NLRP3 inflammasome activation by photooxidative damage provides a novel link between hallmark pathogenic features of age-dependent macular degeneration

Abstract

In the developed world, age-related macular degeneration (AMD) is the most common cause for severe visual loss and legal blindness in the elderly. In this progressive disease, degeneration of the retinal pigment epithelium (RPE) results in the death of photoreceptors and secondary loss of central vision. Two late manifestations of AMD can be distinguished, neovascular AMD and atrophic AMD. Neovascular AMD is characterized by rapid visual loss secondary to VEGF-mediated ingrowth of choroidal neovascularizations (CNV). In contrast, atrophic AMD causes slowly progressive central visual decline by RPE cell degeneration known as geographic atrophy. Anti-VEGF treatment has proven highly effective in neovascular AMD and is now widely used clinically. In contrast, there is still no treatment available for atrophic AMD. Several lines of evidence indicate that oxidative and lipofuscinmediated photooxidative damage play an important role in AMD pathology. Further characteristics of the disease include the formation of extracellular deposits called drusen, and chronic low-grade immune processes including complement activation in the sub-RPE space. Inflammation is recognized as a major driving force in atrophic AMD. However, the mechanisms triggering and maintaining the retinal inflammation remain incompletely understood. Recent studies have shown that the NLRP3 inflammasome, a key mediator of the innate immune system, is activated in the RPE of patients with atrophic and neovascular AMD. The NLRP3 inflammasome is a multiprotein complex, which induces caspase-1 activation resulting in secretion of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and Interleukin-18 (IL-18). An initial priming signal in combination with a subsequent activation signal, such as reactive oxygen species or lysosomal membrane permeabilization (LMP), leads to assembly of the NLRP3 inflammasome. Studies demonstrated that phototoxic damage mediated by intralysosomal accumulation of photoreactive lipofuscin leads to LMP in RPE cells. The present thesis investigated whether lipofuscin-mediated phototoxicity activates the NLRP3 inflammasome in RPE cells in vitro and aimed to identify a novel mechanism of inflammasome activation by light damage that may provide new treatment targets for blinding diseases such as AMD. For this purpose, lipofuscin-loaded primary human RPE cells and human RPE cell line ARPE-19 cells were irradiated with blue light (dominant wavelength 448 nm, irradiance 0.8 mW/cm2 , duration 6 h) to induce photooxidative stress. The obtained results, presented in chapter IV demonstrate that accumulation of lipofuscin rendered RPE cells in vitro susceptible to phototoxic destabilization of lysosomes and cytosolic leakage of lysosomal enzymes. This resulted in NLRP3 inflammasome activation as evidenced by caspase-1 activation, processing and release of IL-1β and IL-18. Investigating the secretion profile of inflammatory cytokines after inflammasome activation revealed predominantly apical secretion of inflammatory cytokines, that corresponds to the neuoretinal side in vivo. In addition, secreted cytokines exerted a chemotactic effects on microglia cells as well as reduced constitutive secretion of VEGF (chapter V). In contrast to inflammasome activation, the mechanism of inflammasome priming in AMD has been little investigated so far. A recent study in patients with early or intermediate AMD demonstrated the complement factor H risk genotype to be associated with significantly increased plasma levels of the inflammasome-regulated cytokine IL-18, suggesting a role for activated complement components in inflammasome activation in AMD. Inflammasome priming by complement activation products has also been proposed in the context of other diseases such as atherosclerosis and gout. The thesis further reveales the capacity of activated complement components to prime human RPE cells for inflammasome activation. Obtained results, presented in chapter VI demonstrate that incubation of ARPE-19 cells with complement-competent normal human serum (NHS) induced expression of pro-IL- 1β and enabled secretion of IL-1β in response to lipofuscin phototoxicity, indicating inflammasome priming by NHS. For further delineation of the relevant priming competent component in NHS, complement factors were heat inactivated or blocked by complement inhibitors which enabled the identification of complement activation product C5a as the priming signal for RPE cells. Likewise, conditioned media of inflammasome-activated RPE cells provided a priming effect that was mediated by the IL-1 receptor, thus suggesting a paracrine amplification loop of inflammasome activation. Further investigation demonstrated that cell priming by IL-1α or C5a increased susceptibility of RPE cells to oxidative/photooxidative damage-mediated cell death (chapter VII). Morphological observations and investigation of the underlying cell death mechanism revealed a change in cell death mode from apoptosis to pyroptosis. Moreover, conditioned media of pyroptotic ARPE-19 cells increased cell death by photooxidative damage in other cells in an interleukin- 1 receptor (IL-1R) dependent fashion. These results make it conceivable that in situations of localized RPE cell death such as in atrophic AMD, this mechanism could result in increased susceptibility of immediate bystander RPE cells to inflammasome-mediated cell death, thus contributing to the centrifugal progression pattern of RPE cell loss in AMD. In summary, this study identified a novel mechanism of inflammasome activation by light damage and provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration. Thereby, this study provide new potential treatment targets for blinding diseases such as AMD.