Presentation Title

Ultrafine Carbon Black Promotes Lysosomal Membrane Permeabilization And Nlrp3 Inflammasome Activation

Faculty Mentor

Jay L. Brewster

Start Date

23-11-2019 12:45 PM

End Date

23-11-2019 1:00 PM

Location

Markstein 209

Session

oral 3

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Carbon black (CB) particulates are a component of diesel exhaust, and ultra-fine airborne pollutant particulate matter. CB exposure contributes to the adverse health effects of pollutants upon the lungs, heart, and nervous system. One source of damage is the chronic inflammation induced by exposure to airborne particulates. CB particles were hypothesized to enter immune cells by endocytosis where they accumulate. The accumulation of CB in RAW264.7 mouse macrophages was evaluated with doses of 50, 100, and 200 ug/ml at time points of 0, .5, 1, 2, 4, 6, 12, 24, and 48 hours. After washing, cells were lysed to release cellular components. Accumulated particulates were purified via centrifugation, then quantified via absorbance readings (A595). Particles were shown to accumulate gradually over time, with significant accumulation detected after 2 hours of exposure. Additional studies used THP-1 human monocytes to evaluate inflammasome signaling. THP-1 monocytes were stimulated with phorbol-12-13-acetate (PMA) to induce differentiation and adherence. To evaluate the hypothesis that CB accumulation impacts lysosomal membranes, we assessed Cathepsin B release into the cytosol and Galectin-3 localization to lysosomal vesicles. Assays provided support for a model of CB-induced lysosomal membrane permeabilization (LMP). We also hypothesized that CB stimulates the assembly of the inflammasome and subsequent production of IL-1β. Differentiated THP-1 cells were stimulated with lipopolysaccharide (100ng/ml) to upregulate expression of inflammasome components such as NLRP3 and ASC proteins. Assessment of inflammasome assembly was determined by measuring ASC oligomerization, formation of cytosolic ASC, and accumulation of extracellular IL-1β. Analysis of ASC oligomerization and fluorescent tagging of ASC oligomer complexes provided evidence for CB activation of the inflammasome, though potential for LPS to stimulate the inflammasome was observed. The future direction of this work will focus on duplicating experiments to solidify results and determine whether upstream mechanisms of inflammasome signaling are affected by CB accumulation.

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Nov 23rd, 12:45 PM Nov 23rd, 1:00 PM

Ultrafine Carbon Black Promotes Lysosomal Membrane Permeabilization And Nlrp3 Inflammasome Activation

Markstein 209

Carbon black (CB) particulates are a component of diesel exhaust, and ultra-fine airborne pollutant particulate matter. CB exposure contributes to the adverse health effects of pollutants upon the lungs, heart, and nervous system. One source of damage is the chronic inflammation induced by exposure to airborne particulates. CB particles were hypothesized to enter immune cells by endocytosis where they accumulate. The accumulation of CB in RAW264.7 mouse macrophages was evaluated with doses of 50, 100, and 200 ug/ml at time points of 0, .5, 1, 2, 4, 6, 12, 24, and 48 hours. After washing, cells were lysed to release cellular components. Accumulated particulates were purified via centrifugation, then quantified via absorbance readings (A595). Particles were shown to accumulate gradually over time, with significant accumulation detected after 2 hours of exposure. Additional studies used THP-1 human monocytes to evaluate inflammasome signaling. THP-1 monocytes were stimulated with phorbol-12-13-acetate (PMA) to induce differentiation and adherence. To evaluate the hypothesis that CB accumulation impacts lysosomal membranes, we assessed Cathepsin B release into the cytosol and Galectin-3 localization to lysosomal vesicles. Assays provided support for a model of CB-induced lysosomal membrane permeabilization (LMP). We also hypothesized that CB stimulates the assembly of the inflammasome and subsequent production of IL-1β. Differentiated THP-1 cells were stimulated with lipopolysaccharide (100ng/ml) to upregulate expression of inflammasome components such as NLRP3 and ASC proteins. Assessment of inflammasome assembly was determined by measuring ASC oligomerization, formation of cytosolic ASC, and accumulation of extracellular IL-1β. Analysis of ASC oligomerization and fluorescent tagging of ASC oligomer complexes provided evidence for CB activation of the inflammasome, though potential for LPS to stimulate the inflammasome was observed. The future direction of this work will focus on duplicating experiments to solidify results and determine whether upstream mechanisms of inflammasome signaling are affected by CB accumulation.