Once activated, the intracellular receptor NLRP3 assembles an inflammasome protein complex that facilitates the caspase-1-mediated maturation of IL-1β and IL-18. Inactive NLRP3 is guarded by a protein complex containing Hsp90. In response to stress stimuli, Hsp90 is released, and NLRP3 can be activated to promote inflammation. In this study, we blocked Hsp90 with geldanamycin and studied the fate of NLRP3 in human retinal pigment epithelial (RPE) cells. RPE cells play a central role in the development of age-related macular degeneration (AMD), a progressive eye disease causing severe vision loss in the elderly. IL-1α-primed ARPE-19 cells, human embryonal stem cell (hESC)-derived RPE cells, and primary human RPE cells were exposed to MG-132 and bafilomycin A to activate NLRP3 via the inhibition of proteasomes and autophagy, respectively. Additionally, RPE cells were treated with geldanamycin at different time points and the levels of NLRP3 and IL-1β were determined. Caspase-1 activity was measured using a commercial assay. Geldanamycin prevented the activation of the inflammasome in human RPE cells. NLRP3 released from its protective complex became degraded by autophagy or secreted from the cells. Controlled destruction of NLRP3 is a potential way to regulate the inflammation associated with chronic diseases, such as AMD.

Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.

Mitochondrial dysfunction has been implicated in a wide variety of degenerative diseases, including age-related macular degeneration. Damage to mitochondria and mitochondrial DNA accumulates with age in the postmitotic retinal pigment epithelium (RPE), which could lead to RPE cell death and trigger disease. One possible mechanism for cells to avoid cell death is mitophagy, the targeted clearance of damaged mitochondria by autophagy. Here, we induced mitochondrial damage in human RPE cells (ARPE-19 and hRPE), using antimycin A, an inhibitor of complex III of the electron transport chain, and investigated cellular viability, mitochondrial structure and function, and autophagy activity. We observed that antimycin A evoked dose-dependent cell death, a rapid loss in mitochondrial membrane potential, and a collapse of oxidative phosphorylation. Mitochondria appeared swollen and there was clear damage to their cristae structure. At the same time, cells were undergoing active autophagy and were sensitive to autophagy inhibition by bafilomycin A1 or chloroquine. These results indicate that mitochondrial dysfunction can cause significant RPE damage and that autophagy is an important survival mechanism for cells suffering from mitochondrial damage.

Background/Aims: Previously, we demonstrated that blockade of the intracellular clearance systems in human retinal pigment epithelial (RPE) cells by MG-132 and bafilomycin A1 (BafA) induces NLRP3 inflammasome signaling. Here, we have explored the activation mechanisms behind this process. NLRP3 is an intracellular receptor detecting factors ranging from the endogenous alarmins and adenosine triphosphate (ATP) to ultraviolet radiation and solid particles. Due to the plethora of triggers, the activation of NLRP3 is often indirect and can be mediated through several alternative pathways. Potassium efflux, lysosomal rupture, and oxidative stress are currently the main mechanisms associated with many activators. Methods: NLRP3 inflammasomes were activated in human RPE cells by blocking proteasomes and autophagy using MG-132 and bafilomycin A1 (BafA), respectively. P₂X₇ inhibitor A740003, potassium chloride (KCl), and glyburide, or N-acetyl-L-cysteine (NAC), ammonium pyrrolidinedithiocarbamate (APDC), diphenyleneiodonium chloride (DPI), and mito-TEMPO were added to cell cultures in order to study the role of potassium efflux and oxidative stress, respectively. IL-1β was measured using the ELISA method. ATP levels and cathepsin B activity were examined using commercial kits, and ROS levels using the fluorescent dye 2´,7´-dichlorodihydrofluorescein diacetate (DCFDA). Results: Elevated extracellular potassium prevented the priming factor IL-1α from inducing the production of reactive oxygen species (ROS). It also prevented IL-1β release after exposure of primed cells to MG-132 and BafA. Inflammasome activation increased extracellular ATP levels, which did not appear to trigger significant potassium efflux. The activity of the lysosomal enzyme, cathepsin B, was reduced by MG-132 and BafA, suggesting that cathepsin B was not playing any role in this phenomenon. Instead, MG-132 triggered ROS production already 30 min after exposure, but treatment with antioxidants blocking NADPH oxidase and mitochondria-derived ROS significantly prevented IL-1β release after this activating signal. Conclusion: Our data suggest that oxidative stress strongly contributes to the NLRP3 inflammasome activation upon dysfunctional cellular clearance. Clarification of inflammasome activation mechanisms provides novel options for alleviating pathological inflammation present in aggregation diseases, such as age-related macular disease (AMD) and Alzheimer’s disease.