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Updated: Aug 7, 2026
Depletion and Reconstitution of Macrophages in Mice
Published on: August 1, 2012
Exosomal delivery of METTL3 promotes M1 macrophage polarization by inducing miR-155-5p maturation via m6A
Xinyu Wu1, Hongbo Gong1, Jie Zhang1
1Basic Medical Experimental Teaching Center, Institute of Basic Medicine, North Sichuan Medical College, Sichuan, Nanchong, China.
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.
Insights
Exosomes drive M1 macrophage polarization via methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification of miR-155-5p. This process activates inflammatory pathways, offering potential therapeutic targets for inflammation and cancer immunotherapy.
Area of Science:
- Immunology
- Epigenetics
- Molecular Biology
Background:
- Macrophage polarization is crucial for immune responses and is influenced by epigenetic regulators and exosome-transported microRNAs.
- The precise mechanisms by which exosomes modulate macrophage polarization, particularly M1 phenotype induction, remain incompletely understood.
Purpose of the Study:
- To investigate the role of exosomes in regulating miR-155-5p maturation through methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification.
- To determine if this exosome-mediated pathway drives M1 macrophage polarization and explore its potential as a therapeutic target for inflammatory diseases and tumor immunotherapy.
Main Methods:
- THP-1 cells were differentiated into M0 macrophages and polarized to M1 phenotype using LPS and IFN-γ.
- Exosomes were isolated and characterized; METTL3 and miR-155-5p levels were manipulated within exosomes delivered to recipient macrophages or injected into mice.
- Macrophage polarization markers, cytokine levels, and signaling pathway activation (NLRP3/TLR4/caspase-1) were assessed using techniques including flow cytometry, ELISA, RT-qPCR, and western blotting.
Main Results:
- Overexpression of METTL3 in exosomes increased m6A modification and promoted M1 macrophage polarization, evidenced by elevated CD86, iNOS, HLA-DR, IL-1β, IL-6, TNF-α, IL-18, NLRP3, TLR4, and caspase-1 expression.
- Conversely, METTL3 knockdown led to decreased M1 markers and increased M2 markers.
- miR-155-5p levels were directly correlated with METTL3-mediated m6A modification and M1 polarization, with direct intervention confirming its regulatory role. In vivo studies corroborated these findings.
Conclusions:
- Exosomes facilitate miR-155-5p maturation via METTL3-mediated m6A modification, subsequently activating the NLRP3/TLR4/caspase-1 pathway.
- This exosome-driven mechanism promotes M1 macrophage polarization and exacerbates inflammatory responses.
- The identified pathway presents novel therapeutic targets for managing inflammation and advancing tumor immunotherapy.
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