Xinyu Wu, Hongbo Gong, Jie Zhang, Ling Yu, Xiangyu Yan, Wanlong Pan, Chaoyue Liu
Exosomes were extracted from THP-1 cell culture supernatants and identified using dynamic light scattering, transmission electron microscopy, and western blotting. Genes for overexpression or knockdown of METTL3/miR-155-5p were loaded into M0 exosomes, which were then used to deliver them to recipient macrophages or injected into mice. The OE-METTL3 group showed significantly elevated expression of M1 macrophage markers (CD86, iNOS, HLA-DR), cytokines (IL-1β, IL-6, TNF-α, IL-18), signaling molecules (NLRP3, TLR4, caspase-1), and decreased expression of M2 macrophage markers (CD206, CD163, Arg-1, IL-10, TGF-β). Exosomal delivery of METTL3 promotes M1 macrophage polarization by inducing miR-155-5p maturation via m6A modification, providing potential targets for inflammation and tumor immunotherapy.
Objective Macrophage polarization and function are regulated by epigenetic mechanisms and microRNAs transported by exosomes; however, the specific mechanisms are not clear yet. The study aims to investigate whether exosomes regulate miR-155-5p maturation through methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification, thereby driving M1 macrophage polarization and providing potential targets for inflammation and tumor immunotherapy.Method THP-1 cells were differentiated into M0 macrophages by PMA and subsequently polarized to the M1 phenotype with LPS and IFN-γ. Exosomes were extracted from culture supernatants using ultracentrifugation, and identified by dynamic light scattering (DLS), transmission electron microscopy (TEM), and western blotting (WB). Genes for overexpression or knockdown of METTL3/miR-155-5p were loaded into M0 exosomes, respectively, and M0-exos were then used to deliver them to recipient macrophages or injected into mice.Results Dot blot analysis showed an increase in m6A modification levels in the OE-METTL3 group. Flow cytometry (FCM), Enzyme-linked immunosorbent assay (ELISA), RT-qPCR, and WB results indicated that the OE-METTL3 group exhibited significantly elevated expression of M1 macrophage markers CD86, iNOS and HLA-DR, cytokines IL-1β, IL-6, TNF-α, and IL-18, signaling molecules NLRP3, TLR4, and caspase-1, whereas the expression of M2 macrophage markers CD206, CD163, Arg-1, IL-10 and TGF-β was decreased. The SI-METTL3 group showed the opposite trends. Further results demonstrated that miR-155-5p was correspondingly upregulated or downregulated. Additional intervention with miR-155-5p confirmed its regulatory effect on m6A modification and M1 polarization (p < 0.05). In vivo experiments yielded consistent results.Conclusion Exosomes promote miR-155-5p maturation through METTL3-mediated m6A modification, activate the NLRP3/TLR4/caspase-1 signaling pathway, drive M1 macrophage polarization, and exacerbate inflammatory responses. This research may provide novel potential targets for inflammation and tumor immunotherapy.