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Updated: Jul 22, 2025

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line
Published on: August 2, 2021
TRAF3/STAT6 axis regulates macrophage polarization and tumor progression
Jian-Hong Shi1,2,3,4, Li-Na Liu5,6, Dan-Dan Song5,6
1Central Laboratory, Affiliated Hospital of Hebei University, Baoding, 071000, Hebei, China. shijianhong@hbu.edu.cn.
Abstract:
Converting tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype is considered an effective strategy for cancer therapy. TRAF3 is known to regulate NF-κB signaling. However, the role of TRAF3 in TAM polarization has not yet been completely elucidated. Here, we found that ablation of TRAF3 increased M1 markers, iNOS, FGR and SLC4A7, while down-regulated M2 markers, CD206, CD36 and ABCC3, expression levels in macrophages. Moreover, TRAF3 deficiency enhanced LPS-induced M1 and abolished IL-4-induced macrophage polarization. Next, quantitative ubiquitomics assays demonstrated that among the quantitative 7618 ubiquitination modification sites on 2598 proteins, ubiquitination modification of IL-4 responding proteins was the most prominently reduced according to enrichment analysis. STAT6, a key factor of IL-4 responding protein, K450 and K129 residue ubiquitination levels were dramatically decreased in TRAF3-deficient macrophages. Ubiquitination assay and luciferase assay demonstrated that TRAF3 promotes STAT6 ubiquitination and transcriptional activity. Site mutation analysis revealed STAT6 K450 site ubiquitination played a vital role in TRAF3-mediated STAT6 activation. Finally, B16 melanoma mouse model demonstrated that myeloid TRAF3 deficiency suppressed tumor growth and lung metastasis in vivo. Taken together, TRAF3 plays a vital role in M2 polarization via regulating STAT6 K450 ubiquitination in macrophages.
Insights
Tumor-associated macrophage (TAM) reprogramming is key for cancer therapy. TRAF3 deficiency promotes M1 macrophage polarization by regulating STAT6 ubiquitination, suppressing tumor growth and metastasis.
Area of Science:
- Immunology
- Cancer Biology
- Molecular Biology
Background:
- Tumor-associated macrophages (TAMs) often exhibit an M2 phenotype, hindering anti-tumor immunity.
- Reprogramming TAMs to an M1 phenotype is a promising cancer therapy strategy.
- The precise role of TRAF3 (TNF receptor-associated factor 3) in TAM polarization remains unclear.
Purpose of the Study:
- To investigate the role of TRAF3 in macrophage polarization.
- To elucidate the molecular mechanisms by which TRAF3 influences TAM phenotype.
- To assess the therapeutic potential of targeting TRAF3 in cancer.
Main Methods:
- Macrophage polarization assays (M1/M2 markers).
- Quantitative ubiquitomics and Western blotting to analyze protein ubiquitination.
- Luciferase and site-directed mutagenesis assays to study STAT6 activation.
- In vivo B16 melanoma mouse model to evaluate tumor growth and metastasis.
Main Results:
- TRAF3 ablation enhanced M1 markers (iNOS, FGR, SLC4A7) and reduced M2 markers (CD206, CD36, ABCC3).
- TRAF3 deficiency promoted LPS-induced M1 polarization and inhibited IL-4-induced M2 polarization.
- TRAF3 was found to promote STAT6 ubiquitination and transcriptional activity, particularly at the K450 residue.
- Myeloid-specific TRAF3 deficiency suppressed tumor growth and lung metastasis in a mouse model.
Conclusions:
- TRAF3 plays a critical role in promoting M2 macrophage polarization.
- TRAF3 regulates M2 polarization by enhancing STAT6 ubiquitination at the K450 site.
- Targeting TRAF3 may represent a novel therapeutic strategy for suppressing tumor progression and metastasis.
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