METTL3‐mediated m6A methylation of lipopolysaccharide‐binding protein exacerbates periodontitis by promoting M1 macrophage polarization

BACKGROUND: Periodontitis is a chronic inflammatory disease driven by dysregulated immune responses and is closely associated with macrophage polarization. N6-methyladenosine (m6A) methylation represents the most prevalent RNA modification; however, its role in periodontitis remains unclear. This study aimed to investigate the mechanism by which m6A modification regulates macrophage polarization during the progression of periodontitis. METHODS: A periodontitis mouse model was established by ligature induction. RAW264.7 macrophages were treated with Porphyromonas gingivalis-derived lipopolysaccharide (LPS) to simulate inflammatory conditions in vitro. Micro-computed tomography (micro-CT), hematoxylin-eosin and immunohistochemical staining, quantitative real-time polymerase chain reaction (qPCR), immunofluorescence staining, methylated RNA immunoprecipitation (MeRIP), and luciferase reporter assays were used to evaluate alveolar bone loss, macrophage phenotypes, and m6A-dependent regulatory mechanisms. RESULTS: Methyltransferase-like 3 (METTL3) expression was significantly upregulated in periodontitis tissues and in LPS-stimulated macrophages. Knockdown of METTL3 attenuated alveolar bone resorption, reduced pro‑inflammatory M1 markers (tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6]), and increased M2 markers (IL-10, arginase-1 [Arg-1]) in mice. In LPS-treated RAW264.7 cells, METTL3 knockdown suppressed M1 polarization markers (CD86, TNF-α, IL-1β) and enhanced M2 markers (CD206, transforming growth factor-beta [TGF-β]). Mechanistically, METTL3 stabilized LPS-binding protein (LBP) mRNA via m6A modification, and overexpression of LBP reversed the METTL3 knockdown-mediated suppression of M1 polarization. RNA immunoprecipitation (RIP) and dual-luciferase assays identified YTHDF1 as the m6A reader responsible for recognizing LBP mRNA. These results indicate that METTL3 promotes periodontitis by enhancing LBP mRNA stability through m6A methylation, thereby driving M1 macrophage polarization. CONCLUSIONS: This study identifies the METTL3-LBP-YTHDF1 axis as a key regulator of macrophage-driven inflammation in periodontitis, providing new potential therapeutic targets for modulating immune responses in periodontal disease. PLAIN LANGUAGE SUMMARY: Periodontitis is a common chronic inflammatory disease that damages gum tissue and tooth-supporting bones and can lead to tooth loss if untreated. Immune cells called macrophages play a key role here-when they become "pro-inflammatory" (promoting inflammation), the disease worsens. We studied a molecule called methyltransferase-like 3 (METTL3), which is more abundant in periodontitis tissues and inflamed cells. Using mouse models and lab-grown cells, we found that reducing METTL3 levels eased inflammation, protected tooth-supporting bones, and shifted macrophages from pro-inflammatory to anti-inflammatory (a type that helps repair tissue). This happens because METTL3 causes a common chemical change in RNA to stabilize another molecule, lipopolysaccharide-binding protein (LBP), with the help of a protein called YTHDF1. Our findings show that the METTL3-LBP-YTHDF1 pathway drives periodontitis by boosting harmful inflammation, offering a new target for developing treatments to regulate immune responses and improve oral health.