Mitochondrial IRF3 drives pulmonary fibrosis by impairing mitophagy and triggering ferroptosis

Background: Pulmonary fibrosis (PF) is a progressive, lethal lung disease with limited treatments. Although inflammation is involved, how it triggers specific oxidative cell death in epithelial cells remains unclear. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is active in PF, but research has focused on its upstream inflammatory role. The function of its key effector, interferon regulatory factor 3 (IRF3), especially through non-canonical mechanisms, is largely unknown. We hypothesized that activated IRF3 translocates to mitochondria to disrupt quality control and promote Ferroptosis, linking inflammation to fibrosis.

Methods: We employed a bleomycin-induced mouse PF model and TGF-β-stimulated A549 cells. Techniques included molecular analyses (western blot, RT-qPCR, Co-IP), imaging (TEM, immunofluorescence), Mitophagy flux assays, and measurement of Ferroptosis markers (Fe²⁺, MDA). Interventions involved H151, si-IRF3, Ferrostatin-1, and Mdivi-1.

Results: In PF, phosphorylated IRF3 translocated to mitochondria, interacting with PINK1 to impair Mitophagy, shown by decreased PINK1, accumulated p62, and reduced LC3-II/LC3-I ratio. This triggered Ferroptosis, evidenced by upregulated ACSL4, downregulated GPX4, elevated Fe²⁺/MDA, and mitochondrial damage. In TGF-β-stimulated A549 cells, IRF3 knockdown or STING inhibition restored Mitophagy and suppressed Ferroptosis. Mdivi-1 reversed si-IRF3's protection. In vivo, H151 treatment suppressed the IRF3-mitophagy-ferroptosis axis and alleviated PF.

Conclusions: Mitochondrial IRF3 integrates cGAS-STING signaling with mitophagic dysfunction and Ferroptosis to drive PF, revealing a novel therapeutic target.