Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage

Abstract

Photooxidative damage and chronic innate immune activation have been implicated in retinal pigment epithelium (RPE) dysfunction, a process that underlies blinding diseases such as age-related macular degeneration (AMD). To identify a potential molecular link between these mechanisms, we investigated whether lipofuscin-mediated phototoxicity activates the NLRP3 inflammasome in RPE cells in vitro. We found that blue light irradiation (dominant wavelength 448 nm, irradiance 0.8 mW/cm(2), duration 6 h) of lipofuscin-loaded primary human RPE cells and ARPE-19 cells induced photooxidative damage, lysosomal membrane permeabilization (79.5 % of cells vs. 3.8 % in nonirradiated controls), and cytosolic leakage of lysosomal enzymes. This resulted in activation of the inflammasome with activation of caspase-1 and secretion of interleukin-1β (14.6 vs. 0.9 pg/ml in nonirradiated controls) and interleukin-18 (87.7 vs. 0.2 pg/ml in nonirradiated controls). Interleukin secretion was dependent on the activity of NLRP3, caspase-1, and lysosomal proteases cathepsin B and L. These results demonstrate that accumulation of lipofuscin-like material in vitro renders RPE cells susceptible to phototoxic destabilization of lysosomes, resulting in NLRP3 inflammasome activation and secretion of inflammatory cytokines. This new mechanism of inflammasome activation links photooxidative damage and innate immune activation in RPE pathology and may provide novel targets for therapeutic intervention in retinal diseases such as AMD.

Key message: • Visible light irradiation of lipofuscin-loaded RPE cells activates inflammasome. • Inflammasome activation results from lysosomal permeabilization and enzyme leakage. • Inflammasome activation induces secretion of inflammatory cytokines by RPE cells. • Photooxidative damage by visible light as new mechanism of inflammasome activation. • Novel link between hallmark pathogenetic features of retinal degenerative diseases.

Fig. 1

Blue light irradiation of lipofuscin-loaded RPE cells induces photooxidative damage. a Intracellular lipofuscin accumulation (yellow) was documented in ARPE-19 cells by fluorescence microscopy. Nuclei were visualized by DAPI staining (blue). b Lipofuscinogenesis was quantified in pRPE cells and ARPE-19 cells by flow cytometry measurements of mean cellular autofluorescence intensity. An example result is shown as an insert. Cells were left untreated (control) or incubated with the lysosomal inhibitor ammonium chloride (NH ₄ Cl), unmodified photoreceptor outer segments (POS), POS and ammonium chloride (POS + NH ₄ Cl), malondialdehyde-modified POS (MDA-POS), and 4-hydroxynonenal-modified POS (HNE-POS). c Photooxidative damage was assessed in ARPE-19 cells by immunocytochemical detection of protein carbonyls (red). Nuclei were labeled by staining with DAPI (blue). d Mean cellular fluorescence of immunocytochemically labeled protein carbonyls was quantified by flow cytometry. Scale bars, 50 μm

Fig. 2

Blue light irradiation of lipofuscin-loaded RPE cells causes phototoxic cell death. a Photooxidative damage-induced cytotoxicity was documented in ARPE-19 cells by light microscopy. b To quantify the phototoxic effect, both loss of plasma membrane integrity and loss of cellular adhesion were analyzed by measuring LDH release and crystal violet staining, respectively. c Photooxidative damage secondary to irradiation was reduced by incubation with the singlet oxygen scavenger DABCO. Scale bar, 250 μm

Fig. 3

Lipofuscin-mediated photooxidative damage results in lysosomal membrane permeabilization with cytosolic leakage of lysosomal enzymes. a Intact lysosomes (red) and nuclei (green) were visualized in ARPE-19 cells by means on acridine orange staining. b Lysosomal membrane permeabilization resulted in a loss of lysosomal staining that was quantified by flow cytometry. c Digitonin effect on ARPE-19 cells was titrated for maximum plasma membrane permeabilization (release of cytosolic LDH) and at the same time minimal lysosomal membrane permeabilization (release of lysosomal acid phosphatase, AP). d A digitonin concentration of 20 μg/ml was selected for separation of cytosolic and lysosomal cellular fractions, and cytosolic leakage of lysosomal enzymes was assessed by analyzing the activity of lysosomal marker enzyme acid phosphatase in the cytosolic fractions. Scale bar, 50 μm

Fig. 4

Lysosomal membrane permeabilization by lipofuscin phototoxicity induces activation of caspase-1. a Following inflammasome activation, activated caspase-1 was detected in ARPE-19 cells by the FLICA probe FAM-YVAD-FMK (green). b Caspase-1 activation was quantified by flow cytometry. Scale bar, 200 μm

Fig. 5

Lysosomal membrane permeabilization by lipofuscin phototoxicity results in inflammasome activation with secretion of IL-1β and IL-18. Inflammasome-mediated secretion of mature IL-1β (a) and IL-18 (b) was analyzed by ELISA in pRPE cells and ARPE-19 cells. c Lysosomal membrane permeabilization by ciprofloxacin and Leu-Leu-OMe in APRE-19 cells served as positive controls. d To assess the role of photooxidative damage in irradiation-induced inflammasome activation, ARPE-19 cells were incubation with the singlet oxygen scavenger DABCO during blue light treatment

Fig. 6

Inflammasome activation by lipofuscin phototoxicity is dependent on inflammasome priming and activity of caspase-1, cathepsin B, and cathepsin L. Secretion of IL-1β (a) and IL-18 (b) by pRPE cells and ARPE-19 cells was assessed by ELISA in primed cells incubated with HNE-POS (Control), unprimed cells incubated with HNE-POS (Unpr.), primed cells incubated with HNE-POS and caspase-1 inhibitor Z-YVAD-FMK (Casp.-1), primed cells incubated with HNE-POS and cathepsin B inhibitor CA-074 (Cath. B), and primed cells incubated with HNE-POS and cathepsin L inhibitor Z-FF-FMK (Cath. L)

Fig. 7

Inflammasome activity by lipofuscin phototoxicity is mediated by NLRP3. a In ARPE-19 cells incubated with HNE-POS prior to blue light irradiation, the effect of siRNA-mediated NLRP3 knockdown on IL-1β secretion was assessed as compared to control cells transfected with nonspecific siRNA. b Accumulation of lipofuscin-like material (yellow) following incubation with POS was documented by fluorescence microscopy in murine macrophages as a substitute model of RPE lipofuscinogenesis. c Following POS-induced lipofuscin accumulation, inflammasome priming with LPS, and subsequent blue light irradiation for 4 h, secretion of IL-1β by wild-type (NLRP3 +/+) and NLRP3 knockout (NLRP3 −/−) macrophages was analyzed by ELISA. Scale bar, 50 μm