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

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: 448nm, irradiance: 0.8mW/cm(2), duration: 3 to 6h) 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 24h 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-1ß 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.

Complement Component C5a Primes Retinal Pigment Epithelial Cells for Inflammasome Activation by Lipofuscin-mediated Photooxidative Damage.

Complement activation, oxidative damage, and activation of the NLRP3 inflammasome have been implicated in retinal pigment epithelium (RPE) pathology in age-related macular degeneration (AMD). Following priming of RPE cells, the NLRP3 inflammasome can be activated by various stimuli such as lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated whether products of complement activation are capable of providing the priming signal for inflammasome activation in RPE cells. We found that incubation of primary human RPE cells and ARPE-19 cells with complement-competent human serum resulted in up-regulation of C5a receptor, but not C3a receptor. Furthermore, human serum induced expression of pro-IL-1ß and enabled IL-1ß secretion in response to lipofuscin phototoxicity, thus indicating inflammasome priming. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum whereas recombinant C5a likewise induced priming. Conditioned medium of inflammasome-activated RPE cells provided an additional priming effect that was mediated by the IL-1 receptor. These results identify complement activation product C5a as a priming signal for RPE cells that allows for subsequent inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration and may provide novel therapeutic targets in this disease.

Effects of Inflammasome Activation on Secretion of Inflammatory Cytokines and Vascular Endothelial Growth Factor by Retinal Pigment Epithelial Cells.

PURPOSE: Activation of the NLRP3 inflammasome has been implicated in the pathogenesis of AMD. Lipofuscin phototoxicity activates the inflammasome in RPE cells by inducing lysosomal membrane permeabilization (LMP). We investigated the effects of LMP-induced inflammasome activation on the secretion of inflammation-related cytokines and VEGF by RPE cells. METHODS: In primary human RPE cells and ARPE-19 cells, the inflammasome was activated by L-leucyl-L-leucine methyl ester (Leu-Leu-OMe)- or lipofuscin phototoxicity-induced LMP. Cytokine secretion was measured by protein dot blot and enzyme-linked immunosorbent assays. The polarization of cytokine secretion was assessed in RPE monolayers on permeable membranes. We analyzed the chemotactic and angiogenic effects of secreted cytokines on murine embryonic stem cell-derived microglia cells and human umbilical vascular endothelial cells, respectively. RESULTS: Inflammasome activation in RPE cells was associated with caspase-1-dependent secretion of IL-1ß, IL-6, IL-18, GM-CSF, and GRO (CXCL1/2/3), whereas constitutive secretion of VEGF was reduced. Secretion of IL-1ß and IL-18 was highly polarized to the apical cell side. Incubation with conditioned media of inflammasome-activated RPE cells induced directed migration of microglia cells (11.0-fold increase) and diminished vascular endothelial cells proliferation (39.0% reduction) and migration (69.3% reduction) as compared with conditioned media of untreated control RPE cells. CONCLUSIONS: Lysosomal membrane permeabilization-induced activation of the NLRP3 inflammasome in RPE cells results in apical secretion of inflammatory cytokines with chemotactic effects on microglia cells and reduced constitutive secretion of VEGF. Via these mechanisms, lipofuscin phototoxicity may contribute to local immune processes in the outer retina as observed in AMD.

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

UNLABELLED: 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-1ß (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.