E3 ubiquitin ligase Peli3 regulates M2 macrophage-to-myofibroblast transition and fibrosis in wound healing via IRF4

Purpose: M2 macrophage-to-myofibroblast transformation (MMT) contributes to fibrosis. The role of interferon regulatory factor 4 (IRF4), a key transcription factor in M2 macrophage polarization, in M2 MMT remains unclear. This study aimed to elucidate the role of IRF4 in M2 MMT.

Materials and methods: An in vitro M2 MMT model was established using RAW264.7 Macrophages stimulated with transforming growth factor-β1. Cellular phenotypic shift and extracellular matrix (ECM)-associated proteins were detected. The regulatory role of Peli3 in IRF4 ubiquitination modification was assessed. In a full-thickness skin defect mouse model, the regulatory effects of PELI3 on scar fibrosis and the MMT process were evaluated through AAV-mediated overexpression of PELI3.

Results: In M2 MMT, IRF4 protein levels were significantly upregulated while ubiquitination levels decreased. Knockdown of IRF4 inhibited M2 polarization and M2 MMT and reduced expression of ECM-associated proteins. Peli3 was significantly downregulated in M2 MMT, and its overexpression suppressed IRF4 protein levels, disrupting M2 MMT and ECM deposition. To directly validate the role of IRF4 in M2 MMT, functional rescue experiments were conducted in PELI3-overexpressing cells. The results demonstrated that overexpressing IRF4 partially reversed the MMT inhibition induced by PELI3 overexpression. Peli3 accelerated IRF4 degradation by promoting its ubiquitin modification. In vivo, Peli3 overexpression reduced fibrosis and MMT.

Conclusions: IRF4 promotes M2 MMT and fibrosis progression, with its stability regulated by Peli3-mediated ubiquitination.