June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Modulation of monocyte activation by retinal pigment epithelium (RPE)-derived exosomes
Author Affiliations & Notes
  • Jared E. Knickelbein
    Laboratory of Immunology, National Eye Institute, Rockville, MD
  • Susan Hannes
    Laboratory of Immunology, National Eye Institute, Rockville, MD
  • Baoying Liu
    Laboratory of Immunology, National Eye Institute, Rockville, MD
  • Anush Arakelyan
    Section on Intercellular Interactions, National Institute of Child Health and Human Development, Bethesda, MD
  • Jean Charles Grivel
    Section on Intercellular Interactions, National Institute of Child Health and Human Development, Bethesda, MD
  • Arvydas Maminishkis
    Section on Epithelial and Retinal Physiology and Disease, National Eye Institute, Bethesda, MD
  • H Nida Sen
    Laboratory of Immunology, National Eye Institute, Rockville, MD
  • Sheldon S Miller
    Section on Epithelial and Retinal Physiology and Disease, National Eye Institute, Bethesda, MD
  • Leonid Margolis
    Section on Intercellular Interactions, National Institute of Child Health and Human Development, Bethesda, MD
  • Robert B Nussenblatt
    Laboratory of Immunology, National Eye Institute, Rockville, MD
  • Footnotes
    Commercial Relationships Jared Knickelbein, None; Susan Hannes, None; Baoying Liu, None; Anush Arakelyan, None; Jean Charles Grivel, None; Arvydas Maminishkis, None; H Nida Sen, None; Sheldon Miller, None; Leonid Margolis, None; Robert Nussenblatt, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3102. doi:
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      Jared E. Knickelbein, Susan Hannes, Baoying Liu, Anush Arakelyan, Jean Charles Grivel, Arvydas Maminishkis, H Nida Sen, Sheldon S Miller, Leonid Margolis, Robert B Nussenblatt; Modulation of monocyte activation by retinal pigment epithelium (RPE)-derived exosomes. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3102.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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