Date of Award
8-2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
School
Biological, Environmental, and Earth Sciences
Committee Chair
Hao Xu
Committee Chair School
Biological, Environmental, and Earth Sciences
Committee Member 2
Alex Flynt
Committee Member 2 School
Biological, Environmental, and Earth Sciences
Committee Member 3
Shahid Karim
Committee Member 3 School
Biological, Environmental, and Earth Sciences
Committee Member 4
Yanlin Guo
Committee Member 4 School
Biological, Environmental, and Earth Sciences
Committee Member 5
Vijay Rangachari
Committee Member 5 School
Mathematics and Natural Sciences
Abstract
Mast cells are one of the major producers of tumor necrosis factor-alpha (TNF), and mast cell-derived TNF (MC-TNF) is associated with pathological conditions such as rheumatoid arthritis, inflammatory bowel diseases, and allergic asthma. However, the molecular machinery underlying TNF release from mast cells has been unclear, despite the conserved nature of exocytosis. To delineate the exocytic machinery required for TNF release from mast cells, we systematically dissected the exocytic fusion machinery (e.g., Munc13, Munc18, and VAMP families) expressed in mast cells, using a combination of CRISPR-dependent knockout, RNA interference, confocal microscopy, lentiviral rescue experiments, and cell-based assays. Out of the three Munc13 proteins found in mast cells, we have shown that while Munc13-4 knock-out eliminated β-hexosaminidase, histamine, and serotonin release, it was only partially required for TNF release. Of the three Munc18 homologs, we showed that Munc18b is the sole Munc18 homolog required for TNF-α release in mast cells. For the first time, we reported the requirement of distinct exocytic machinery in the presence of different stimuli in mast cells by showing that while VAMP7 is required for TNF release in TLR-dependent mast cell activation, it does not play a role in IgE/allergen activation of mast cells. Furthermore, we delineated the compartmentalization and dynamics of TNF release in mast cells by employing a combination of microscopy and biochemical characterization. We showed that mast cell TNF is associated with multivesicular bodies and can be released in exosomes. Upon this discovery, we generated a tool to specifically monitor and quantify exosomal TNF release which we showed can be employed in quantifying exosomal TNF release and delineating exocytic machinery involved in this process.
In summary, this current study uncovers important aspects of the molecular mechanisms and dynamics of TNF release in mast cells. Our discovery of mast cell TNF release in exosomes and the corresponding design of a tool for its quantification has set the stage for the elucidation of molecular mechanisms underlying the release of exosomal TNF and its roles in intercellular signaling events.
ORCID ID
0000-0002-0618-2998
Copyright
2024, Tolulope E. Ayo
Recommended Citation
Ayo, Tolulope Eunice, "Elucidation of Molecular Mechanisms Underlying Tumor Necrosis Factor (TNF) Release from Mast cells" (2024). Dissertations. 2272.
https://aquila.usm.edu/dissertations/2272