Nlrp3+ Macrophages Epigenetically Regulates Gli1+Mscs During Alveolar Bone Regeneration

Achieving alveolar bone regeneration under the inflammatory microenvironment is one of the main bottlenecks in the field of dental research.This study aims to reveal the mechanism on how NLRP3+ macrophages regulating Gli1+ MSCs osteogenic differentiation during periodontal tissues regeneration. Mice periodontitis models and periodontal regeneration models were constructed. Gli1-Tdt, Nlrp3-/-; Gli1-Tdt mice were used. Micro-CT, Opal multiplexing immunohistochemistry staining, western blot, qRT-PCR, flow cytometry, chromatin immunoprecipitation, DNA hydroxymethylation level detection were applied to disclose the mechanisms. 1.During periodontal inflammation and regeneration process, the numbers of Gli1+ MSCs and NLRP3+macrophages showed opposite trends. 2.During periodontal inflammation process, the osteogenic differentiation ability of Gli1+ MSCs decreased accompanied by increased IL-1β secreted by NLRP3+ macrophages. 3.Periodontal tissue regeneration of Nlrp3-/- mice were better with less periodontal inflammation. 4.Single cell analysis showed that there was an interaction between Gli1+MSCs and NLRP3+macrophages, and in vitro experiments showed that NLRP3+macrophages inhibit osteogenic differentiation of Gli1+MSCs. 5. RNA sequencing results showed that NLRP3+macrophages regulated the Hedgehog pathway in Gli1+MSCs. 6. IL-1β secreted by NLRP3+ macrophages activated the NF -κB pathway, thereby inhibiting TET2 expression and upregulating the methylation level of the key gene Ihh in the Hedgehog pathway, further inhibited the osteogenic differentiation ability of Gli1+MSCs. NLRP3+ macrophages secretes IL-1β to mediate TET2, which epigenetically regulates Ihh to regulate osteogenic differentiation of Gli1+MSCs, targeting NLRP3+ macrophages may be a feasible approach to promote periodontal regeneration.