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.

将肿瘤相关巨噬细胞 (TAM) 从 M2 表型转变为 M1 表型被认为是癌症治疗的有效策略。 TRAF3 已知可调节 NF-κB 信号传导。然而，TRAF3在TAM极化中的作用尚未完全阐明。在这里，我们发现 TRAF3 的消除增加了巨噬细胞中 M1 标记物 iNOS、FGR 和 SLC4A7 的表达水平，同时下调了 M2 标记物 CD206、CD36 和 ABCC3 的表达水平。此外，TRAF3 缺陷增强了 LPS 诱导的 M1 并消除了 IL-4 诱导的巨噬细胞极化。接下来，定量泛素组学分析表明，根据富集分析，在 2598 个蛋白质的 7618 个定量泛素化修饰位点中，IL-4 响应蛋白的泛素化修饰减少最为显着。 TRAF3缺陷型巨噬细胞中IL-4响应蛋白的关键因子STAT6、K450和K129残基泛素化水平显着降低。泛素化实验和荧光素酶实验表明TRAF3促进STAT6泛素化和转录活性。位点突变分析表明 STAT6 K450 位点泛素化在 TRAF3 介导的 STAT6 激活中发挥着至关重要的作用。最后，B16 黑色素瘤小鼠模型证明骨髓 TRAF3 缺陷可抑制体内肿瘤生长和肺转移。总而言之，TRAF3 通过调节巨噬细胞中 STAT6 K450 泛素化在 M2 极化中发挥着至关重要的作用。