IRF7 Activates LCN2 Transcription to Enhance LPS-Induced Acute Lung Injury by Inducing Macrophage Ferroptosis and M1 Polarization

Abstract

Acute lung injury (ALI), a severe pulmonary disorder that poses a significant threat to life, is closely associated with macrophage ferroptosis and polarization. Lipocalin 2 (LCN2) has been previously reported to be implicated in the pathogenesis of ALI. However, the specific role of LCN2 in macrophage ferroptosis and polarization remains undetermined. Lipopolysaccharide (LPS) was used to establish a mouse model of ALI and also to stimulate mouse RAW264.7 cells. H&E staining was used for histopathologic evaluation, and immunohistochemistry analysis was used to determine the 4-HNE-positive cells. The secretion levels of TNF-α, IL-6, and IL-1β were assessed by ELISA. Gene and protein expression assays were performed using quantitative PCR and immunoblotting. The levels of MDA, GSH, ROS, and lipid ROS were detected to evaluate the alteration in ferroptosis. CD86⁺ and CD206⁺ cells were quantified by flow cytometry. The relationship between LCN2 and interferon regulatory factor 7 (IRF7) was confirmed by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. LCN2 expression was upregulated in the lungs of LPS-induced ALI mice and LPS-stimulated RAW264.7 cells. In LPS-induced ALI mice, the depletion of LCN2 alleviated lung injury and ferroptosis, and also inhibited inflammation and macrophage M1 polarization. In LPS-stimulated RAW264.7 cells, the depletion of LCN2 suppressed ferroptosis, inflammation, and M1 polarization. Mechanistically, IRF7 enhanced LCN2 transcription in RAW264.7 cells by binding to its promoter region. More importantly, the silencing of IRF7 inhibited ferroptosis and M1 polarization in LPS-stimulated RAW264.7 cells by downregulating LCN2. Taken together, the IRF7/LCN2 cascade enhances the ferroptosis and M1 polarization of LPS-stimulated macrophages, thereby exacerbating ALI. Anti-IRF7 and anti-LCN2 therapies might potentially be exploited for the prevention and treatment in ALI.

Keywords: Acute lung injury; Ferroptosis; IRF7; LCN2; Macrophage polarization.