Nlrp3 inflammasome drives regulatory T cell depletion to accelerate periapical bone erosion.

AIM: Apical periodontitis is an inflammatory disorder triggered by an immune response to bacterial infection, leading to the periapical tissue damage and alveolar resorption. However, the underlying mechanisms driving this process remain elusive, due to the complex and interconnected immune microenvironment within the local lesion site. In this study, the influence of Nlrp3 inflammasome-mediated immune response on the apical periodontitis was investigated. METHODOLOGY: RNA sequencing, immunohistochemistry and ELISA assay were performed to investigate the activation of Nlrp3 inflammasome signalling pathways in the human periapical tissues, including radicular cysts, periapical granulomas and healthy oral mucosa. A mouse model of apical periodontitis was established to study the role of Nlrp3 knockout in periapical bone resorption and Treg cell stability, and the underlying mechanism was explored through in vitro experiments. In vivo Treg cell adoptive transfer was performed to investigate the effects of Treg cells on the progression of apical periodontitis. RESULTS: Our findings find that the hyperactivated Nlrp3 inflammasome is present in human periapical lesions and plays a vital role in the immune-related periapical bone loss. Using a mouse model of apical periodontitis, we observe that Nlrp3 deficiency is resistant to bone resorption. This protection was accompanied by elevated generation and infiltration of local Treg cells that displayed a notable ability to suppress RANKL-dependent osteoclast differentiation. In terms of the mechanism of action, Nlrp3 deficiency directly inhibits the osteoclast differentiation and bone loss through JNK/MAPK and NF-κB pathways. In addition, Nlrp3 induces pyroptosis in the stem cells from apical papilla (SCAPs), and the subsequent release of cytokines affects the stability of Treg cell in periapical lesions, leading indirectly to enhanced bone resorption. In turn, adoptive transfer of both Nlrp3-deficient and wild-type Treg cells effectively prevent the bone erosion during apical periodontitis. CONCLUSIONS: Together, our data identify that the Nlrp3 inflammasome modulates the Treg cell stability and osteoclastogenesis in the periapical inflammatory microenvironment, thus determining the progression of bone erosion.

An update on periodontal inflammation and bone loss.

Periodontal disease is a chronic inflammatory condition that affects the supporting structures of the teeth, including the periodontal ligament and alveolar bone. Periodontal disease is due to an immune response that stimulates gingivitis and periodontitis, and its systemic consequences. This immune response is triggered by bacteria and may be modulated by environmental conditions such as smoking or systemic disease. Recent advances in single cell RNA-seq (scRNA-seq) and in vivo animal studies have provided new insight into the immune response triggered by bacteria that causes periodontitis and gingivitis. Dysbiosis, which constitutes a change in the bacterial composition of the microbiome, is a key factor in the initiation and progression of periodontitis. The host immune response to dysbiosis involves the activation of various cell types, including keratinocytes, stromal cells, neutrophils, monocytes/macrophages, dendritic cells and several lymphocyte subsets, which release pro-inflammatory cytokines and chemokines. Periodontal disease has been implicated in contributing to the pathogenesis of several systemic conditions, including diabetes, rheumatoid arthritis, cardiovascular disease and Alzheimer's disease. Understanding the complex interplay between the oral microbiome and the host immune response is critical for the development of new therapeutic strategies for the prevention and treatment of periodontitis and its systemic consequences.

Type 17 immune response promotes oral epithelial cell proliferation in periodontitis.

OBJECTIVES: This study aims to investigate the effects of type 17 immune response on the proliferation of oral epithelial cells in periodontitis. DESIGN: A time-dependent ligature induced periodontitis mouse model was utilized to explore gingival hyperplasia and the infiltration of interleukin 17A (IL-17A) positive cells. Immunohistochemistry and flow cytometry were employed to determine the localization and expression of IL-17A in the ligature induced periodontitis model. A pre-existing single-cell RNA sequencing dataset, comparing individuals affected by periodontitis with healthy counterparts, was reanalyzed to evaluate IL-17A expression levels. We examined proliferation markers, including proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription (STAT3), Yes-associated protein (YAP), and c-JUN, in the gingival and tongue epithelium of the periodontitis model. An anti-IL-17A agent was administered daily to observe proliferative changes in the oral mucosa within the periodontitis model. Cell number quantification, immunofluorescence, and western blot analyses were performed to assess the proliferative responses of human normal oral keratinocytes to IL-17A treatment in vitro. RESULTS: The ligature induced periodontitis model exhibited a marked infiltration of IL-17A-positive cells, alongside significant increase in thickness of the gingival and tongue epithelium. IL-17A triggers the proliferation of human normal oral keratinocytes, accompanied by upregulation of PCNA, STAT3, YAP, and c-JUN. The administration of an anti-IL-17A agent attenuated the proliferation in oral mucosa. CONCLUSIONS: These findings indicate that type 17 immune response, in response to periodontitis, facilitates the proliferation of oral epithelial cells, thus highlighting its crucial role in maintaining the oral epithelial barrier.

Th17-to-Tfh plasticity during periodontitis limits disease pathology.

Th17 cell plasticity is crucial for development of autoinflammatory disease pathology. Periodontitis is a prevalent inflammatory disease where Th17 cells mediate key pathological roles, yet whether they exhibit any functional plasticity remains unexplored. We found that during periodontitis, gingival IL-17 fate-mapped T cells still predominantly produce IL-17A, with little diversification of cytokine production. However, plasticity of IL-17 fate-mapped cells did occur during periodontitis, but in the gingiva draining lymph node. Here, some Th17 cells acquired features of Tfh cells, a functional plasticity that was dependent on IL-6. Notably, Th17-to-Tfh diversification was important to limit periodontitis pathology. Preventing Th17-to-Tfh plasticity resulted in elevated periodontal bone loss that was not simply due to increased proportions of conventional Th17 cells. Instead, loss of Th17-to-Tfh cells resulted in reduced IgG levels within the oral cavity and a failure to restrict the biomass of the oral commensal community. Thus, our data identify a novel protective function for a subset of otherwise pathogenic Th17 cells during periodontitis.

Toll-like receptor 4 deficiency affects the balance of osteoclastogenesis and osteoblastogenesis in periodontitis.

Toll-like receptor 4 (TLR4) acts as a double-edged sword in the occurrence and development of periodontitis. While the activation of TLR4 in macrophages aids in clearing local pathogens, it can also disrupt innate immune responses, upsetting microecological balance and accelerating the destruction of periodontal bone tissues. To date, the effects of TLR4 on osteogenesis and osteoclastogenesis in periodontitis have not been comprehensively studied. In this study, we investigated the development of periodontitis in the Tlr4-/- mice by ligating their second molars with silk threads. Compared to wild-type (WT) mice, Tlr4-/- mice demonstrated increased resistance to periodontitis-associated bone destruction, as evidenced by decreased bone resorption and enhanced bone regeneration. Mechanistically, the deletion of Tlr4 not only inhibited osteoclast formation by reducing the expression of NFATc1, CTSK and TRAP, but also enhanced osteogenic abilities through increased expression of OCN, OPN and RUNX2. In conclusion, TLR4 tips the balance of osteoclastogenesis and osteogenesis, thereby promoting periodontal bone destruction in periodontitis.

IRE1α regulates macrophage polarization in type 2 diabetic periodontitis through promoting endoplasmic reticulum stress.

OBJECTIVES: The aim of this study was to investigate the effect of 4µ8c, an inhibitor targeting the endoplasmic reticulum stress-associated factor IRE1α, on macrophage polarization in an experimental model of diabetic periodontitis through ex vivo experiments. MATERIALS AND METHODS: Local alveolar bone parameters were evaluated using Micro-CT following intraperitoneal administration of 4µ8c in mice with experimental diabetic periodontitis. Surface markers indicating macrophage polarization were identified using immunofluorescence. In vitro experiments were performed employing bone marrow-derived macrophages and gingival fibroblasts. Macrophage polarization was determined using flow cytometry. Principal impacted signaling pathways were identified through Western blot analysis. RESULTS: Results from both in vitro and in vivo experiments demonstrated that 4µ8c mitigated alveolar bone resorption and inflammation in mice with diabetic periodontitis. Furthermore, it modulated macrophage polarization towards the M2 phenotype and augmented M2 macrophage polarization through the MAPK signaling pathway. CONCLUSIONS: These findings suggest that inhibiting IRE1α can modulate macrophage polarization and alleviate ligature-induced diabetic periodontitis via the MAPK signaling pathway. This unveils a novel mechanism, offering a scientific foundation for the treatment of experimental diabetic periodontitis.