Modulation of Immune Responses by Extracellular Vesicles From Retinal Pigment Epithelium

Abstract

PurposeExtracellular vesicles (EV), such as exosomes, are important mediators of intercellular communication and have been implicated in modulation of the immune system. We investigated if EV released from retinal pigment epithelium (RPE) modulate immune responses in vitro.MethodsExtracellular vesicles were isolated from ARPE-19 cultures stimulated or not with the inflammatory cytokines IL-1β, IFN-γ, and TNF-α. Isolated EV were characterized by nanoparticle flow and Western blot analyses. Retinal pigment epithelium–derived EV were cultured with human peripheral blood mononuclear cells, which were assayed for T-cell proliferation by 3H-thymidine incorporation. Retinal pigment epithelium–derived EV were also independently cultured with enriched lymphocytes or monocytes. Cell phenotype and cell death were evaluated by flow cytometric analysis. Cytokine levels were assayed in culture supernatants by multiplex bead analysis.ResultsThe concentration of ARPE-derived EV from cytokine-stimulated cultures was slightly higher than from nonstimulated cultures. The size of EV was approximately 100 nm in both groups. Extracellular vesicles from both nonstimulated and cytokine-stimulated ARPE-19 significantly inhibited T-cell proliferation without affecting T-cell viability. Culture of EV from nonstimulated ARPE-19 with undifferentiated human monocytes induced an immunoregulatory phenotype with a significantly higher percentage of CD14++CD16+ monocytes and upregulation of TGF-β1. Culture of EV from cytokine-stimulated ARPE-19 cells with human monocytes induced upregulation of several proinflammatory cytokines and monocyte death.ConclusionsRetinal pigment epithelium cells constitutively secrete EV in the size range of exosomes, with increased release from RPE cells stimulated with inflammatory cytokines. Extracellular vesicles from both nonstimulated and cytokine-stimulated RPE inhibited T-cell stimulation. Extracellular vesicles from nonstimulated ARPE-19 cells promoted an immunoregulatory CD14++CD16+ phenotype in human monocytes, while EV from cytokine-stimulated ARPE-19 cells caused human monocyte death. These findings suggest that RPE cells use EV to induce a downregulatory immune environment under homeostatic conditions. In an inflammatory milieu, RPE-derived EV may mitigate a potentially harmful inflammatory response through killing of monocytes.

Effect of aging and lifestyle on photoreceptors and retinal pigment epithelium: cross-sectional study in a healthy Danish population

“…RPE cells and photoreceptors are sensitive to inflammatory stimulation [29,30], which may explain the differences in thicknesses between the physically active and inactive. Kim et al investigated these mechanisms in an experimental model, where they evaluated age-related changes on mice allocated either to regular aerobic training (treadmill training) or sedentary controls [31].…”

Roles of exosomes in the normal and diseased eye

“…However, a recent in vitro study found that small EVs released from cultures of the spontaneously immortalized retinal pigmented epithelium (RPE) cell line ARPE-19, promoted an immunoregulatory phenotype in monocytes (Knickelbein et al, 2016). Interestingly, when ARPE-19 cultures were stimulated with inflammatory cytokines, they released EVs that induced monocyte death.…”

“…For example, mesenchymal stem cell-derived exosomes possess anti-inflammatory properties that may be applicable to inflammatory eye diseases (Blazquez et al, 2014; Zhang et al, 2014). As mentioned previously, RPE cells are thought to use exosomes to modulate local immune responses by killing monocytes (Knickelbein et al, 2016). Exosomes also appear to deliver anti-inflammatory drugs to microglial cells to suppress neuroinflammation (Zhuang et al, 2011) or αB-crystallin to the neural retina, which could act as neuroprotection to photoreceptors (Sreekumar et al, 2010).…”

Recent advances of exosomes in immune-mediated eye diseases

“…RPE cells have been revealed to have immunosuppressive properties, including induction of Tregs and inhibition of Th17 and Th22 cell differentiation [91]. Knickelbein et al reported that exosomes released by both resting and cytokine-stimulated RPE cells suppressed the proliferation of T lymphocytes isolated from the peripheral blood of noninfectious uveitis patients, and these nanosized vesicles could also regulate human monocyte phenotype and viability [59]. The above results indicate that exosome secretion may be a crucial mechanism for RPE cells to perform their immunoregulatory effects.…”