Year of Award

2014

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Toxicology

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Andrij Holian

Commitee Members

Kevan Roberts, Howard Beall, Christopher Migliaccio, Steven Lodmell

Keywords

inflammation, pulmonary, silica-induced

Abstract

Inhalation of crystalline silica for an extended period of time results in inflammation in the lungs, which plays a pivotal role in the development of silicosis, a progressive, inflammatory and fibrotic pulmonary disease. Inhaled silica particles are encountered by alveolar macrophages (AM) in the lungs. Previous studies have indicated the role of scavenger receptors in binding of environmental particles and also demonstrated that AM recognize and bind silica particles through class A scavenger receptors (SRA) expressed on their surface. MARCO (Macrophage Receptor with Collagenous Structure) is a SRA family receptor and the predominant receptor for recognition and binding of silica particles by alveolar macrophages (AM). Respirable silica particles are recognized by MARCO on AM and are internalized through phagocytosis by AM and subsequently contained in phagosomes. The lysosomes fuse with the phagosomes forming phagolysosomes and deliver the lysosomal enzymes for degradation of the particulates. However, silica cannot be degraded and induce lysosomal membrane permeabilization (LMP), by as yet unknown mechanism. LMP results in the release of lysosomal proteases, to the cytoplasm. This triggers the assembly of the NLRP3 (Nucleotide-binding domain, and Leucine-rich Repeat containing family, Pyrin domain containing) inflammasome assembly and further inflammation as a consequence. This project has focused on determining the relationship between MARCO and NLRP3 inflammasome activity and consequent downstream inflammation. Silica increased NLRP3 inflammasome activation and release of the pro-inflammatory cytokine, IL-1β, to a greater extent in MARCO-/- AM compared to wild type (WT) AM. A similar increase in NLRP3 inflammasome activation by silica was observed using MARCO Ab to block MARCO receptors in WT AM. Cathepsin B Inhibition (CA-074-Me), attenuated pro-inflammatory cytokine release. WT AM treated with cholesterol trafficking modifier U18666A decreased silica-induced NLRP3 inflammasome activation and MARCO null AM had reduced ability to sequester cholesterol following silica exposure. Taken together, this study demonstrates the critical role played by lysosomal membrane permeabilization (LMP) in triggering silica-induced inflammation and the contribution of MARCO in normal cholesterol uptake in macrophages, Therefore, in absence of MARCO, macrophages are susceptible to greater inflammatory response following silica exposure due to increased LMP. This project also determined the anti-inflammatory potential of imipramine to inhibit pulmonary inflammation following acute and chronic silica exposure. Anti-inflammatory potential of imipramine was evaluated in vitro in isolated AM from C57Bl/6 WT mice, and in acute and chronic in vivo silica exposure model. The in vitro results demonstrated that pretreatment with imipramine significantly attenuated cathepsin B activity and IL-1β release. In acute in vivo model, pretreatment with imipramine alleviated silica-induced inflammation, as was evident from the decrease in the chemokine KC and neutrophil infiltration. In the chronic in vivo exposure model used in the co-administration study, the protective effect of imipramine was validated by the histopathological analysis of lung tissues and the hydroxyproline quantification of silica-induced lung fibrosis. In the chronic exposure model used in the post-administration study, though imipramine treatment after four weeks of silica exposure attenuated the silica-induced lung injury, imipramine treatment after four weeks of silica exposure reduced the chronic pathological condition. These experiments establish that the pretreatment or concomitant treatment of imipramine was effective in alleviating silica-induced lung pathology, signifying that the early and acute phase of silica-induced inflammation is a critical phase where potential therapeutics interventions could be effective. Taken together, this project elucidates the

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© Copyright 2014 Rupa Biswas