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Carolina Brandstetter, Susannah M Kleeberger, Frank G Holz, Tim U Krohne; Photooxidative damage-induced NLRP3 inflammasome activation in eye cup organ cultures of Abca4 -/- mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2295.
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Activation of the NLRP3 inflammasome has been reported in the RPE of patients with age-related macular degeneration and may contribute to disease pathogenesis. In RPE cells in vitro, inflammasome activation is induced by lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated this mechanism in an eye cup organ culture model.
Pigmented mouse strains with increased lipofuscin accumulation within the RPE (Abca4 -/-) and deficiency of NLRP3 inflammasome function (Nlrp3 -/-) were employed. Eyes were harvested from 6 months-old wildtype, Abca4 -/-, Nlrp3 -/-, and Abca4 -/- /Nlrp3 -/- mice. Following removal of anterior segment and retina, the resulting posterior eye cups (RPE, choroid, sclera) were cultured in phenol red-free DMEM/F12 medium with 1 % FBS. Photooxidative stress was induced by blue LED light irradiation (448 nm, 0.8 mW/cm2) for 90 min. Photooxidative damage and inflammasome activation were assessed by means of release of LDH and IL-1β, respectively. Small molecule NLRP3 inhibitor MCC950 was applied in control experiments.
In wildtype eye cups, blue light irradiation induced photooxidative damage as evident from disruption of ZO-1 pattern of the RPE and release of LDH into the medium (16.5-fold increased compared to non-irradiated controls; p<0.001). LDH release after irradiation was amplified in Abca4 -/- eye cups (36.0-fold increased compared to controls; p<0.001). RPE cells of irradiated Abca4 -/- eye cups exhibited lysosomal membrane permeabilisation (loss of acridine orange staining) and caspase-1 activation (FAM-YVAD-FMK staining). Irradiation resulted in inflammasome activation in wildtype eye cups with release of IL-1β into the medium (77.3 pg vs. 1.8 pg in non-irradiated controls; p=0.007) that was further increased in irradiated Abca4 -/- eye cups (174.4 pg; p<0.001). Irradiation-induced IL-1β release was suppressed in both MCC950-treated Abca4 -/- eye cups (8.8 pg) and Abca4 -/- /Nlrp3 -/- eye cups (7.8 pg).
In blue light-irradiated Abca4 -/- eye cups, increased lipofuscin accumulation within the RPE is associated with augmented photooxidative damage that results in lysosomal membrane permeabilisation, NLRP3 inflammasome activation, and release of IL-1β. This organ culture model can be employed for preclinical evaluation of pharmacological inhibitors of inflammasome activation in the RPE.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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