June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Microarray transcriptome analysis of the inflammatory response of retinal pigment epithelial cells
Author Affiliations & Notes
  • William Samuel
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute / National Institutes of Health, Bethesda, Maryland, United States
  • R Krishnan Kutty
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute / National Institutes of Health, Bethesda, Maryland, United States
  • Cynthia Jaworski
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute / National Institutes of Health, Bethesda, Maryland, United States
  • Todd Duncan
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute / National Institutes of Health, Bethesda, Maryland, United States
  • T. Michael Redmond
    Laboratory of Retinal Cell and Molecular Biology, National Eye Institute / National Institutes of Health, Bethesda, Maryland, United States
  • Paul Wong
    Dept of Opthalmology, Emory University, Atlanta, Georgia, United States
  • Footnotes
    Commercial Relationships   William Samuel, None; R Kutty, None; Cynthia Jaworski, None; Todd Duncan, None; T. Michael Redmond, None; Paul Wong, None
  • Footnotes
    Support  NEI Intramural Research Program
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 628. doi:
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    • Get Citation

      William Samuel, R Krishnan Kutty, Cynthia Jaworski, Todd Duncan, T. Michael Redmond, Paul Wong; Microarray transcriptome analysis of the inflammatory response of retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):628.

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

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Abstract

Purpose : Dysfunction of retinal pigment epithelium (RPE) triggered by inflammatory responses may contribute to the pathology of age-related macular degeneration (AMD). We have shown earlier by qRT-PCR that RPE-derived ARPE-19 cells respond to the proinflammatory cytokines (PIC) IFN-Υ, TNF-α and IL-1β by decreasing the expression of many RPE-characteristic genes while altering the expression of many cytokines, chemokines, miRNAs and lncRNAs. We used the microarray technique to obtain a more comprehensive and global analysis of the response of RPE cells to PIC to better understand the inflammatory response.

Methods : ARPE-19 cells, grown for 4 months, displaying epithelial morphology and pigmentation, were treated with and without PIC: IFN-γ, 100 u/ml; IL-1β, 10 ng/ml; and TNF-α, 10 ng/ml. After 4 days cells were harvested and RNA was isolated. Transcript levels were evaluated using Affymetrix microarray chips and analyzed with Partek Genomic Suite. Differentially expressed genes were defined as those having greater than 2-fold changes in expression levels and p-values < 0.05.

Results : Expression changes in genes up-regulated by PIC treatment largely corresponded to well-known actions of chemokines. Analysis of enriched Gene Ontology terms for cell components shows a marked up-regulation in genes of both MHC class I and class II protein complexes. Numerous up-regulated genes are among canonical receptor signaling pathways involving upstream CC chemokine receptors (CCRs) and Toll-like receptors (TLRs) coupled to STATs, IRF, and NF-kB. Genes that activate JAK-STAT cascades are also up-regulated. The biological process most down-regulated by PIC treatment is melanin biosynthesis; similarly, in Gene Ontology the most diminished cell component is the melanosome membrane. Significant but less dramatic down-regulation is seen for axon elements, photoreceptor outer segment, and Golgi lumen categories. Transcript levels for RPE65 and other visual cycle components are also broadly reduced.

Conclusions : In this study of differentiated ARPE-19 cells treated with PIC, previously described down-regulation of RPE signature genes is corroborated and expanded. With the identification of additional up- and down-regulated genes, this approach provides an efficient means for exploring mechanisms underlying inflammation-induced loss of RPE features and identifies further candidates for in-depth analysis by other approaches.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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