Figure 8. Type 3 innate lymphoid cells (ILC3s) are critical for cervical alveolar bone resorption in the ligature-induced periodontitis development.

(A) Proportion plots showing the relative amounts of T cell subpopulations in gingival tissue during periodontitis development. Treg, T regulatory cells; ILC, innate lymphoid cells; Th, T helper cells; Tc, cytotoxic T cells. (B) Violin plots showing expression levels of the T cell marker genes Cd8 (Tc), Cd4 (Th), Zbtb16 (ILC), and Foxp3 (Treg) on day 7 following ligature placement. (C) Violin plots showing expression levels of Nfil3, Rorγ, Il17a, Il17f, Tbx21, and Gata3 on day 7 following ligature placement. These gene signatures indicate that gingival ILCs primarily comprise ILC3s. (D) Representative micro-computed tomography (microCT) images of the maxilla taken from the lateral view for the ligated side and from the contralateral view for the unligated side. (E) Alveolar bone loss was determined from the total distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) of the buccal bone or palatal bone at six sites in the ligated side (n = 6). (F) HE staining of the periodontal tissue on day 7. gCT, gingival connective tissue; Bone, alveolar bone; PDL, periodontal ligament; scale bars, 100 µm. (G) Tartrate-resistant acid phosphatase (TRAP) staining of periodontal tissue from WT mice on day 7; scale bar, 100 µm. Total number of TRAP-positive cells in a 0.01 mm² area of the buccal and palatal bone in the cervical PDL site (H) and apical PDL site (I) (n = 6). Significance was determined by ANOVA, with Tukey’s multiple-comparison test. Data are presented as mean values ± SD; p<0.05 was considered significant.

Figure 8—figure supplement 1. Effects of innate lymphoid cell (ILC) deletion on alveolar bone loss in the mouse periodontitis model.

(A) Alveolar bone loss was assessed at six sites by measuring the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) on the buccal or palatal side of the alveolar bone of wild-type (WT), Rag2^-/-, and Rag2^-/-Il2rg^-/- mice on day 7 following ligature placement (n = 6). (B) Bone volume/total volume (BV/TV), bone surface, trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the buccal side of alveolar bone of the second molar were measured on day 7 (n = 6). (C) Representative intra-oral photographs of maxilla from WT, Rag2^-/-, and Rag2^-/-Il2rg^-/- mice taken on day 7 following ligature placement. (D) The gingival defect area was measured and normalized to the circumferential area of M1 (n = 5). Significance was determined by ANOVA, with Tukey’s multiple-comparison test. Data are presented as mean values ± SD; p<0.05 was considered significant.

Figure 8—figure supplement 2. Human periodontitis phenotype.

(A) Panoramic radiograph of a human patient diagnosed with generalized periodontitis. The alveolar bone resorption was only noted at the cervical alveolar bone (arrows), whereas the apical alveolar bone was well maintained and not affected by periodontitis. (B) Periapical dental radiograph and the clinical pictures during the periodontal surgery. A localized alveolar bone loss (arrows) was noted at the cervical periodontal ligament area.