Date of Completion

11-10-2015

Embargo Period

11-9-2018

Keywords

Silica, silicosis, NADPH oxidase, apoptosis, cell death, ROS, phagolysosomal leakage, macrophages, imaging

Major Advisor

David Knecht

Associate Advisor

Andrea Hubbard

Associate Advisor

Adam Zweifach

Field of Study

Cell Biology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Silicosis is a lung disease characterized by pulmonary fibrosis that arises from chronic inhalation of silica dust in various occupational settings. Inhaled silica particles get deposited in the alveoli where macrophages engulf them leading to inflammation and macrophage cell death. The presence of apoptotic macrophages in the lungs is sufficient to cause inflammation and fibrosis whereas; inhibition of macrophage cell death prevents fibrosis. The goal of this study is therefore to identify the processes within an alveolar macrophage leading to cell death after uptake of silica particles. In order to accomplish this goal, we have utilized various fluorescent probes and microscopy based methods to visualize and quantify the processes of particle phagocytosis, phagosome maturation, phagolysosome damage, reactive oxygen species (ROS) generation and a role of various apoptotic proteins in cell death.

We show that following silica particle phagocytosis by a macrophage, a phagolysosome containing a silica particle leaks after an average of 25 minutes. A leaky phagolysosome is then resealed 10 minutes later and stops leaking. Toxic silica particles were able to generate phagosomal ROS independent of NADPH oxidase whereas non-toxic latex particles could not. Latex particles also did not cause phagolysosomal leakage or cell death. Release of cathepsins during phagolysosomal leakage is hypothesized to activate proapoptotic proteins to cause cell death. We did not observe activation of pro-apoptotic proteins such as Bid and Bax immediately after phagolysosomal leakage but only hours later close to the time of apoptosis. A unique increase in mitochondrial membrane potential was observed during apoptosis where as cells undergoing necrosis showed a decrease in mitochondrial membrane potential. In apoptotic cells mitochondrial hyperpolarization, caspase -9 and -3 activation and cell blebbing were observed together in a 5-minute temporal window. The order of events leading to cell death was same in all cells but there was a temporal heterogeneity between these events in cells.

Available for download on Friday, November 09, 2018

Share

COinS