Changes in proportions of major cell types during periodontitis development in mice. (A) A ligature (5.0 silk suture) was placed around the maxillary second molar (M2) of wild-type (WT) mice. Representative intra-oral photographs of the maxilla on Day 0, prior to ligature placement (healthy gingiva), and on Days 1, 3, and 7 after ligature placement. (B) The gingival defect area was measured and normalized to the circumferential area of the maxillary first molar (M1) (n = 6). Gingival defects appeared on Day 3. (C) Representative micro-computed tomography (microCT) images of the maxilla taken from the lateral view. (D) Alveolar bone loss was determined from the total distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) of the buccal or palatal bone at six sites in the ligated side (n = 6). Alveolar bone loss was apparent on Day 7. (E) Single-cell RNA sequencing (scRNA-seq) t- distributed stochastic neighbor embedding (t-SNE) projection plots showing the major cell types present within gingival tissue during periodontitis development on Day 0, 1, 4, and 7. Colors indicate cell type, as follows: green, epithelial cells; blue, fibroblasts; pink, endothelial cells; yellow, B cells; red, T cells; and purple, myeloid cells. (F) Proportion plots showing the relative amounts of each major cell type on Days 0, 1, 4, and 7. Significance was determined by analysis of variance (ANOVA), with Tukey’s multiple-comparison test (B, D). Data are presented as mean values ± standard deviation (SD); p < 0.05 was considered statistically significant.

Myeloid cell composition and activity in gingival tissue during in periodontitis development. (A) t-SNE projection plots showing myeloid cell subpopulations in gingival tissue during periodontitis development on Days 0, 1, 4, and 7. Colors indicate cell type, as follows: green, macrophages and red, neutrophils. (B) Proportion plots showing the relative amounts of neutrophils and macrophages on Days 0, 1, 4, and 7. (C) Violin plots showing Trem1 and Mmp9 expression levels in myeloid cells on Days 0, 1, 4, and 7; both genes are upregulated in neutrophils after ligature placement. (D) Violin plots showing Tgfb1 and Tnfa expression in myeloid cells on Days 0, 1, 4, and 7; no obvious induction is observed in response to ligature placement. Dot plots depicting expression levels of the C motif chemokine ligand (CCL) genes Ccl2, Ccl3, Ccl4, Ccl6, Ccl9 (E), and the CXC motif chemokine ligand (CXCL) genes Cxcl2, Cxcl3, Cxcl4, and Cxcl9 (F). Chemokine expression in myeloid cells was unrelated to progression of gingival inflammation from Day 1 to 7.

Fibroblasts activated to guide leukocyte migration (AG fibroblasts) are one of three fibroblast subpopulations in gingival tissue during periodontitis development. (A) Violin plots showing gene expression levels of type I collagen (Col1a1), type XIV collagen (Col14a1), and smooth muscle aortic actin 2 (Acta2) in gingival fibroblast subpopulations during periodontitis development. AG, AG fibroblasts; KT, ‘keep typical’ (KT) fibroblasts; MF, myofibroblasts. Gene ontology (GO) enrichment analysis of the biological functions of AG fibroblasts and KT fibroblasts on Day 0 without ligature placement (B) and on Day 1 (C) and Day 7 (D) after ligature placement. Gene clusters related to immune regulation (red) were identified in AG fibroblasts, and these clusters dominate after ligature placement. (E) t-SNE projection plots showing fibroblast subpopulations in gingival tissue during periodontitis development. Colors indicate cell type, as follows: blue, KT fibroblasts; red, AG fibroblasts; and yellow, MFs. (F) Proportion plots showing the relative amounts of each fibroblast subpopulation on Days 0, 1, 4, and 7.

AG fibroblasts and immune surveillance in periodontitis development. (A) Dot plots depicting expression levels of the CCL genes Ccl8, Ccl11, Ccl19, Cxcl1, Cxcl9, Cxcl11, and Cxcl12 in gingival fibroblast subpopulations during periodontitis development. (B) Dot plots depicting expression levels of the Toll-like receptor and related genes Tlr2, Tlr3, Tlr4, Myd88, Irak1, Map3k7, and Rela in gingival fibroblast subpopulations during periodontitis development. Upregulation of chemokines and TLR-related molecules is predominantly observed in the AG fibroblast subpopulation. (C) Hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining for COL14A1 and CXCL12 in periodontal tissue on Day 1; scale bars, 100 µm (HE) and 20 µm, (IHC). Yellow arrows indicate COL14A1- and CXCL12-positive cells in the connective tissue papillae and periodontal ligament (PDL).

Role of AG fibroblasts in myeloid cell activation. Interaction between chemokine ligands expressed by AG fibroblasts and their putative chemokine receptors expressed by myeloid cells during periodontitis development. Dot plots depicting expression levels of the CC chemokine receptor (CCR) and the CXC chemokine receptor (CXCR) genes Ccr1, Ccr2, Ccr5, Ccr7, Cxcr2, Cxcr3, and Cxcr4 in myeloid cell subpopulations on Day 1 (A) and Day 7 (B) following ligature placement. NicheNet ligand–target matrix indicating the regulatory potential between active ligands expressed in fibroblasts and target genes expressed in myeloid cells from the p-EMT program on Day 1 (C) and Day 7 (D). (E) Dot plot depicting expression levels of active ligand genes from panel (C) in fibroblast subpopulations on Day 1. (F) Dot plots depicting expression levels of active ligand genes from panel (D) in fibroblast subpopulations on Day 7.(G) Dot plot depicting expression levels of target genes from panel (C) in myeloid cell subpopulations on Day 1. (H) Dot plot depicting expression levels of target genes from panel (D) in myeloid cell subpopulations on Day 7. Results suggest a strong intercellular communication network from AG fibroblasts to neutrophils.

T cell subpopulations in 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 type 3 ILCs (ILC3s). (D) Violin plots showing expression levels of the macrophage-colony stimulating factor (M-CSF)-encoding gene, Csf1 (M-CSF), in each major cell type. (E) Violin plots showing expression levels of Csf1 in fibroblast subpopulations, myeloid cell subpopulations, and T cell subpopulations. (F) Violin plots showing expression levels of the nuclear factor kappa-Β ligand (RANKL)-encoding gene, Tnfsf11, in each major cell type. (G) Violin plots showing expression levels of Tnfsf11 in fibroblast subpopulations, myeloid cell subpopulations, and T cell subpopulations.

ILC3s are critical for cervical alveolar bone resorption in the mouse periodontitis model. (A) Representative intra-oral photographs of maxilla from WT, Rag2-/-, and Rag2-/-γc-/- mice taken on Day 7 following ligature placement. (B) The gingival defect area was measured and normalized to the circumferential area of M1 (n = 5). (C) Representative microCT images of the maxilla taken from the lateral view for the ligated side and from the contralateral view for the unligated side. (D) Alveolar bone loss was determined from the total distance between the CEJ and the ABC of the buccal bone or palatal bone at six sites in the ligated side (n = 6). (E) HE staining of the periodontal tissue on Day 7. gCT, gingival connective tissue; Bone, alveolar bone; PDL, periodontal ligament; scale bars, 100 µm. (F) 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 (G) and apical PDL site (H) (n = 6). Significance was determined by ANOVA, with Tukey’s multiple-comparison test (D, G, H). Data are presented as mean values ± SD; p < 0.05 was considered significant.

The role of AG fibroblasts and neutrophils in ILC3 development in periodontitis. Violin plots showing expression levels of the genes encoding interleukin (IL)-6 (Il6) (A) and IL-23 (Il23a) (B) in each major cell type, fibroblast subpopulations, and myeloid cell subpopulations during periodontitis development. (C) Interaction between chemokine ligands strongly expressed by AG fibroblasts and their putative chemokine receptors expressed by T cells, including ILCs. Dot plots depict gene expression levels of Ccr1, Ccr2, Ccr5, Ccr7, Ccr8, Cxcr2, Cxcr3, and Cxcr4 in T cell subpopulations on Day 7 following ligature placement. (D) Interaction between chemokine ligands strongly expressed by neutrophils and their putative chemokine receptors expressed by T cells. Dot plots depicting gene expression levels of Ccr1, Ccr4, and Ccr5 in T cell subpopulations on Day 7 following ligature placement. (E) NicheNet ligand–target matrix denoting the regulatory potential between active ligands in fibroblasts and target genes in T cells from the p-EMT program on Day 7 following ligature placement. (F) NicheNet ligand– target matrix denoting the regulatory potential between active ligands in myeloid cells and target genes in T cells from the p-EMT program on Day 7 following ligature placement. (G) Dot plot depicting expression levels of active ligand genes from panel (E) in fibroblast subpopulations. (H) Dot plot depicting expression levels of active ligand genes from panel (F) in myeloid cell subpopulations. (I) Dot plot depicting expression levels of target genes from pane (E) in T cell subpopulations. (J) Dot plot depicting expression levels of target genes from panel (F) in T cell subpopulations.

Schematic overview of the newly proposed AG fibroblast–neutrophil–ILC3 axis. We propose that periodontal inflammation is initiated by the activation of AG fibroblasts, which secrete chemokines and cytokines that recruit neutrophils to sites of tissue damage. Activated neutrophils and AG fibroblasts, in turn, activate ILC3s, leading to production of proinflammatory IL-17 cytokines. Ultimately, cervical alveolar bone resorption is facilitated by a localized osteoclastogenic environment, induced by activated ILC3s, together with AG fibroblasts, neutrophils, myofibroblasts, and gingival epithelial cells, including those with an epithelial–mesenchymal transition (EMT) phenotype.

Alveolar bone loss in the ligature-induced mouse periodontitis model. Alveolar bone loss was assessed at the mesiobuccal cusp (M1-1), distobuccal cusp (M1-2), and distal cusp (M1-3) of the first molar, the mesiobuccal cusp (M2-1) and distobuccal cusp (M2-2) of the second molar, and the buccal cusp (M3) of the third molar 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 (n = 6). Significance was determined by analysis of variance (ANOVA), with Tukey’s multiple-comparison test. Data are presented as mean values ± standard deviation (SD); p < 0.05 was considered significant.

Identification of major cell types in mouse gingival tissue during periodontitis development by single-cell RNA sequencing (scRNA-seq). Violin plots showing expression levels of cell-type marker genes in each major cell type: epithelial cells, cadherin 1 (Cdh1) and type XVII collagen (Col17a1); fibroblasts, type I collagen (Col1a1) and lumican (Lum); endothelial cells, selectin P (Selp) and selectin E (Sele); B cells, membrane spanning 4 domains A1 (Ms4a1) and cluster of differentiation 79A (Cd79a); T cells, epsilon subunit of T cell receptor complex (Cd3e) and cluster of differentiation 5 (Cd5); and myeloid cells, lysozyme 2 (Lyz2) and integrin subunit alpha M (Itgam).

Identification of myeloid cell subpopulations in mouse gingival tissue during periodontitis development by scRNA-seq. Violin plots showing expression levels of macrophage and neutrophil marker genes in myeloid cell subpopulations: macrophages, cluster of differentiation 86 (Cd86) and integrin subunit alpha X (Itgax); and neutrophils, CXC motif chemokine receptor 2 (Cxcr2) and G0/G1 switch gene 2 (G0s2).

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 CEJ to the ABC on the buccal or palatal side of the alveolar bone of wild-type (WT), Rag2-/-, and Rag2-/-γc-/- 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). Significance was determined by ANOVA, with Tukey’s multiple-comparison test. Data are presented as mean values ± SD; p < 0.05 was considered significant.

Epithelial cell subpopulations involved in periodontitis development in mice. (A) t-distributed stochastic neighbor embedding (t-SNE) projection plots showing epithelial cell subpopulations in gingival tissue on Days 1, 4, and 7 following ligature placement. Colors indicate cell type, as follows: dark green, epithelial cell population 1 (Epi 1); sea green, epithelial cell population 2 (Epi 2); pale green, epithelial cell population 3 (Epi 3); light green, epithelial cell population 4 (Epi 4); and light blue, epithelial cell population expressing epithelial–mesenchymal transition (EMT) genes. (B) Heatmap showing expression of the top-10 differentially expressed genes in Epi 1, Epi 2, Epi 3, Epi4, and EMT. (C) Violin plots showing gene expression levels of keratin 5 (Krt5), keratin 14 (Krt14), cadherin 11 (Cdh11), Col1a1, tenascin C (Tnc), and smooth muscle aortic actin 2 (Acta2) in epithelial cell subpopulations. (D) Violin plots showing gene expression levels of interleukin 6 (Il6) and interleukin 23A (Il23a) in epithelial cell subpopulations.