Abstract
Purpose:
Photooxidative damage of the retinal pigment epithelium (RPE) is associated with the pathogenesis of age-related macular degeneration (AMD). In addition, involvement of a chronic immune response in the sub-RPE space including activation of the complement system has been demonstrated in AMD. To identify a molecular link between these mechanisms we investigated the capability of activated complement components to prime RPE cells for activation of the NLRP3 inflammasome by lipofuscin phototoxicity.
Methods:
Lipofuscinogenesis was induced in primary human RPE cells and ARPE-19 cells by incubation with isolated photoreceptor outer segments following modification with lipid peroxidation products. For inflammasome priming, lipofuscin-loaded cells were incubated in serum-free media or media supplemented with full human serum, C5-deficient serum, or isolated C5a. Specific C5a receptor (CD88) antibodies were used to block C5a binding. Control cells were primed with IL-1α. Following priming, cells were irradiated with blue light for up to 6 hours. NLRP3 inflammasome activation was assessed by measuring IL-1β and IL-18 secretion. Pyroptotic cell death was analyzed using LDH release assay, TUNEL staining, and DNA/histone-specific ELISA.
Results:
Priming of RPE cells with full human serum or isolated complement component C5a resulted in a lipofuscin load- and light dose-dependent activation of the NLRP3 inflammasome with secretion of IL-1β and IL-18. Complement heat-inactivation, C5 depletion, or C5a receptor inhibition suppressed the priming effect of human serum. Specific inhibition of caspase-1 or cathepsin B, L, or D likewise prevented NLRP3 activation. Inflammasome activation was followed by RPE cell death by pyroptosis as identified by morphological and molecular characteristics.
Conclusions:
Complement component C5a is capable of providing the priming signal for subsequent activation of the NLRP3 inflammasome by phototoxic effects of lipofuscin. This molecular pathway may represent a functional link between hallmark features of AMD such as lipofuscin accumulation, photooxidative damage, chronic immune response, and progressive degeneration of the RPE and may provide a novel target for therapeutic intervention in AMD.
Keywords: 582 ipofuscin •
634 oxidation/oxidative or free radical damage •
557 inflammation