Tolerance Induced by Porphyromonas gingivalis Altered Inflammatory Responses in Mice

ABSTRACT Tolerance refers to a hyporesponsiveness toward repeated stimulations with bacteria and their virulence factors, which might exist in the development of periodontitis. To identify the roles of tolerance induced by Porphyromonas gingivalis ( P. gingivalis ) in periodontitis, an original tolerized mice model was established by high‐dose of oral P. gingivalis inoculation following a primary infection. The alveolar bone loss of maxillae was detected by Micro‐CT. The infiltration of neutrophils and macrophages, and macrophage polarization were detected by IHC and flow cytometry, respectively. Residual P. gingivalis in subgingival plaque with and without macrophage/neutrophil depletion was measured by real‐time PCR. Moreover, a real‐time PCR chip and bioinformatic analysis were then employed to explore the cytokine expression profiles in gingivae. The abundance of TNF‐α, Toll‐like receptor 2 (TLR2), and TLR4 were further verified by western blot. In comparison with the non‐tolerance group, TNF‐α protein levels, alveolar bone loss, and the infiltration of neutrophils and macrophages in the tolerance group were significantly suppressed ( p < 0.05), while the quantities of residual P. gingivalis in subgingival plaque were increased ( p < 0.05). Moreover, the depletion of macrophages by liposomal clodronate weakened the inhibitory effect of tolerance, as evidenced by the lack of differences in the quantities of residual bacteria between the tolerance and non‐tolerance groups ( p > 0.05). Macrophages in gingivae of tolerized mice were more likely to polarize into M2 type. In addition, the expressions of cytokines related to neutrophil and macrophage infiltration and recruitment and the protein levels of TLR2 and TLR4 were decreased in tolerized mice ( p < 0.05). Tolerance induced by repeated P. gingivalis stimulations suppressed inflammatory responses in periodontal tissues, and the established periodontal tolerance model provided a reliable tool for the further study on periodontal tolerance in vivo.