Inflammasome priming increases retinal pigment epithelial cell susceptibility to lipofuscin phototoxicity by changing the cell death mechanism from apoptosis to pyroptosis

https://doi.org/10.1016/j.jphotobiol.2016.05.018Get rights and content

Highlights

  • RPE cell death by photooxidative damage increases following inflammasome priming.

  • Mechanism of cell death changes after priming from apoptosis to pyroptosis.

  • Priming can occur by IL-1α, C5a, or conditioned media of pyroptotic cells.

  • Inflammasome inhibition decreases photooxidative cell death in primed cells.

Abstract

Progressive death of retinal pigment epithelium (RPE) cells is a hallmark of age-related macular degeneration (AMD), the leading cause of blindness in all developed countries. Photooxidative damage and activation of the NLRP3 inflammasome have been suggested as contributing factors to this process. We investigated the effects of inflammasome activation on oxidative damage-induced RPE cell death. In primary human RPE cells and ARPE-19 cells, lipofuscin accumulated following incubation with oxidatively modified photoreceptor outer segments. Oxidative stress was induced by blue light irradiation (dominant wavelength: 448 nm, irradiance: 0.8 mW/cm2, duration: 3 to 6 h) of lipofuscin-loaded cells and resulted in cell death by apoptosis. Prior inflammasome priming by IL-1α or complement activation product C5a altered the cell death mechanism to pyroptosis and resulted in a significant increase of the phototoxic effect. Following IL-1α priming, viability 24 h after irradiation was reduced in primary RPE cells and ARPE-19 cells from 65.3% and 56.7% to 22.6% (p = 0.003) and 5.1% (p = 0.0002), respectively. Inflammasome-mediated IL-1β release occurred only in association with pyroptotic cell lysis. Inflammasome priming by conditioned media of pyroptotic cells likewise increased cell death. Suppression of inflammasome activation by inhibition of caspase-1 or cathepsins B and L significantly reduced cell death in primed cells. In summary, inflammasome priming by IL-1α, C5a, or conditioned media of pyroptotic cells increases RPE cell susceptibility to photooxidative damage-mediated cell death and changes the mechanism of induced cell death from apoptosis to pyroptosis. This process may contribute to RPE degeneration in AMD and provide new targets for intervention.

Introduction

Age-related macular degeneration (AMD) is the leading cause of blindness in all industrialized countries [1]. For the late-stage atrophic form of the disease (geographic atrophy), there is currently no effective treatment available. Geographic atrophy secondary to AMD is characterized by progressive degeneration of the retinal pigment epithelium (RPE), resulting in corresponding secondary photoreceptor loss and visual impairment. The mechanism of RPE cell death in AMD has not yet been fully elucidated. Several lines of clinical and experimental evidence indicate that oxidative and lipofuscin-mediated photooxidative damage plays an important pathophysiological role [2]. Recent studies suggest that the NLRP3 inflammasome also contributes to RPE cell death secondary to AMD [3], [4]. Indeed, NLRP3 inflammasome activation has been demonstrated in RPE cells affected by AMD [3], [5], and increased intravitreal and systemic levels of the inflammasome activation products IL-1β and IL-18 have been reported in AMD patients [6], [7].

We have identified a mechanism that links oxidative/photooxidative damage and inflammasome activation in RPE cells by demonstrating that lipofuscin phototoxicity results in oxidative damage to lysosomal membranes with subsequent cytosolic leakage of lysosomal enzymes and activation of the NLRP3 inflammasome [8]. Inflammasome activation in RPE cells requires a prior priming signal that can be provided by complement activation product C5a [9]. Inflammasome activation can be accompanied by pyroptosis, a recently described type of programmed cell death that is distinct from other cell death mechanisms including apoptosis and necrosis. Pyroptosis is characterized by a combination of several features including caspase-1 dependence, DNA fragmentation, rapid loss of cell membrane integrity, and inflammatory cytokine release [10].

Against the background of the interrelations between oxidative damage, inflammasome activation, and RPE cell death, we sought to elucidate the effects of inflammasome priming on mechanism and extent of photooxidative damage-induced cytotoxicity in RPE cells.

Section snippets

Cell Culture

Human fetal primary RPE (pRPE) cells (Clonetics H-RPE; Lonza, Cologne, Germany) were cultured in medium provided by the manufacturer (Clonetics RtEGM; Lonza) containing 2% heat-inactivated fetal bovine serum and were used in experiments for a maximum of 6 cell culture passages. The spontaneously immortalized, non-transformed human RPE cell line ARPE-19 (CRL-2302; ATCC, Rockville, MD, USA) was maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium

Inflammasome Priming Increase Cell Death by Lipofuscin Phototoxicity

The cell culture model used in this study was characterized previously in detail. Briefly, we demonstrated in pRPE cells and ARPE-19 cells that incubation with HNE-modified POS induces lipofuscin accumulation [12], [14] and that subsequent blue light irradiation results in photooxidative damage to lysosomal membranes and cell death [8]. Substances such as IL-1α or C5a represent priming signals for the inflammasome in RPE cells that induce expression of pro-IL-1β [5], [9]. In primed RPE cells,

Discussion

Blue light irradiation of RPE cells in vitro results in lipofuscin-dependent generation of reactive oxygen species [21], LMP by oxidative damage [22], [23], and cell death [24]. We have demonstrated that LMP by lipofuscin-mediated photooxidative damage not only results in oxidative-damage dependent cell death but also in activation of the NLRP3 inflammasome in primed RPE cells [8], [9]. This mechanism may underlie the inflammasome activation observed in the RPE of AMD patients [3], [5] and may

Conflict of interest

CB, none. JP, none. FGH, research grants: Acucela, Alcon, Allergan, Bayer, Carl Zeiss Meditec, Genentech, Heidelberg Engineering, Novartis, Optos, Roche; consultancy honoraria, lecture fees, travel grants: Acucela, Alcon, Allergan, Bayer, Genentech, GlaxoSmithKline, Heidelberg Engineering, Novartis, Roche. TUK, research grants: Alcon, Novartis; consultancy honoraria, lecture fees, travel grants: Alimera Sciences, Bayer, Heidelberg Engineering, Novartis.

Acknowledgment

The authors thank Claudine Strack, BTA, for expert technical assistance. The study was supported by German Research Foundation (DFG), Bonn, Germany, grant KR 2863/7-1; Pro Retina Foundation, Bonn, Germany, grant Q-037.0162; University of Bonn, Bonn, Germany, BONFOR Program, SciMed Program, and Bonn Graduate School of Neuroimmunology (BonnNI), grants O-137.0014, O-137.0017, and Q-611.0554; and Dr. Eberhard and Hilde Rüdiger Foundation, Bonn, Germany, grant Q-037.0133 (all to TUK). The paper was

References (29)

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