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Carolina Brandstetter, Frank G Holz, Tim U Krohne; Complement component C5a primes the NLRP3 inflammasome in retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3444.
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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.
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.
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.
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.
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