July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
Retinal Pigment Epithelial Exosomes Mediate Macrophage Activity and Survival
Author Affiliations & Notes
  • Andrew W Taylor
    Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States
  • Nayan Sanjiv
    Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States
  • Sarah Nocco
    Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States
  • Araz Chiloyan
    Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States
  • Tat Fong Ng
    Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Andrew Taylor, None; Nayan Sanjiv, None; Sarah Nocco, None; Araz Chiloyan, None; Tat Fong Ng, None
  • Footnotes
    Support  PHS Grant from NEI of the NIH EY025961, and the Massachusetts Lions Eye Research Foundation
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2531. doi:
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    • Get Citation

      Andrew W Taylor, Nayan Sanjiv, Sarah Nocco, Araz Chiloyan, Tat Fong Ng; Retinal Pigment Epithelial Exosomes Mediate Macrophage Activity and Survival. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2531.

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

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Abstract

Purpose : Neuropeptides produced by healthy retinal pigment epithelial cells (RPE) program activated macrophages to be anti-inflammatory, and promote immune tolerance. When the neuropeptide alpha-Melanocyte Stimulating Hormone (α-MSH) is neutralized the RPE induce apoptosis in macrophages. Since soluble mediators of apoptosis are not effective inducers of apoptosis, we examined the possibility that RPE exosomes are the source of the apoptotic signal.

Methods : Posterior eyecups were dissected from enucleated eyes of C57BL/6 mice with or without autoimmune uveitis. The neural retina was removed, and the RPE eyecups were placed into wells of a 96-well culture plate, and submerged in serum-free media. The conditioned media (CM) was removed 24 hours later. Also, CM was collected from in vitro cultures of established ARPE-19 cell monolayers in serum free media. From the conditioned media, exosomes were isolated using the precipitation and centrifugation method. The isolated exosomes were washed, resuspended in PBS, and added to cultures of resting and LPS activated macrophages. The macrophage cultures were assayed for Nitric Oxide, IL-1β, and TNF-α. In addition, the cultures were assayed for Caspase 3 activity. The isolated exosomes were assayed by immunoblot for FasL and TRAIL, and by PCR for miR204 and miR155, which are microRNA that can influence apoptotic signaling, and pro-inflammatory activity in macrophages

Results : Only the exosomes from the RPE eyecups of healthy and uveitic eyes activated Caspase-3 activity, which is suppressed by α-MSH treatment. Immunoblotting showed that the exosomes from only the RPE eyecups have membrane FasL and TRAIL. The exosomes from RPE eyecups contain the pro-apoptotic miR204, which is also a constitutively expressed miRNA of differentiated RPE cells. In contrast, the anti-apoptotic, pro-inflammatory miR155 was found only in the exosomes from uveitic RPE eyecups, but as pre-miRNA. The exosomes suppressed Nitric Oxide, and IL-1β, but not TNF-α production by activated macrophages.

Conclusions : RPE release exosomes with proteins and microRNA that can influence the activity of macrophages. In addition, the ability of RPE to deliver an apoptotic signal to activated macrophages suggests a potential mechanism in the retina that selects for macrophages and microglial cells programed for memory-tolerance.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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