Interferon Regulatory Factor 3 as a Mediator and Therapeutic Target in Innate Immune-Driven Corneal Stromal Inflammation and Opacity.

Abstract

PURPOSE: Interferon regulatory factor 3 (IRF3) is classically recognized for antiviral immunity, yet emerging evidence implicates it in sterile inflammation. This study investigated whether IRF3 regulates macrophage-stromal signaling driving corneal pathology and evaluated the therapeutic potential of targeting IRF3. METHODS: In vitro assays utilized human macrophages stimulated with lipopolysaccharide (LPS) and interferon-γ (IFNγ) to generate an inflammatory macrophage population (Mϕ-LPS/IFNγ cells), along with primary human corneal stromal fibroblasts (HCFs) stimulated with LPS. IRF3 was silenced by small interfering RNA (siRNA), and inflammatory mediators and toll-like receptor 4 (TLR4)/MyD88 transcripts were quantified. Macrophage-HCF interactions were assessed in Transwell cocultures. In vivo, Irf3-/- and wild-type mice underwent corneal epithelial debridement followed by topical LPS to evaluate corneal opacity, fibrotic markers, cytokine expression, and myeloid-cell subsets. Piceatannol, a stilbenoid compound known to inhibit IRF3, was tested in vitro and as topical eye drops. RESULTS: IRF3 expression was upregulated in both macrophages and HCFs under inflammatory conditions. Silencing IRF3 significantly reduced inflammatory cytokines, including CXCL10, and decreased TLR4-associated transcripts in HCFs. In cocultures, IRF3-silenced macrophages partially attenuated HCF activation. In vivo, Irf3 deficiency markedly reduced LPS-induced corneal opacity, stromal inflammatory transcripts, monocyte recruitment, and macrophage-associated markers. Piceatannol suppressed IRF3-dependent inflammatory responses in vitro and, when applied topically, reduced corneal Irf3 expression and myeloid/inflammatory signatures. CONCLUSIONS: IRF3 is a pivotal regulator of innate immune-driven stromal inflammation and opacity. Both genetic and pharmacologic inhibition of IRF3 attenuated cytokine and myeloid responses, suggesting that targeting IRF3, potentially via piceatannol (PIC), offers a therapeutic strategy for preserving corneal transparency.