hBMSC-derived Exosomes Mitigate Th17/Treg Homeostasis in Periodontitis via miR-1246

Abstract: Background Chronic inflammatory bone loss in periodontitis is closely related to helper T cell 17 (Th17) / regulatory T cell (Treg) imbalance. The therapeutic function of mesenchymal stem cells is mainly attributed to the paracrine exosomes. We aimed to investigate the immunomodulatory effect of human bone marrow mesenchymal stem cells (hBMSC)-derived exosomes in Th17/Treg homeostasis in periodontitis. Methods Peripheral blood was collected from periodontitis patients or healthy donors. The level of Th17-related biomarker interleukin-17A (IL-17A) and Treg-related forkhead box protein 3 (FOXP3) was analyzed by ELISA, and mRNA expression level of RAR-related orphan receptor C (RORC) and FOXP3 was detected by qRT-PCR. Naïve CD4⁺ T cells extracted from the peripheral blood of periodontitis patients were co-cultured with hBMSC-derived exosomes from healthy subjects. The ratio of Th17/Treg was examined by flow cytometry and the expression of inflammatory cytokines was determined by qRT-PCR. In vivo, exosomes-loaded hydrogel was injected into periodontal pockets of mice with experimental periodontitis. The periodontal inflammation and bone regeneration was evaluated by histological staining, immunofluorescence staining and micro-CT. Furthermore, the differentially expressed miRNAs in exosomes stimulated by P.g. LPS were investigated by miRNA sequencing and bioinformatics analysis. The interaction between candidate miRNA and its target gene in CD4⁺ T cells was verified by dual luciferase activity assay. Lastly, the downstream Yes-related protein 1(YAP1)/Hippo pathway was evaluated by western blotting. Results The ratio of Th17/Treg is significantly increased in the peripheral blood of periodontitis patients. hBMSC-derived exosomes decreased Th17/Treg ratio in CD4⁺ T cells in vitro and ameliorated inflammation and bone loss in periodontitis mice. Mechanistically, the enrichment of miR-1246 in exosomes showed the enhanced effects of down-regulating Th17/Treg ratio, which could be reversed by miR-1246 inhibitor. Furthermore, exosomal miR-1246 suppressed the expression of the target protein angiotensin converting enzyme2 (ACE2) and upregulated the expression ratio P-YAP1/YAP1 in CD4⁺ T cells. Conclusions hBMSC-derived exosomes could alleviate periodontal inflammation through modulating the balance of Th17/Treg via targeting ACE2 and downregulating YAP1/Hippo signaling pathway, which shed light on a novel cell-free immunotherapy for periodontal regeneration.