[Preliminary study on the role of IL-1β+ neutrophils in bone regulation of periodontitis mice].

Objective: To investigate the role and mechanism of interleukin-1β+ (IL-1β+) neutrophils in bone regulation of periodontitis in mice. Methods: The periodontitis model was established by local oral infection with Porphyromonas gingivalis (Pg). Twenty 8-week-old male C57BL/6J mice were randomly divided into 2 groups (10 mice in each group) using a random number table method: the control group [con group, 2% carboxymethylcellulose (CMC) without Pg applied topically to both buccal sides of the maxilla in mice] and the experimental group [periodontitis (PD) group, 2% CMC with Pg applied topically to both buccal sides of the maxilla in mice]. Micro-CT was used to evaluate maxillary alveolar bone. Single-cell transcriptome sequencing was employed to analyze the content of different immune cells in the diseased maxilla of mice and to screen for differentially expressed genes. HE staining and immunohistochemical staining were used to observe changes in the maxillary periodontal tissues of mice. Flow cytometry and immunofluorescence staining were applied to detect the content and expression of IL-1β+ neutrophils in the maxilla. In vitro experiments involved stimulating neutrophils with Pg to detect the expression of IL-1β, followed by sorting IL-1β+ neutrophils and constructing a co-culture model with mouse embryo osteoblast precursor cells (MC3T3-E1 cells). Alkaline phosphatase staining and alizarin red staining were used to assess the osteogenic differentiation of MC3T3-E1 cells. Western blotting was used to analyze changes in the Toll-like receptor 4-nuclear factor-κB (TLR4-NF-κB) signaling pathway. Results: The periodontitis model was successfully established in vivo. The height from the cementoenamel junction to the alveolar crest (CEJ-ABC) on the buccal side of mice in the PD group [(232.90±25.29) μm] was significantly increased compared with that in the con group [(149.50±16.69) μm, P<0.001]. The palatal CEJ-ABC [(190.70±10.67) μm] was also significantly higher than that of mice in the con group [(151.80±11.29) μm, P<0.001]. Single-cell transcriptome sequencing of mouse maxillary alveolar bone revealed that the distribution ratio of neutrophils in the PD group (0.62±0.04) was significantly higher than that in the con group (0.44±0.05, P=0.010), and the expression of IL-1β in neutrophils was significant (P=0.003). Immunohistochemical staining showed that the height of the maxillary alveolar bone in the PD group was reduced than that in the con group, as well as the bone formation ability. Flow cytometry revealed that the proportion of IL-1β+ neutrophils in the diseased maxilla of the PD group [(13.66 ± 1.14)%] was significantly higher than that in the con group [(4.40±0.47)%, P<0.001], and immunofluorescence staining also indicated an increase of IL-1β+ neutrophils in the maxillary alveolar bone of the PD group. After in vitro stimulation of neutrophils with Pg, the expression of IL-1β increased (P=0.047). After co-culturing IL-1β+ neutrophils with MC3T3-E1 cells, alizarin red staining showed a decrease in mineralized nodules, and the alkaline phosphatase staining became lighter. The protein expression levels of TLR4 and pp65 increased. All the above changes could be partially or completely reversed by TLR4 inhibitors. Conclusions: IL-1β⁺ neutrophils are significantly increased in the diseased maxillary alveolar bone of mice with periodontitis, which inhibit the osteogenic differentiation of MC3T3-E1 cells and are associated with the TLR4-NF-κB signaling pathway, providing a scientific basis for immunotherapy for bone regeneration in periodontitis.