Suppressive Regulation of Interferon Regulatory Factor 3 by Piceatannol in the Macrophage Microenvironment to Attenuate Corneal Inflammation

초록

Purpose : We identified 17 candidate proteins from M1 macrophage-derived exosomes as potential targets for suppressing immune inflammation, with interferon regulatory factor 3 (IRF3) being a key target linked to lipopolysaccharide (LPS)-induced inflammatory activation in myeloid cells. Using the Repurposing Data Portal, we discovered piceatannol (PIC) as an effective IRF3 inhibitor. This study examines the impact of PIC-mediated IRF3 suppression on alleviating inflammation in an in vitro keratocyte and M1 macrophage co-culture model and an in vivo LPS-induced cornea inflammatory mouse model. Methods : THP-1 monocytes were differentiated into M1 macrophages, and LPS-stimulated keratocytes from peripheral corneas were used to create inflammatory human corneal fibroblasts (HCFs). The anti-inflammatory effects of PIC were tested in three models: 1) direct in vitro PIC treatment on each M1 and HCF, 2) PIC treatment in an insert co-culture system of M1 and HCFs, and 3) PIC eye drops in a mouse model of corneal inflammation induced by LPS. QRT-PCR analyzed pan-macrophage, M1 and M2 markers, innate immunity-associated inflammatory cytokines, IRF3 pathway factors, and inflammasome markers. Results : In HCFs, PIC treatment downregulated STING1, TLR4, MYD88, and IRF3. It also suppressed inflammatory cytokines IL1B, IL6, IL15, CXCL10, and inflammasome markers NLRP1, PYCARD, and CASP1. In co-culturing with M1, PIC inhibited IRF3, IL1B, and CXCL10 in HCFs. This anti-inflammatory effect was IRF3-dependent, as PIC was ineffective in siIRF3-M1 and siIRF3-HCFs. In a murine model, PIC eye drops reduced the expression of pan-macrophage markers ADGRE1, CD68 and M1 marker CD86. It also lowered the expression of TLR4, MYD88, IRF3, and CXCL10, along with inflammasome markers PYCARD, AIM2, and CASP1. Conclusions : M1 macrophages can induce a distinct innate immune inflammation in keratocytes. By using piceatannol to inhibit IRF3 expression in the microenvironment of LPS-induced inflammatory keratocytes and M1 macrophages, it is expected that excessive inflammation in corneal innate immune responses involving macrophages can be properly regulated.