Periodontitis, a common oral disease, endangers dental and general health, worsening the global chronic disease burden and linking it to various diseases. Gingival tissue with immunomodulatory fibroblasts resists pathogens, but periodontitis mechanisms are unclear and treatments are limited.

N6-methyladenosine (m⁶A), catalyzed by a METTL3-containing complex, is a major eukaryotic mRNA modification. METTL3 is involved in many processes, and its m⁶A modification matters in inflammation, yet its role in periodontitis is unclear. Previous m⁶A changes in periodontitis need further exploration of METTL3's role.

Recently, Bin Shao&Quan Yuan's team from China published an article titled "Inhibition of METTL3 Alleviates NLRP3 Inflammasome Activation via Increasing Ubiquitination of NEK7" in Advanced Science. This study focused on exploring Mettl3's effects on periodontal destruction and inflammation, finding an important mechanism that METTL3 affects periodontitis progression via regulating NLRP3 inflammasome activation by NEK7, and screening a small-molecule natural inhibitor targeting METTL3.

Materials and Methods

In this study, multiple materials and methods were used. For antibodies and reagents, various primary and secondary antibodies, along with inhibitors like disulfiram and COP, were employed. Mice were bred in a pathogen-free lab at Sichuan University, with genetically targeted ones created by CRISPR-Cas9. Periodontitis models were induced on anesthetized mice. Then, procedures like anesthesia, fixation, and CBCT scanning were done. Micro-CT analysis, histological staining, Western Blot, etc., were carried out with specific steps. Human gingival fibroblasts were isolated and cultured with other cell lines. Gene knockdown in HGFs by siRNA transfection and TNFAIP3 knockout by plasmid transfection was achieved. RNA sequencing, MeRIP data analysis, and quantitative real-time PCR were also performed. Additionally, assays like ELISA of IL-1β, LDH release, PI Uptake Staining, LC-MS/MS, and Dot Blot were conducted for different analyses.

Results

Firstly, they utilized Gli1 cells (multipotent stem cells that play an important role in maintaining the homeostasis of periodontal tissues) to generate Gli1-cre^ER; Mettl3^fl/fl (CKO) mice for deleting Mettl3 in periodontal stromal tissues. The 5-week-old mice were intraperitoneally injected with tamoxifen for three consecutive days before oral ligation (Fig. 1A). Subsequently, the mice were subjected to oral ligation and then the ligated mice were treated with STM2457 (a selective small-molecule inhibitor of METTL3).

The results showed that after ligation, wild-type mice exhibited severe alveolar bone destruction, while CKO mice had less bone resorption and more remaining bone. Moreover, the deletion of Mettl3 led to a reduction in the secretion of IL-1β by gingival fibroblasts (Fig. 1B-F). Treatment with STM2457 could ameliorate the alveolar bone loss and decrease the expression of GSDMD and the production of IL-1β in periodontal tissues.

Fig. 1 Deletion of Mettl3 in periodontal mesenchymal cells ameliorates periodontal destruction.

Next, RNA sequencing analysis, relevant pathway detection, and various experiments were conducted on METTL3-deficient human gingival fibroblasts (HGFs) mediated by small interfering RNAs (siRNAs), along with histological analysis on mouse periodontitis models.

RNA sequencing revealed transcripts in these cells were up/downregulated. GO enrichment analysis showed METTL3 deficiency downregulated genes related to inflammatory diseases and the NF-κB pathway (Fig. 2A-C). GSEA verified that genes associated with inflammasome activation were downregulated in METTL3-deficient HGFs under proinflammatory stimuli. Immunoblotting and other assays confirmed silencing METTL3 changed protein expression and impaired inflammasome assembly/activation (Fig. 2D-F). Changes in pyroptosis-related indicators suggested METTL3 knockdown could prevent cells from pyroptotic death. Affected cytokines and other factors led to the downregulation of IL-1β secretion and LDH release. In mouse periodontitis models, NLRP3 and GSDMD expression was downregulated in CKO mice (Fig. 2G-M).

Fig. 2 METTL3 regulates inflammasome assembly and pyroptosis.

To assess if blockade of pyroptosis could improve periodontal destruction, ligated mice were treated with disulfiram, a pyroptosis inhibitor (Fig. 3A). Results showed that oral ligation dropped mice weights, and disulfiram-treated WT mice's weights rose on day 7, while not in CKO mice (Fig. 3B). Micro-CT showed it alleviated WT mice's periodontal bone absorption (Fig. 3C-E). It also reduced related protein expression/maturation in WT mice (Fig 3F, G). In CKO mice, no difference in bone loss or pyroptosis level with/without disulfiram, indicating its effect on periodontitis is METTL3-dependent (Fig. 3C–G).

Fig. 3 Administration of disulfiram relieves periodontal inflammation.

The subsequent experiments were designed to explore the role of METTL3-mediated m⁶A modification and TNFAIP3. Analyzing MeRIP data showed that there were m⁶A peaks near the stop codon of TNFAIP3 and on its 3'UTR upon LPS stimulation, which were affected by Mettl3 deletion (Fig. 4A).

Mettl3 deletion increased TNFAIP3 levels in ligature mice and METTL3-deficient HGFs, and proinflammatory stimulation further enhanced its expression with METTL3 depletion. RNA decay assays showed TNFAIP3 degraded slower in METTL3-deficient HGFs (Fig. 4B-F). Deleting TNFAIP3 in such HGFs raised the expression of GSDMD's activated N-terminal and LDH release. In DKO mice with Mettl3 and Tnfaip3 deleted in periodontal tissues, micro-CT, and HE staining revealed aggravated alveolar bone crest destruction, more GSDMD-positive cells around it, and elevated IL-1β positive granules in fibroblasts' cytoplasm (Fig. 4G-N).

Fig. 4 METTL3-mediated m⁶A promotes TNFAIP3 degradation.

Multiple assays were conducted to validate TNFAIP3-NEK7 interaction and TNFAIP3's effect on NEK7 stability. Assays confirmed TNFAIP3 binds NEK7 (Fig. 5A-D). Overexpressing TNFAIP3 sped up NEK7-HA degradation, MG132 inhibited its proteasome-dependent one, and it increased NEK7 ubiquitination (more with LPS and ATP), indicating TNFAIP3 directly catalyzed the ubiquitination of NEK7. Silencing TNFAIP3 upregulated NEK7 and GSDMD, indicating an augmented response to inflammation on account of increased NEK7 expression (Fig. 5E-I).

Fig. 5 TNFAIP3 interacts with NEK7 and promotes its ubiquitination.

To explore METTL3 as a periodontitis target, they screened for an inhibitor. Virtual screening led to 30 agents, with BER showing good results. Six BER analogs were retrieved, and Coptisine chloride (COP), a SAM competitive inhibitor, was the most active (Fig. 6A-E). In AML cell line MOLM-13, COP decreased mRNA m⁶A level, down-regulated certain proteins, didn't affect METTL3/METTL14, inhibited cell survival, and induced cell differentiation (Fig. 6F-I).

Fig. 6 Discovery of a specific inhibitor of METTL3.

Finally, they evaluated COP's therapeutic efficacy for preventing periodontal inflammation. They found it decreased HGFs' RNA m⁶A levels without cytotoxicity (Fig. 7A, B). COP dose-dependently downregulated NEK7, suppressing related factors' cleavage, and reduced IL-1β and LDH release (Fig. 7D, E). Immunofluorescence showed fewer ASC specks (Fig. 7F). In periodontitis mice, daily local injections of COP improved periodontal bone loss, with less tissue destruction and alveolar bone resorption. COP also attenuated IL-1β and GSDMD expression, relieving pyroptosis (Fig. 7G-L).

Fig. 7 Therapeutic effect of Coptisine chloride on periodontitis.

Conclusion

The study explored METTL3's role in periodontitis, focusing on inflammasome activation and pyroptosis in oral mucosal immunity. Key findings include METTL3 regulating NLRP3 inflammasome via m⁶A modification of TNFAIP3, with Mettl3 deletion alleviating periodontal issues. A novel METTL3 inhibitor, COP, was found effective in vitro and in vivo. Significantly, it uncovers a new post-transcriptional regulation mechanism and offers a potential treatment. Future work could focus on the clinical application of COP and explore applicability to other inflammatory diseases like gastric cancer.

• Human Treg ELISA Kit
• Periodontitis Animal Models Establishment
• Single-Cell Sequencing