Tlr2/4-Mediated Hyperinflammation Promotes Cherubism-Like Jawbone Expansion in Sh3bp2 (P416R) Knockin Mice

Abstract

Cherubism (CBM), characterized by expansile jawbones with multilocular fibrocystic lesions, is caused by gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2; mouse orthologue Sh3bp2). Loss of jawbone and dental integrity significantly decrease the quality of life for affected children. Treatment for CBM is limited to multiple surgeries to correct facial deformities. Despite significant advances made with CBM knockin (KI) mouse models (Sh3bp2 ^KI/KI ), the activation mechanisms of CBM lesions remain unknown because mutant mice do not spontaneously develop expansile jawbones. We hypothesize that bony inflammation of an unknown cause triggers jawbone expansion in CBM. To introduce jawbone inflammation in a spatiotemporally controlled manner, we exposed pulp of the first right mandibular molar of 6-week-old Sh3bp2 ^+/+ , Sh3bp2 ^KI/+ , and Sh3bp2 ^KI/KI mice. Bacterial invasion from the exposed pulp into root canals led to apical periodontitis in wild-type and mutant mice. The pathogen-associated molecular patterns (PAMPs)-induced inflammation of alveolar bone resulted in jawbone expansion in Sh3bp2 ^KI/+ and Sh3bp2 ^KI/KI mice. CBM-like lesions developed exacerbated inflammation with increased neutrophil, macrophage, and osteoclast numbers. These lesions displayed excessive neutrophil extracellular traps (NETs) compared to Sh3bp2 ^+/+ mice. Expression levels of IL-1β, IL-6, and TNF-α were increased in periapical lesions of Sh3bp2 ^+/+ , Sh3bp2 ^KI/+ , and Sh3bp2 ^KI/KI mice and also in plasma and the left untreated mandibles (with no pulp exposure) of Sh3bp2 ^KI/KI mice, suggesting a systemic upregulation. Ablation of Tlr2/4 signaling or depletion of neutrophils by Ly6G antibodies ameliorated jawbone expansion induced by PAMPs in Sh3bp2 ^KI/KI mice. In summary, successful induction of CBM-like lesions in jaws of CBM mice is important for studying initiating mechanisms of CBM and for testing potential therapies. Our findings further emphasize a critical role of host immunity in the development of apical periodontitis and the importance of maintaining oral health in CBM patients. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: CHERUBISM; GENETIC ANIMAL MODELS; JAW ABNORMALITIES; NEUTROPHIL EXTRACELLULAR TRAPS; OSTEOIMMUNOLOGY.

Fig. 1

CBM‐like jawbone expansion in Sh3bp2 mutant mice. (A) Timeline of mandible collection with or without pulp exposure of right 1st molar. Faxitron image confirmed the pulp access (yellow arrows). (B) Representative Faxitron images of expansile jawbones in Sh3bp2 ^KI/+ and Sh3bp2 ^KI/KI female mice. Yellow arrows indicate jawbone expansion. (C) Coronal planes and (D) axial planes of μCT images. Scale bar = 1 mm. (E) Quantification of jawbone dimensions by measuring distances from furcation of 1st molars to the top margin of incisors (FA′, FB′, FC′) and normalizing to distances from furcation to the end of root tips (FA, FB, FC) along lines A, B, and C. Data are presented as mean ± SD. Each dot in graphs represents a single biological sample (n = 5 per group). *p < 0.05, **p < 0.01 indicate statistical significance by two‐way ANOVA followed by Sidak correction. F = furcation.

Fig. 2

Tlr2/4‐mediated inflammatory bone resorption is exacerbated in Sh3bp2 ^KI/KI mice. Immunohistochemistry of paraffin‐embedded Sh3bp2 ^KI/KI mandibular sections show (A) rapid infiltration (3 days after pulp exposure, middle panel) and interspersed influx (14 days after pulp exposure, right panel) of neutrophils stained with NIMPR14 antibody. Neutrophils in Sh3bp2 ^+/+ mice were confined to the periapex of the roots. Scale bar = 500 μm. (B) Increased macrophages stained by CD68 antibody. Scale bar = 200 μm. (C) Increased bone resorption and multinucleated osteoclasts stained by CatK antibody. Scale bar = 500 μm (left) and 50 μm (right panel). (D) Quantification of macrophage numbers normalized to bone surface (mm²) and (E) CatK‐positive area normalized to bone perimeter (mm). n = 3–9 per group. (F) Coronal planes and (G) axial planes of μCT images from Sh3bp2 ^+/+ Tlr2/4 ^KO/KO and Sh3bp2 ^KI/KI Tlr2/4 ^KO/KO mandibles with or without pulp exposure. (H) Quantitation of jawbone dimensions. No significant jawbone expansion 14 days after pulp exposure in Sh3bp2 ^KI/KI mice when Tlr2/4 were knocked out although periapical lesions could still be observed. Data are presented as mean ± SD. Each dot in graphs represents a single biological sample (n = 4–5 per group). Measurements were analyzed using two‐way ANOVA followed by Sidak correction. *p < 0.05, **p < 0.01 indicate statistical significance. CatK = cathepsin K.

Fig. 3

Increased local and systemic levels of inflammatory cytokines in Sh3bp2 ^KI/KI mice after pulp exposure. qPCR showing gene expression of IL‐1β, IL‐6, and TNF‐α with or without pulp exposure (A) in treated right mandibles; (B) in left undrilled mandibles of Sh3bp2 ^+/+ , Sh3bp2 ^KI/+ , and Sh3bp2 ^KI/KI experimental mice (n = 3–6 per group). (C) IL‐1β and IL‐6 plasma levels with or without pulp exposure in Sh3bp2 ^+/+ , Sh3bp2 ^KI/+ , and Sh3bp2 ^KI/KI mice (n = 4–12 per group). No: naïve mice (no pulp exposure). Each dot in graphs represents a single biological sample. Data presented as mean ± SD. *p < 0.05, ** < 0.01 indicate significant difference by two‐way ANOVA followed by Sidak correction.

Fig. 4

CBM mutation in Sh3bp2 enhances neutrophil‐mediated events that contribute to PAMPs‐induced jawbone expansion. (A) Increased SH3BP2 expression in sorted Ly6G‐positive cells from Sh3bp2 ^KI/KI bone marrow shown by immunoblotting. (B) Increased percentages of blood neutrophils (Ly6G⁺CD115⁻) and monocytes (Ly6G⁻CD115⁺) in Sh3bp2 ^KI/KI mice shown by flow cytometry (n = 5 per group). (C) Detection of MPO (green) and citrullinated H3 (pink) in Sh3bp2 ^+/+ and Sh3bp2 ^KI/KI right mandibles 14 days after pulp exposure. Scale bar = 200 μm. Histograms show quantification of MPO, citH3, and colocalization of MPO and citH3 signals normalized to DAPI (blue) from nine periapical and furcation areas in three Sh3bp2 ^+/+ and Sh3bp2 ^KI/KI mice. (D) Coronal and axial planes of μCT images of Sh3bp2 ^KI/KI mice treated with Ly6G antibody or isotype control prior to and after pulp exposure. (E) Quantification of mandible expansion shows significant amelioration after Ly6G antibody treatment in Sh3bp2 ^KI/KI mice compared to no antibody or isotype control (arrows) antibody. Scale bar = 1 mm. Each dot in graphs represents a single biological sample (n = 3–7 per group). Data are presented as mean ± SD. *p < 0.05, **p < 0.01 indicate significant difference by Student's t test (B), (C) and one‐way ANOVA (E).