and bone‐resorbing capacity of osteoclasts. (A) Tartrate‐resistant acid phosphatase (TRAP) staining of alveolar bone in males between two buccal roots of the ligated second molar. Scale bar = 100 μm. (B) Histomorphometric analysis for TRAP‐positive cells on the alveolar bone surface. (C) qPCR analysis for Rankl and Opg expression and their ratio. RNA was isolated from the alveolar bone. Average levels in unligated WT mice were set as 1. (D) TRAP staining of male bone marrow‐derived M‐CSF‐dependent macrophages (BMMs) stimulated with 50 ng/mL RANKL for 5 days and quantitation of the TRAP‐positive multinucleated cells (MNCs) per well. n = 4. Scale bar = 100 μm. (E) Resorption assay for calcium phosphate. BMMs from male mice stimulated with 50 ng/mL RANKL were cultured for 7 days. Cells were removed, and remaining calcium phosphate was stained with silver nitrate to visualize the nonresorbed area. Resorbed areas were measured by ImageJ software (NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/). n = 4. Scale bar = 100 μm. (F) Resorption assay using dentin slices. BMMs from male mice stimulated with RANKL were cultured for 7 days. Cells were removed, and dentin slices were stained with toluidine blue for observation by stereomicroscope. Dentine slices were further decalcified and sectioned for H&E staining. Pit depth was measured with cross‐section H&E images of the dentin slice using ImageJ software. n = 100 in males and 100 in females. Scale bar = 100 μm (toluidine blue staining) and 10 μm (H&E staining). Mean ± SD. *p < 0.05. NS = not significant. ANOVA with Tukey–Kramer post hoc test for (B,C), Student's t test for (D,E), Mann–Whitney U test for (F).

Figure 3

Spleen tyrosine kinase (SYK) deletion in myeloid cells decreases susceptibility to bone loss in Sh3bp2KI/+ mice with ligature‐induced periodontitis. (A) 3D μCT images surrounding the maxillary second molar (left) and 2D coronal plane μCT images in the middle of the maxillary second molar (right). (B) Alveolar bone volume and percentage of bone loss against the contralateral unligated side. (C) TRAP staining of alveolar bone in males between two buccal roots of the ligated second molar. Scale bar = 100 μm. (D) Histomorphometric analysis for TRAP‐positive cells. ANOVA with Tukey–Kramer post hoc test. Mean ± SD. *p < 0.05. NS = not significant; + = +/+; cre = cre/cre; fl = fl/fl.

Figure 4

Antibiotic treatment suppresses alveolar bone loss in ligature‐induced periodontitis both in WT and Sh3bp2KI/+ mice. (A) 3D μCT images surrounding the maxillary second molar (left) and 2D coronal plane μCT images in the middle of the maxillary second molar (right). P = Palatal side; B = buccal side. (B) Alveolar bone volume between two buccal roots underneath the maxillary second molar. (C) Percentage of bone loss against the contralateral unligated side. ANOVA with Tukey–Kramer post hoc test. Mean ± SD. *p < 0.05. NS = not significant.