Proinflammatory Cytokines Induce Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells

Abstract

Purpose: Proinflammatory cytokines secreted by infiltrating macrophages and lymphocytes may play a role in retinal pigment epithelial (RPE) dysfunction associated with age-related macular degeneration (AMD). Proinflammatory cytokines are also known to promote epithelial-mesenchymal transition (EMT) during pathological conditions such as carcinogenesis and fibrosis. The purpose of the present study is to elucidate the role of EMT in proinflammatory cytokines-induced RPE dysfunction. We employed ARPE-19 cells in culture exposed to IFN-γ, TNF-α and IL-1β as a model system.

Methods: ARPE-19 cells were cultured in DME medium containing 4.5 g/L glucose, 1% fetal bovine serum,1 mM sodium pyruvate and antibiotics. The cells kept in culture for 4 months with media exchange twice a week exhibited epithelial morphology and expressed mRNA for visual cycle genes RPE65 and RDH5. The differentiated cells were treated with a mixture containing proinflammatory cytokines IFN-γ (10 u/ml), TNF-α (1 ng/ml) and IL-1β (1 ng/ml) in a serum free medium for 20 hours. Real-time PCR analysis was employed for the analysis of gene expression using TaqMan reagents from Life Technologies, Grand Island, NY.

Results: Real-time PCR analysis showed that cultured ARPE-19 cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) by greatly increasing the expression of chemokines and cytokines and markedly decreasing the expression of RPE-specific genes RPE65, RDH5, RDH10, MITF, TYR and MERTK. These changes were associated with decreased expression of epithelial marker gene CDH1 and increased expression of mesenchymal marker genes CDH2, VIM and CCND1. The proinflammatory cytokines also increased the expression of ZEB1 and SNAI1, two transcription factor genes known to promote EMT.

Conclusions: Proinflammatory cytokines markedly decreased expression of key genes involved in visual cycle, phagocytosis and pigment synthesis in treated RPE cells. The loss of these RPE characteristics was associated with EMT like changes possibly mediated by transcription factor genes ZEB1 and SNAI1. Thus, proinflammatory cytokines can potentially induce RPE dysfunction via EMT, and this process may play a role in the pathology of AMD.