Modulation of monocyte activation by retinal pigment epithelium (RPE)-derived exosomes

Abstract

Purpose: Exosomes are important mediators of intercellular communication and have been implicated in modulation of the immune system. We sought to investigate if exosomes secreted from retinal pigment epithelial (RPE) cells could alter the activation status of immune cells in vitro.

Methods: ARPE-19 cells were stimulated or not with the inflammatory cytokines interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), and interleukin 1 beta (IL-1b) for 48 hours. Supernatants were harvested for isolation of exosomes with the ExoQuick TC isolation kit, and ARPE-19 cells were assayed for expression of surface and intracellular CD81 and CD107b (known exosomal proteins) by flow cytometry. Isolated exosomes were quantified with a NanoSight NS300 nanoparticle analyzer and cultured for 24 hours with either THP-1 or enriched human donor monocytes, which were assayed for expression of ICAM-1 (THP-1) or the phenotypic markers CD14 and CD16 (human monocytes) by flow cytometry.

Results: Stimulation of ARPE-19 cells with IFN-g, TNF-a, and IL-1b reduced the expression of surface and intracellular CD81, while levels of intracellular CD107b (LAMP-2) were unaltered, compared to non-stimulated controls. The number of exosomes secreted from ARPE-19 did not differ between stimulated and non-stimulated cultures. THP-1 monocytes upregulated ICAM-1 expression upon exposure to exosomes isolated from non-stimulated and cytokine-stimulated ARPE-19 cells compared to THP-1 cells not exposed to exosomes. However, exosomes from cytokine-stimulated ARPE-19 cells caused significantly higher ICAM-1 expression per THP-1 cell compared to those from non-stimulated ARPE-19. Exposure to exosomes from non-stimulated ARPE-19 cells induced undifferentiated human monocytes into a more regulatory phenotype with a significantly higher percentage of CD14⁺⁺CD16⁺ cells compared to human monocytes exposed to exosomes from ARPE-19 cells stimulated with cytokines.

Conclusions: RPE constitutively secrete exosomes. The quality but not the quantity of exosomes secreted from ARPE-19 cells is altered with cytokine stimulation. Exosomes from cytokine-stimulated ARPE-19 cells activated THP-1 cells to express high levels of ICAM-1, while exosomes from non-stimulated ARPE-19 cells induced human monocytes toward a regulatory phenotype. These findings suggest that the inflammatory milieu of the RPE can influence monocyte activation in a paracrine fashion through exosomes.