IADR Abstract Archives
Bacterial EDNA Initiates Periodontitis Through the TLR9/Ctsk Axis
Objectives: Prevention of periodontitis and peri-implantitis is essential to achieve long-term success in prosthodontic treatment. The understanding of mechanisms that drive initial gingival degradation and the specific role of oral commensal bacteria is critical to achieve our goal. The objective of this study was to examine the role of oral microbial components and the interacting host pathways in the initiation of periodontitis.
Methods: A mouse ligature-induced periodontitis model was used to establish single cell RNA sequencing (scRNA-seq) for characterization of inflammatory tissue responses. Periodontitis induction was confirmed by histology and microCT imaging. Cathepsin K (Ctsk) activity was determined by immunohistochemistry (IHC) and a newly developed bone-binding FRET probe (OFS). Furthermore, we established an in vivo model, in which separate oral microbial components such as P. gingivalis LPS, bacterial unmethylated CpG oligonucleotide (ODN) and extracellular bacterial DNAs (eDNA) isolated from in vitro grown human oral microbial biofilm were topically applied to the mouse palatal gingiva. Gingival responses and Ctsk activation were examined by scRNA-seq and OFS.
Results: The ligature placement immediately activated Ctsk from Day1, while pro-inflammatory responses started from Day3, along with alveolar bone resorption. Gingival fibroblasts, associated with collagen matrix degradation, were determined as the early cellular source of Ctsk and Toll-like receptor 9 (TLR9) by IHC and scRNA-seq. Furthermore, topical application of eDNA and ODN induced gingival inflammation and a much stronger OFS fluorescent signal was detected in the DNA treated groups compared to the LPS group.
Conclusions: Our results indicate that bacterial eDNA, a TLR9 specific ligand, and Ctsk in gingival fibroblasts are the previously unrecognized inducers of periodontitis and periodontal pocket formation. This new finding should provide a critical clue in determining the optimal property and design of dental biomaterials in order to inhibit eDNA and TLR9/Ctsk axis for long-term stability of prosthodontic treatment.
Methods: A mouse ligature-induced periodontitis model was used to establish single cell RNA sequencing (scRNA-seq) for characterization of inflammatory tissue responses. Periodontitis induction was confirmed by histology and microCT imaging. Cathepsin K (Ctsk) activity was determined by immunohistochemistry (IHC) and a newly developed bone-binding FRET probe (OFS). Furthermore, we established an in vivo model, in which separate oral microbial components such as P. gingivalis LPS, bacterial unmethylated CpG oligonucleotide (ODN) and extracellular bacterial DNAs (eDNA) isolated from in vitro grown human oral microbial biofilm were topically applied to the mouse palatal gingiva. Gingival responses and Ctsk activation were examined by scRNA-seq and OFS.
Results: The ligature placement immediately activated Ctsk from Day1, while pro-inflammatory responses started from Day3, along with alveolar bone resorption. Gingival fibroblasts, associated with collagen matrix degradation, were determined as the early cellular source of Ctsk and Toll-like receptor 9 (TLR9) by IHC and scRNA-seq. Furthermore, topical application of eDNA and ODN induced gingival inflammation and a much stronger OFS fluorescent signal was detected in the DNA treated groups compared to the LPS group.
Conclusions: Our results indicate that bacterial eDNA, a TLR9 specific ligand, and Ctsk in gingival fibroblasts are the previously unrecognized inducers of periodontitis and periodontal pocket formation. This new finding should provide a critical clue in determining the optimal property and design of dental biomaterials in order to inhibit eDNA and TLR9/Ctsk axis for long-term stability of prosthodontic treatment.