June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
7-Ketocholesterol Induces Interleukin(IL)-1 beta and IL-18 Production in RPE Cells via Activation of the Inflammasome
Author Affiliations & Notes
  • Guangpu Shi
    National Eye Institute, Bethesda, MD
  • Osato Ogbeifun
    National Eye Institute, Bethesda, MD
  • Lindsey Nugent
    National Eye Institute, Bethesda, MD
  • Ignacio Rodriguez
    National Eye Institute, Bethesda, MD
  • Igal Gery
    National Eye Institute, Bethesda, MD
  • Footnotes
    Commercial Relationships Guangpu Shi, None; Osato Ogbeifun, None; Lindsey Nugent, None; Ignacio Rodriguez, None; Igal Gery, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 150. doi:
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      Guangpu Shi, Osato Ogbeifun, Lindsey Nugent, Ignacio Rodriguez, Igal Gery; 7-Ketocholesterol Induces Interleukin(IL)-1 beta and IL-18 Production in RPE Cells via Activation of the Inflammasome. Invest. Ophthalmol. Vis. Sci. 2013;54(15):150.

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

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Abstract

Purpose: 7-Ketocholesterol is a form of oxidized cholesterol. It has been found to be associated with lipoprotein deposits in Bruch’s membrane, choriocapillaris, and RPE cells in the primate retina as well as a component of drusen in human eyes affected by AMD. The purpose of this study was to assess whether 7-Ketocholesterol could activate inflammasomes in RPE cells to induce IL-1 beta and IL-18 production.

Methods: Primary cultured RPE cells from an eighty-year-old human donor, or ARPE-19 cells, were primed using LPS plus IL-1 alpha for 6 hours, followed by incubation with 7-Ketocholesterol for 16 hours. The production of IL-1 beta and IL-18 was assessed in cell culture supernatant using enzyme linked immunosorbant assay. Total protein extracted from treated ARPE-19 cells was subjected to western blot assay to detect cleaved caspase-1 p20, using ATP treatment as a positive control. Glyburide, an inflammasome inhibitor, was added into cell culture together with 7-Ketocholesterol to confirm signaling pathway involvement.

Results: RPE cells from an eighty-year-old human donor incubated with 10 µM 7-Ketocholesterol produced a considerable amount of IL-1 beta, but a lower amount of IL-18. Treatment with glyburide reduced the release of the IL-1 beta by about 60%, but had no effect on IL-18 production. Incubation of 7-Ketocholesterol with ARPE19 cells produced similar amounts of IL-1 beta and IL-18. Western blot of 7-Ketocholesterol-treated APRE-19 cells showed elevated caspase-1 p20 production to levels similar to those stimulated by ATP-treatment.

Conclusions: 7-Ketocholesterol induces IL-1 beta and IL-18 production from RPE cells. Accumulation of 7-Ketocholesterol in drusen of AMD patients may therefore have a pathogenic role in the disease process through the release of the proinflammatory cytokine IL-1 beta and IL-18.

Keywords: 412 age-related macular degeneration • 557 inflammation • 490 cytokines/chemokines  
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