Accelerated cellular senescence along with periodontitis progression.

(A and B) IHC staining and semi-quantification of P16 and P21 in healthy and periodontitis patient gingiva (n=3). (C) Strategy of ligature-induced periodontitis (LIP) mouse model. (D) IHC staining and semi-quantification of P16 in mouse gingiva of health and LIP post 3D, 7D and 14D (n=3). (E) Western blot images and semi-quantification of P16 protein levels in control and LIP post 7D mouse gingiva (n=3). (F) qPCR analysis of P16, P21and P53 in control and LIP post 7D mouse gingiva (n=3). (G) Volcano plots displaying the differentially expressed genes in mouse gingiva among LIP 7D compared to the control. Representative aging related genes are indicated as green. blue dots indicate differentially down-regulated genes; red dots indicate differentially up-regulated genes. Significantly different expression gene with log2FC > 1 and FDR < 0.05. (H) GSEA enrichment analysis of aging gene sets in differentially expressed genes in mouse gingiva. (I) GO enrichment analysis with genes upregulated (red) and downregulated (blue) genes shown in (G). Data are mean ± SD. *p<0.05, **p<0.01, ***p<0.001. ****p<0.0001.

Inhibition of cellular senescence alleviates bone damage in ligation induced periodontitis (LIP) murine models.

(A) Establishment of a mouse model of periodontitis treated with metformin. (B) Micro-CT images and 3-D visualization of the maxilla. and quantified by the BV/TV ratio (Ga) the CEJ-ABC distance (Gb) (C) Representative images of HE staining. (D) Representative images of Masson staining and quantification of collagen fiber(Gc), in which collagen fibers were stained into blue (E and F) IHC staining and semi-quantification (Gd) of P16. (F) IHC staining and semi-quantification (Ge) of P21. N = 6, *P <= 0.05, **P<= 0.01, ***P<= 0.001, ****P<= 0.0001.

Gingival fibroblasts are the main senescent cell type in periodontitis.

(A) UMAP diagram and single-cell annotation of cells clusters for the healthy and periodontitis samples from GSE164241. (B) Histogram of gingival tissue cell ratio in healthy and periodontitis patients. (C) GSEA enrichment analysis of aging pathway in differentially expressed genes in fibroblasts among periodontitis and healthy gingiva. (D) The violin plot showing aging score of subgroups in healthy and periodontitis gingiva. (E) The violin plot showing aging score in subgroups in gingiva of healthy, mild and severe periodontitis patient from GSE152042. (F and G) IF staining of P16 (red), VIM (green), and nuclei (blue) in healthy and periodontitis patient gingiva or in control and LIP mouse gingiva. (H) β-Galactosidase staining and semi-quantification of HGF stimulated by different concentrations of pg-LPS. White arrow indicates positive cells. (I) qPCR analysis of P16 expression of HGF stimulated by different concentrations of pg-LPS. *P <= 0.05, **P<= 0.01, ***P<= 0.001, ****P<= 0.0001.

CD81 is identified as the representative marker of senescent gingival fibroblast.

(A) UMAP diagram illustrated the cell clusters of fibroblasts. (B) Histogram of fibroblasts subclusters ratio in healthy and periodontitis gingiva. (C) The violin plot showing aging score of fibroblasts subclusters in healthy and periodontitis gingiva. (D) Enrichment analysis on Hallmark Gene-sets from GSEA. (E) GO enrichment analysis of fibroblasts subclusters. (F) Localization of the top 20 marker gene in fibroblasts subcluster 1. (G) Density map of CD81 expression. (H) Single-cell annotation of fibroblasts subclusters. (I, J) IF staining of CD81 (red), VIM (green) and nuclei (blue) in healthy and periodontitis patient gingiva or in healthy and LIP mouse gingiva. White arrow indicates double positive cells. (K) IF staining of P16 (red), VIM (green), CD81 (purple), and nuclei (blue) in LIP mouse gingiva. gray arrow indicates triple positive cells. (L) Western blot image and semi-quantification of human P16, P21 and CD81 protein levels of HGF stimulated by different concentrations of pg-LPS. * P <= 0.05, **P<= 0.01, ***P<= 0.001, ****P<= 0.0001.

CD81+ gingival fibroblasts are terminally differentiational cell with high SASP expression and metabolism alteration.

(A) Heatmap showing the relative expression for SASP genes in each fibroblast subclusters. (B) The heatmap representing metabolic pathways in each fibroblast subclusters. (C) The heatmap representing metabolic pathways between healthy and periodontitis gingiva. (D) The flow chart representing the metabolism of arachidonic acid. (E) The dot plot representing that COX1 and COX2 are significantly increased in CD81+ gingival fibroblasts. (F) Trajectory reconstruction of all gingival fibroblast clusters (G) Monocle pseudotime analysis revealing the progression of gingival fibroblast clusters. (H) Upper panel: Heatmap showing the scaled expression of differently expressed genes in trajectory as in (G), cataloged into four gene clusters (labels on left). Bottom panel: GO analysis of expressed genes whose expression increases as the differentiation trajectory progresses. (I) SASP-related genes with increased expression as the differentiation trajectory progresses. * P <= 0.05, **P<= 0.01, ***P<= 0.001, ****P<= 0.0001.

CD81+fibroblasts recruit neutrophils via C3/C3aR1 axis.

(A) The relative number and intensity of interactions between fibroblasts and neutrophil in periodontitis gingiva. (B)Significant increased (left) and decreased (right) Ligand-Receptor interaction derived from CD81+fibroblasts. (C) The heatmap showing the communication patterns of the complement signaling pathway between fibroblasts and immune cell type. (D) The expression level of four representative genes in complement signaling pathway. (E) IHC staining and semi-quantification of C3 in healthy and periodontitis gingiva. (F) IF staining of CD81 (red), C3 (green), and nuclei (blue) in control and LIP mouse gingiva. (G) IF staining of CD81 (red), MPO (green), and nuclei (blue) in control and LIP mouse gingiva. (H) H&E image of the representative inflamed oral mucosa from GSE152042. (I) Representative spatial mapping of CD81 and SOD2 in health and periodontal disease showing co-localization in CD81+ fibroblasts and neutrophil in the periodontal disease. (J and K) IHC staining and semi-quantification of C3 and MPO in periodontitis treated by metformin. *P <= 0.05, **P<= 0.01, ***P<= 0.001, ****P<= 0.0001.

Schematic overview of the CD81+ senescent gingival fibroblast-neutrophil axis in periodontitis progression.

We propose that the initial periodontal inflammation is triggered by the CD81+ senescent gingival fibroblast induced by bacterial virulence like Pg-LPS. CD81+ senescent gingival fibroblast could exaggerate inflammation in the periodontal tissue via secreting SASPs and recruiting neutrophils by C3. In addition, metformin could alleviate the cellular senescence of the fibroblast and rescue the uncontrolled inflammation and bone resorption.