Presentation Type

Poster

Abstract

Lysosomal Membrane Permeablization (LMP) has long been known as a significant cause of programmed cell death. LMP causes the release of hydrolytic enzymes from the endosomal compartments of cells which signal the cell to die. Certain hydrolytic enzymes, such as the cathepsins, cause the formation of a large protein complex called the inflammasome and subsequent activation of caspase-1 induces the release of pro-inflammatory cytokines and a type of cell death called pyroptosis. In recent years, many studies have suggested that altering cholesterol levels in lysosomes can affect the severity of LMP and its effect on programmed cell death. Two drugs, U18666A and methyl beta cyclodextran (MbCD), are known to have opposing effects on cholesterol trafficking in the endosomal compartments. U18666A impedes cholesterol trafficking in the endosomal system, causing accumulation of cholesterol and hindering programmed cell death by LMP. MbCD, a cholesterol-chelating drug, extracts cholesterol from cells and thereby increases cell sensitivity to LMP. In this study, we will use the NLRP3KO cell line to visualize the formation of the inflammasome when cells are treated with either U18666A or MbCD and certain nanomaterials which are known to induce LMP in certain cell lines. We expect to see that inflammasome formation induced by nanomaterial is decreased by U18666A and increased with MbCD due to cholesterol modification in lysosomal membranes. Along with testing the effects of certain cholesterol modifiers on NLRP3KO cells, we also attempted to optimize the conditions and treatments for utilizing these cells as a model for inflammasome formation and inflammation by nanoparticles. To do this, we performed dosage response tests to nanoparticles using cell viability and IL-1β production assays, looked at a time course response to particulates in order to determine the timeline of inflammasome formation, and endeavored to create a protocol for quantifying inflammasome formation in particle-treated cells.

Category

Life Sciences

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Apr 11th, 3:00 PM Apr 11th, 4:00 PM

Utilizing the NLRP3KO cell line to visualize inflammasome formation in the presence of cholesterol-trafficking modifiers

Lysosomal Membrane Permeablization (LMP) has long been known as a significant cause of programmed cell death. LMP causes the release of hydrolytic enzymes from the endosomal compartments of cells which signal the cell to die. Certain hydrolytic enzymes, such as the cathepsins, cause the formation of a large protein complex called the inflammasome and subsequent activation of caspase-1 induces the release of pro-inflammatory cytokines and a type of cell death called pyroptosis. In recent years, many studies have suggested that altering cholesterol levels in lysosomes can affect the severity of LMP and its effect on programmed cell death. Two drugs, U18666A and methyl beta cyclodextran (MbCD), are known to have opposing effects on cholesterol trafficking in the endosomal compartments. U18666A impedes cholesterol trafficking in the endosomal system, causing accumulation of cholesterol and hindering programmed cell death by LMP. MbCD, a cholesterol-chelating drug, extracts cholesterol from cells and thereby increases cell sensitivity to LMP. In this study, we will use the NLRP3KO cell line to visualize the formation of the inflammasome when cells are treated with either U18666A or MbCD and certain nanomaterials which are known to induce LMP in certain cell lines. We expect to see that inflammasome formation induced by nanomaterial is decreased by U18666A and increased with MbCD due to cholesterol modification in lysosomal membranes. Along with testing the effects of certain cholesterol modifiers on NLRP3KO cells, we also attempted to optimize the conditions and treatments for utilizing these cells as a model for inflammasome formation and inflammation by nanoparticles. To do this, we performed dosage response tests to nanoparticles using cell viability and IL-1β production assays, looked at a time course response to particulates in order to determine the timeline of inflammasome formation, and endeavored to create a protocol for quantifying inflammasome formation in particle-treated cells.