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.

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.

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.

Abbreviations: AMD, age-related macular degeneration; ASC, adaptor protein apoptosis-associated speck-like protein containing a CARD; ATP, adenosine triphosphate; CPD, cyclobutane pyrimidine dimer; ELISA, enzyme-linked immunosorbent assay; H 2 DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; IL-18, interleukin-18; IL-1α, interleukin-1α; IL-1β, interleukin-1β; KCl, potassium chloride; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NAC, n-acetyl-cysteine; NLRP3, nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3; qRT-PCR, quantitative real-time polymerase chain reaction; RFU, relative fluorescence unit; ROS, reactive oxygen species; RPE, retinal pigment epithelium; siRNA, small interfering RNA; UVB, ultraviolet B radiation.

Retinal pigment epithelial (RPE) cells maintain homeostasis at the retina and they are under continuous oxidative stress. Cigarette smoke is a prominent environmental risk factor for age-related macular degeneration (AMD), which further increases the oxidant load in retinal tissues. In this study, we measured oxidative stress and inflammatory markers upon cigarette smoke-derived hydroquinone exposure on human ARPE-19 cells. In addition, we studied the effects of commercial Resvega product on hydroquinone-induced oxidative stress. Previously, it was observed that Resvega induces autophagy during impaired protein clearance in ARPE-19 cells, for which it has the potential to alleviate pro-inflammatory pathways. Cell viability was determined while using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the cytokine levels were measured using the enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) production were measured using the 2′,7′-dichlorofluorescin diacetate (H2DCFDA) probe. Hydroquinone compromised the cell viability and increased ROS production in ARPE-19 cells. Resvega significantly improved cell viability upon hydroquinone exposure and reduced the release of interleukin (IL)-8 and monocytic chemoattractant protein (MCP)-1 from RPE cells. Resvega, N-acetyl-cysteine (NAC) and aminopyrrolidine-2,4-dicarboxylic acid (APDC) alleviated hydroquinone-induced ROS production in RPE cells. Collectively, our results indicate that hydroquinone induces cytotoxicity and increases oxidative stress through NADPH oxidase activity in RPE cells, and resveratrol-containing Resvega products prevent those adverse effects.