IRF4 exacerbates pulmonary inflammation in bronchopulmonary dysplasia mice model by regulating macrophage polarization and phagocytosis.

Abstract

This study investigates the critical function of interferon regulatory factor 4 (IRF4) during bronchopulmonary dysplasia (BPD) progression by regulating alveolar macrophages (AMs) polarization and phagocytic function. We developed IRF4 knockout mice using CRISPR/Cas9 technology and established an animal model for neonatal bronchopulmonary dysplasia (BPD) by hyperoxia exposure. Lung tissue pathology was analyzed by Hematoxylin and eosin staining (HE). The concentrations of TNF-α, IL-1β, IL-10, and TGF-β were ascertained by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to investigate M1/M2 macrophage polarization in bronchoalveolar lavage fluid (BALF) and lung tissue, while Western blotting and quantitative real-time PCR (qRT-PCR) were employed to detect IRF4, iNOS, and Arg-1 protein and mRNA expression. At cellular level, we silenced IRF4 in murine alveolar macrophage cell lines using IRF4 siRNA to investigate its effect on inflammatory cytokine secretion, polarization, and phagocytic function. To assess the effect of IRF4 on alveolar macrophage phagocytosis after hyperoxia, we utilized flow cytometry to ascertain the mean fluorescence intensity of engulfed fluorescent microspheres and fluorescence microscopy to quantify phagocytic cells. Hyperoxia-exposed mice showed markedly upregulated IRF4 expression, increased M1 macrophage and iNOS levels, and decreased M2 macrophages and Arg-1 expression. This cytokine shift was characterized by a marked upregulation of inflammatory factors such as TNF-α and IL-1β, accompanied by a notable decline in anti-inflammatory factors encompassing IL-10 and TGF-β, indicating an imbalance towards a pro-inflammatory state. IRF4 knockout or siRNA-mediated silencing attenuated the inflammatory response, promoting M2 macrophage differentiation while suppressing M1 differentiation. Phagocytic assays showed that hyperoxia impaired the phagocytic activity of AMs, while transfection of IRF4 siRNA restored the phagocytic activity of macrophage. IRF4 functions as a crucial regulator in the progression of hyperoxia-induced bronchopulmonary dysplasia, exerting its effects by influencing the polarization state and phagocytic function of alveolar macrophages. Deletion of IRF4 promotes an M2-dominant anti-inflammatory response, attenuates hyperoxia-induced inflammation, and increases the phagocytosis capacity of macrophages.