A novel lncRNA-mediated epigenetic regulatory mechanism in periodontitis

Abstract

Periodontitis is a highly prevalent chronic inflammatory disease with an exaggerated host immune response, resulting in periodontal tissue destruction and potential tooth loss. The long non-coding RNA, LncR-ANRIL, located on human chromosome 9p21, is recognized as a genetic risk factor for various conditions, including atherosclerosis, periodontitis, diabetes, and cancer. LncR-APDC is an ortholog of ANRIL located on mouse genome chr4. This study aims to comprehend the regulatory role of lncR-APDC in periodontitis progression. Our experimental findings, obtained from lncR-APDC gene knockout (KO) mice with induced experimental periodontitis (EP), revealed exacerbated bone loss and disrupted pro-inflammatory cytokine regulation. Downregulation of osteogenic differentiation occurred in bone marrow stem cells harvested from lncR-APDC-KO mice. Furthermore, single-cell RNA sequencing of periodontitis gingival tissue revealed alterations in the proportion and function of immune cells, including T and B cells, macrophages, and neutrophils, due to lncR-APDC silencing. Our findings also unveiled a previously unidentified epithelial cell subset that is distinctively presenting in the lncR-APDC-KO group. This epithelial subset, characterized by the positive expression of Krt8 and Krt18, engages in interactions with immune cells through a variety of ligand-receptor pairs. The expression of Tff2, now recognized for its role in chronic inflammatory conditions, exhibited a notable increase across various tissue and cell types in lncR-APDC deficient mice. Additionally, our investigation revealed the potential for a direct binding interaction between lncR-APDC and Tff2. Intra-gingival administration of AAV9-lncR-APDC was shown to have therapeutic effects in the EP model. In conclusion, our results suggest that lncR-APDC plays a critical role in the progression of periodontal disease and holds therapeutic potential for periodontitis. Furthermore, the presence of the distinctive epithelial subpopulation and significantly elevated Tff2 levels in the lncR-APDC-silenced EP model offer new perspectives on the epigenetic regulation of periodontitis pathogenesis.

Keywords: bone loss; immune response; long-noncoding RNA; periodontitis; single-cell RNA sequencing.

Figure 1

EP model on APDC deficiency mice and control mice. a) The digital (.stl) file and 3D printed copy of the surgical tools. b) Morphology analysis showed the distance from the alveolar bone crest (ABC) to the cementoenamel junction (CEJ) on the buccal sides and palatal sides (n=7). c) the expression level of APDC in wildtype and APDC KO mouse. d) HE staining of the periodontal tissues. The zoom-ins show the inflamed periodontal tissues. Green triangles show cementum resorption (discontinuous cementum). e) TRAP staining shows the TRAP positive cells (red) on the alveolar bone surface in APDC KO and control group at 3- and 6- weeks. Original magnification 400×. Scale bars = 200 μm. Values are shown as mean± SD. * p < 0.05, **p < 0.01.

Figure 2

The impact of APDC on inflammation related cytokines and bone metabolism. a) the heatmap of untreated (day0) BMSCs (upper) and osteogenic differentiated (day3) BMSCs (lower) comparing between APDC KO and control mice. b) the volcano plot with top genes marked (WT vs. KO). c) the GO enrichment results at untreated (upper) and osteogenesis-induced (lower) groups. d) the mRNA expression level of the osteogenic markers. e) ALP and ARS staining results (upper) and the mRNA expression level of osteogenesis related genes (lower) for APDC KO and control mice. f) the serum level of cytokines (IL-1β, IL-6, CXCL1, TNF-α, IL-10, IL-2, IL-5 and IFN-γ) (n=5). Values are shown as mean± SD. * p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 3

Overview of the 25,161 single cells from periodontal tissue of APDC-KO mice and wildtype mice (n=4 per group). a) Uniform Manifold Approximation and Projection (UMAP) representation of the 25,161 cells, colored by cell type annotation. b) stacked violin plots showing the expression scores of selected marker gene sets across all 10 clusters. c) the bar plots indicating the percentage of each cell type. d) UMAP plot representation the distribution of 11,917 cells of WT group (left) and 13,244 cells of APDC-KO group (right) from the gingival tissue in the 10 clusters. e) the correlation matrix of the 10 cell types.

Figure 4

The proportion and function changes of the immune cells in the periodontitis gingival tissue due to the APDC deficiency. (a-d) T cell: a) the subclusters of T cells. b) the violin plots showed the expression of the marker genes. c) the WT and KO group of T cells. d) the DEGs of CD4+ and CD8+ cells. e-f B cell. (e-f) B cell: e) the UMAP (left) and pie chart (right) showed the proportion of WT and KO B cells. f) the violin plots showed the gene expression level of KO and WT at different stages of B cells. (g-j) Macrophage: g) the subclusters of macrophages. h) the WT and KO group of macrophages. i) the highly expressed genes of each subcluster. j) the M1 and M2 macrophages marker genes expression in the subclusters. (k-l) Neutrophil: k) the UMAP of all neutrophils and divided by WT and KO. l) dot plot showed highly expressed genes of cluster 1 and 0.

Figure 5

Sub-clustering of the epithelial cells. a) the subclusters of epithelial cells. b) epithelial cells divided by WT and KO (right). c) the classic distinct markers of epithelium. d) Epi_1 cluster highly expressed Krt 8 and Krt 18. e) Epi_2 cluster highly expressed Krt 14 and Krt5. f) the DEGs of Epi_1 and Epi_2. g) the GO enrichment results of Epi_1 and Epi_2.

Figure 6

The mRNA and protein expression of Tff2 and its predicted binding site with APDC. a) the expression of Tff2 in WT and KO group across various cell types. b) the volcano plot illustrated the expression level of DEGs. c) the predicted binding site of Tff2 (top) and APDC (bottom). d) the heatmap showed the lowest binding energy and highest portability at the location of 1985 to 2026 on APDC. e) the Tff2 expression level in EP maxilla and gingival. f) the immunohistochemistry staining showed the Tff2 protein level in WT and KO groups.

Figure 7

The cell-cell communications among different cell types. a) the plot results of top ligand-receptor pairs between T cell and other cell types in WT and KO group. b) the plot results of top ligand-receptor pairs between Neutrophil and other cell types in WT and KO group, c) the plot results of top ligand-receptor pairs between macrophage and other cell types in WT and KO group. d) the plot results of top ligand-receptor pairs between epithelial cells and other cell types in WT and KO group. e) The expression of Ilb1 in WT and KO respectively. f) The expression of Pglyrp1 in WT and KO respectively.

Figure 8

The AAV9-CAG-APDC attenuates periodontal bone destruction. a) the AAV-APDC was administrated through microinjection on gingiva. b) the timeline of the treatment. c) the IVIS imaging of the RFP expression in AAV and PBS group. d) the APDC expression level in wild type groups. e) the APDC expression level in APDC KO groups. f) Micro-CT showed the bone resorption level with and without AAV treatment in WT mice. g) Micro-CT showed the bone resorption level with and without AAV treatment in KO mice (n=4-6). h) The mRNA expression of Tff2 after AAV-APDC treatment in WT group. i) The mRNA expression of Tff2 after AAV-APDC treatment in KO group. * p < 0.05.