Investigative ophthalmology & visual science · 2026
TNFAIP3 in M2 Macrophage Attenuates Subretinal Fibrosis in Laser-Induced Murine Model.
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Zhao Qin, Gong Yajun, Zhang Longji, Chen Chiayuan, Li Longhui, Ma Zijian, Chen Feier, Cai Shiyin, Sovannkiri Neauv, Zhou Xiaolai, Lai Kunbei
Аннотация
PURPOSE: Although the anti-inflammatory role of tumor necrosis factor alpha-induced protein 3 (TNFAIP3) in M2 macrophages has been reported, its function in subretinal fibrosis remains unclear. This study aimed to investigate the role of TNFAIP3 in M2 macrophages in subretinal fibrosis and to elucidate its mechanism.
METHODS: A model of subretinal fibrosis of choroidal neovascularization (CNV) lesions was established in C57BL/6 mice using a two-stage laser photocoagulation protocol. TNFAIP3 of M2 macrophages in subretinal fibrosis of CNV lesions was analyzed by single-cell RNA sequencing (scRNA-seq), immunofluorescence, and western blot. In vitro, RAW264.7 cells polarized to M2 phenotype using IL-4 (40 ng/mL) and IL-13 (40 ng/mL) were co-cultured with primary mouse retinal pigment epithelial cells. The effects of Tnfaip3 genetically modulated in M2 macrophages on epithelial-mesenchymal transition and the mechanisms in M2 macrophages were analyzed by immunofluorescence, western blot, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Peripheral blood monocyte-derived macrophages in mice were depleted through intraperitoneal administration of clodronate liposomes. Subsequently, intravitreal injection of M2 macrophages, M2 macrophages with Tnfaip3-overexpression, or PBS was performed, and the areas of CNV and subretinal fibrosis of CNV lesions were assessed by imaging examinations and immunofluorescence staining.
RESULTS: Our findings revealed a transient upregulation followed by subsequent downregulation of TNFAIP3 in M2 macrophages in subretinal fibrosis of CNV lesions and in M2 polarized macrophages. Mechanistically, TNFAIP3 of macrophages inhibits M2 polarization by promoting degradation of nuclear factor kappa B (NF-κB), which reduces Spi1 and subsequently suppresses CCAAT/enhancer-binding protein beta (C/EBPβ). Macrophage depletion led to a significant reduction in both the area of CNV and subretinal fibrosis, with areas measuring (2.23 ± 0.22) × 104 µm2 versus (0.11 ± 0.02) × 104 µm2 for CNV and (6.04 ± 0.64) × 104 µm2 versus (1.17 ± 0.15) × 104 µm2 for fibrosis. Furthermore, intravitreal injection of Tnfaip3-overexpressing M2 macrophages in macrophage-depleted mice attenuated the area of CNV and subretinal fibrosis compared with control M2 macrophages, with areas measuring (0.62 ± 0.08) × 104 µm2 versus (0.11 ± 0.02) × 104 µm2 for CNV and (2.12 ± 0.20) × 104 µm2 versus (1.30 ± 0.17) × 104 µm2 for fibrosis.
CONCLUSIONS: Our findings demonstrated that TNFAIP3 in macrophages modulates subretinal fibrosis of CNV lesions by regulating M2 polarization through the NF-κB/Spi1/C/EBPβ signaling pathway. These results suggest that targeting TNFAIP3 in M2 macrophages might serve as a promising therapeutic strategy for CNV and subretinal fibrosis.
METHODS: A model of subretinal fibrosis of choroidal neovascularization (CNV) lesions was established in C57BL/6 mice using a two-stage laser photocoagulation protocol. TNFAIP3 of M2 macrophages in subretinal fibrosis of CNV lesions was analyzed by single-cell RNA sequencing (scRNA-seq), immunofluorescence, and western blot. In vitro, RAW264.7 cells polarized to M2 phenotype using IL-4 (40 ng/mL) and IL-13 (40 ng/mL) were co-cultured with primary mouse retinal pigment epithelial cells. The effects of Tnfaip3 genetically modulated in M2 macrophages on epithelial-mesenchymal transition and the mechanisms in M2 macrophages were analyzed by immunofluorescence, western blot, and chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). Peripheral blood monocyte-derived macrophages in mice were depleted through intraperitoneal administration of clodronate liposomes. Subsequently, intravitreal injection of M2 macrophages, M2 macrophages with Tnfaip3-overexpression, or PBS was performed, and the areas of CNV and subretinal fibrosis of CNV lesions were assessed by imaging examinations and immunofluorescence staining.
RESULTS: Our findings revealed a transient upregulation followed by subsequent downregulation of TNFAIP3 in M2 macrophages in subretinal fibrosis of CNV lesions and in M2 polarized macrophages. Mechanistically, TNFAIP3 of macrophages inhibits M2 polarization by promoting degradation of nuclear factor kappa B (NF-κB), which reduces Spi1 and subsequently suppresses CCAAT/enhancer-binding protein beta (C/EBPβ). Macrophage depletion led to a significant reduction in both the area of CNV and subretinal fibrosis, with areas measuring (2.23 ± 0.22) × 104 µm2 versus (0.11 ± 0.02) × 104 µm2 for CNV and (6.04 ± 0.64) × 104 µm2 versus (1.17 ± 0.15) × 104 µm2 for fibrosis. Furthermore, intravitreal injection of Tnfaip3-overexpressing M2 macrophages in macrophage-depleted mice attenuated the area of CNV and subretinal fibrosis compared with control M2 macrophages, with areas measuring (0.62 ± 0.08) × 104 µm2 versus (0.11 ± 0.02) × 104 µm2 for CNV and (2.12 ± 0.20) × 104 µm2 versus (1.30 ± 0.17) × 104 µm2 for fibrosis.
CONCLUSIONS: Our findings demonstrated that TNFAIP3 in macrophages modulates subretinal fibrosis of CNV lesions by regulating M2 polarization through the NF-κB/Spi1/C/EBPβ signaling pathway. These results suggest that targeting TNFAIP3 in M2 macrophages might serve as a promising therapeutic strategy for CNV and subretinal fibrosis.