H2O2-induced oxidative stress modulates methotrexate and dexamethasone-dependent regulation of NLRP3-associated transcription and HLA class I expression in primary human RPE cells

Abstract

Purpose: Oxidative stress-driven inflammation is a central mechanism in retinal diseases. This study investigated how methotrexate (MTX) and dexamethasone (DEX), alone or in combination, modulate NLRP3-related gene expression and HLA class I expression in primary human retinal pigment epithelial (RPE) cells under basal and oxidative stress conditions.<br /> Methods: Primary human RPE cells were isolated from cadaveric donor eyes and treated with MTX (100 & micro;g/mL), DEX (50 & micro;g/mL), or their combination for 24 h under normal culture conditions or following H2O2-induced oxidative stress (100 & micro;M). mRNA expression of NLRP3, PYCARD, CASPASE-1, HLA-A, and HLA-B was quantified by qPCR. ELISA measured IL-1(3 and IL-18 secretion.<br /> Results: Under basal conditions, MTX and DEX downregulated NLRP3 and HLA-A/B mRNA expression while increasing PYCARD and CASPASE-1 transcripts. Combination treatment reduced IL-1(3 secretion, whereas MTX and combination therapy decreased IL-18 levels. In oxidative stress conditions, MTX and combination therapy upregulated NLRP3 and CASPASE-1 mRNA, while HLA-A/B and PYCARD transcripts were primarily increased by combination treatment. IL-1(3 levels remained unchanged, and IL-18 was reduced only in the combination group.<br /> Conclusions: MTX and DEX modulate NLRP3-associated and HLA class I transcriptional responses in RPE cells in a redox-dependent manner. Although oxidative stress modulated drug-associated gene expression profiles, these transcriptional changes were not paralleled by corresponding alterations in secreted inflammatory cytokine levels, suggesting that the context-dependent nature of inflammatory signaling and cytokine release in RPE cells should be taken into consideration.