Author ORCID Identifier
https://orcid.org/0009-0004-9146-9898
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Adam Hawkridge
Abstract
Macrophages are integral components of the immune system, involved in pathogen clearance, wound healing, and the modulation of inflammatory responses. They achieve these functions through the detection and phagocytosis of pathogens, as well as the secretion of pro-inflammatory and antimicrobial mediators that recruit other immune cells. A novel defensive mechanism employed by macrophages is the formation of macrophage extracellular traps (METs), which are released in response to various stimuli, including microbial exposure and inflammatory signals. METs consist of DNA strands complexed with chromatin binding proteins and granule proteins, capable of entrapment and neutralization of pathogens via a process known as ETosis. Despite advances in understanding METs, significant gaps remain regarding their proteomic characterization under varying physiological conditions and macrophage polarization states. This dissertation comprises two major projects aimed at deepening our understanding of macrophage functions under different physiological conditions, particularly in the context of inflammation and immune responses. The first project investigates how macrophage polarization influences the formation of METs when stimulated by neutrophil elastase (NE), elucidating whether different polarization states (M0, M1, M2) result in differential MET formation and composition. Results indicate that NE induces MET formation in all three macrophage phenotypes and that MET formation is concentration-dependent, with higher concentrations of NE inducing more METs. The second project employs a mass spectrometry-based proteomics approach to conduct a comprehensive characterization of the MET proteome along with cell lysates and conditioned media, identifying and analyzing protein components under different polarization states and experimental conditions. Following NE treatment, we observed distinct changes in histone composition and ribosomal protein enrichment across M0, M1, and M2 macrophages. A total of 105, 123, and 117 differentially expressed METassociated proteins were identified in M0, M1, and M2 macrophages, respectively, with specific enrichment in pathways related to RNA metabolism in M2 METs and altered lysosomal and extracellular matrix (ECM) pathways in METs from M0 macrophages. Additionally, NE treatment resulted in the downregulation of antigen processing and immune signaling pathways in M1 macrophages, as well as shifts in chromatin dynamics and DNA replication processes across all polarization states. Notably, differential expression of RNA helicases and ligases was also identified, highlighting their functional implications in NE-induced METs formation. Together, these complementary projects aim to reveal critical insights into how macrophage polarization and proteomic alterations influence MET formation, with broader implications for chronic inflammation and disease progression.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-12-2024
Included in
Amino Acids, Peptides, and Proteins Commons, Analytical Chemistry Commons, Biochemistry Commons, Bioinformatics Commons, Biostatistics Commons, Data Science Commons, Immunology and Infectious Disease Commons, Respiratory Tract Diseases Commons