June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Mechanism of RPE cell death following activation of the NLRP3 inflammasome by lipofuscin photoreactivity
Author Affiliations & Notes
  • Carolina Brandstetter
    Department of Ophthalmology, University of Bonn, Bonn, Germany
  • Lena Mohr
    Department of Ophthalmology, University of Bonn, Bonn, Germany
  • Frank Holz
    Department of Ophthalmology, University of Bonn, Bonn, Germany
  • Tim Krohne
    Department of Ophthalmology, University of Bonn, Bonn, Germany
  • Footnotes
    Commercial Relationships Carolina Brandstetter, None; Lena Mohr, None; Frank Holz, Acucela (C), Allergan (C), Genentech (F), Heidelberg Engineering (F), Zeiss (F), Novartis (F), Novartis (C), Optos (F), Merz (C), Bayer (F), Bayer (C), Boehringer Ingelheim (C); Tim Krohne, Novartis Pharma GmbH, Nuremberg, Germany (F), Novartis Pharma GmbH, Nuremberg, Germany (C), Novartis Pharma GmbH, Nuremberg, Germany (R)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1802. doi:
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    • Get Citation

      Carolina Brandstetter, Lena Mohr, Frank Holz, Tim Krohne; Mechanism of RPE cell death following activation of the NLRP3 inflammasome by lipofuscin photoreactivity. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1802.

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

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Abstract

Purpose: Abnormal lipofuscin accumulation in the retinal pigment epithelium (RPE) as well as chronic local inflammation in the sub-RPE space has been implicated in progressive RPE dysfunction and degeneration in age-related macular degeneration (AMD). We have previously demonstrated that lysosomal destabilization by lipofuscin photoreactivity activates the NLRP3 inflammasome in RPE cells. Here we analyze the molecular mechanisms of cell death induced by lipofuscin-mediated inflammasome activation.

Methods: Lipofuscinogenesis was induced in human RPE-derived ARPE-19 cells and fetal human RPE cells by incubation with isolated photoreceptor outer segments following modification with lipid peroxidation products. Lysosomal membrane permeabilization was induced by blue light irradiation (wavelength 455-460 nm; irradiance 0.8 mW/cm2). NLRP3 inflammasome activation was assessed by means of IL-1β secretion following priming with IL-1α. Cell death was quantified using cell detachment and LDH release assays. Specific inhibition of caspase 1 and cathepsin B was achieved by Z-YVAD-FMK and CA-074, respectively.

Results: Blue light irradiation for up to 3 hours resulted in lysosomal membrane permeabilization and cytotoxicity in lipofuscin-loaded cells but not in control cells. Cell death was associated with NLRP3 inflammasome activation with secretion of IL-1β and IL-18. Dying cells exhibited cellular swelling in light microscopy, early loss of plasma membrane integrity (LDH release), loss of mitochondrial membrane potential, and positive TUNEL staining. Oligonucleosomal DNA fragmentation (DNA laddering) and apoptosis labeling (annexin V+/PI-) in flow cytometry were not detectable, although positive in UV light-irradiated controls. Caspase-1 inhibition prevented both IL-1β release and cell death. This combination of features identifies the induced cell death mechanism as pyroptosis, a cell death pathway distinct from apoptosis and necrosis.

Conclusions: Lipofuscin photoreactivity results in activation of the NLRP3 inflammasome with induction of pyroptotic cell death, a pro-inflammatory cell death mechanism that can perpetuate chronic local inflammation. This molecular pathway could represent a functional link between several hallmark features of AMD, i.e. lipofuscin accumulation, chronic inflammation, and progressive degeneration of the RPE, and may provide a novel target for therapeutic intervention in AMD.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 582 ipofuscin  
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