Glut3/Tnfaip3-Mediated Pad4 Deubiquitination In Nets Formation During Periodontitis

ScRNA-seq identified GLUT3 as a pivotal molecule within the NETs-related subpopulation, while its regulatory role and mechanism in the excessive formation of NETs during periodontitis remain unclear. We analyzed GLUT3 expression in healthy, gingivitis, and periodontitis integrating scRNA-seq data from GSE266897 and our data. Immunofluorescence staining, qRT-PCR and WB were performed to detect the expression and localization of GLUT3 and NETs in tissue samples, neutrophils, and dHL-60 cells. The role of GLUT3 and PAD4-mediated NETs was investigated through gingival localized GLUT3 knockdown and PAD4 inhibitor treatment in periodontitis mice. Differentially expressed genes in NETs-related subpopulation were cross-referenced with the iUUCD 2.0 database to screen ubiquitin-related genes with maximal differences. The regulatory effects of candidate molecules on PAD4 ubiquitination degradation and GLUT3-mediated modulation on this process were explored via in vitro and in vivo experiments. Bioinformatic data and tissue samples indicated that compared to healthy gingival tissues, GLUT3 expression was slightly downregulated in gingivitis but significantly upregulated in periodontitis, where it co-localized with NETs. Specifically, GLUT3 mediated the increased formation of NETs by upregulating the protein level of PAD4, which was found to be attributed to TNFAIP3(a deubiquitinating enzyme)inhibiting ubiquitization degradation, rather than mRNA upregulation. GLUT3 exacerbates periodontitis by inducing excessive NETs formation through upregulating TNFAIP3, which inhibits ubiquitination-mediated degradation of PAD4. These findings provide novel insights into the immunopathological mechanisms of periodontitis and suggest potential therapeutic targets.