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dc.contributor.authorPiippo, Niina
dc.contributor.authorKorhonen, Eveliina
dc.contributor.authorHytti, Maria
dc.contributor.authorKinnunen, Kati
dc.contributor.authorKaarniranta, Kai
dc.contributor.authorKauppinen, Anu
dc.date.accessioned2018-09-25T08:34:27Z
dc.date.available2018-09-25T08:34:27Z
dc.date.issued2018
dc.identifier.urihttps://erepo.uef.fi/handle/123456789/6955
dc.description.abstractBackground/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.
dc.language.isoenglanti
dc.publisherS. Karger AG
dc.relation.ispartofseriesCellular physiology and biochemistry
dc.relation.urihttp://dx.doi.org/10.1159/000492886
dc.rightsCC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
dc.titleOxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy
dc.description.versionpublished version
dc.contributor.departmentSchool of Pharmacy, Activities
dc.contributor.departmentSchool of Medicine / Clinical Medicine
uef.solecris.id55668372en
dc.type.publicationTieteelliset aikakauslehtiartikkelit
dc.rights.accessrights© Authors
dc.relation.doi10.1159/000492886
dc.description.reviewstatuspeerReviewed
dc.format.pagerange359-367
dc.publisher.countrySveitsi
dc.relation.issn1015-8987
dc.relation.issue1
dc.relation.volume49
dc.rights.accesslevelopenAccess
dc.type.okmA1
uef.solecris.openaccessOpen access -julkaisukanavassa ilmestynyt julkaisu


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