Macrophages Polarization Promote Alveolar Bone Destruction In Periodontitis

Periodontitis,one of the most prevalent infectious diseases in oral cavity,is the sixth most common epidemic in the world.Local bacterial and bacterial products trigger an immune response in the host,leading to inflammatory cell infiltration and connective tissue degeneration and increased tooth mobility or loss,which has a significant impact on the patient’s oral and general health.Macrophages are important components of the innate immune system to protect against infection.They could be polarized and acquire specific phenotypes,such as the M1-type and the M2-type,which are closely related to the progression of alveolar bone destruction and teeth loss in periodontitis.Objective: To analyze the infiltration of M1 and M2 in the periodontal alveolar bone destruction,and explore its role in the imbalance of osteoblasts and osteoclasts and analyze its possible mechanism.Methods: C57BL/6 mice aged 6-8 weeks were ligated by sterile surgical silk to establish periodontitis model.Micro-CT was used to determine and analyze the alveolar bone destruction in the control group(left upper jaw)and experimental group(right upper jaw),including SMI,Tb.Sp,BMD,BVF,Tb.T and Tb.N.Osteoblasteogenesis and osteoclasteogenesis were detected by Masson and TRAP staining,and macrophage infiltration in the periodontal tissue was analyzed by HE staining and immunohistochemistry.In vitro,RAW264.7 was differentiated into M1 and M2 phenotypes to stimulate the osteoblasts.Western blotting was used to detect the expression of Runx2 and Ocn.Bone marrow mononuclear cells were aslo differentiated into M1 and M2.Runx2 and Ocn were detected in the osteoblasts after condition medium stimulation.ALP activity and ALP staining were also analyzed.TRAP staining and immunofluorescence were used to evaluate the osteoclasts differentiation ability.Based on these observations,the MC and MC-M1 cells were subjected to RAN-seq detection,and the differential gene expression in the Toll-like receptor signaling pathway and the accociated mechanism of M1 in the inhibition of osteogenesis was analyzed.Results: After 7 days of silk wire ligation,the micro-CT showed that the alveolar bone destruction was significantly aggravated in the experimental group,the SMI and Tb.SP were increased and the BMD,BVF,Tb.Th and Tb.N was decreased in the P group.Masson and TRAP staining showed that the osteogenesis was weakened and the bone was destroyed in the experimental group.The HE staining showed that the periodontal fibers were disordered,the inflammatory cells infiltrated,and the alveolar bone was absorbed.The results of immunohistochemistry showed that the bones destruction was associated with M1 and M2 macrophages infiltration.And the M1 may play a critical role in the process.In vitro,the expression of Runx2 and Ocn was decreased after M1/M2 macrophage stimulation,while ALP activity and ALP staining was decreased,M1 was more effective than M2 in the inhibition of osteogenesis.Meanwhile,M1 macrophages play a more prominent role in the osteoclast formation than M2.RNA-sequencing revealed that M1 promotes IL-6 expression by regulating TLR4/AP1 in osteoblasts and plays an important role in inhibiting osteogenesis.While the role of M1 macrophages in the inhibition of osteogenesis was decreased after TLR4 was inhibited,the expression of Runx2 and Ocn was down-regulated,the AP1 expression and IL-6 expression was also down-regulated.At the same time,RANKL/OPG in osteoblasts was significantly increased after M1 sitimulation,and the expression of NFATc1 was also increased.While TLR4 was inhibited,RANKL/OPG ratio and the expression of NFATc1 were down-regulated after M1 situmulaion.Conclusions: Macrophage infiltration is closely associated with alveolar bone destruction.M1 macrophages are bone destructors via reduction of osteoblastogenesis and induction of osteoclast differentiation,while M2 weakly than the M1 in the process.More importantly,we found that the activation of TLR4/AP1 signaling pathway may contribution to the effects of M1 macrophages on the bone loss in periodontitis.