Social Networking: Extracellular Vesicles and MicroRNA Messages Coordinate Inflammation in Severe Asthma
Open Access
- Author:
- Lambert, Kristin Anne
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 20, 2019
- Committee Members:
- Todd Schell, Dissertation Advisor/Co-Advisor
Faoud Ishmael, Committee Chair/Co-Chair
James Robert Connor, Committee Member
Charles H Lang, Committee Member
Rebecca Phaeton, Outside Member
Faoud Ishmael, Dissertation Advisor/Co-Advisor
Todd Schell, Committee Chair/Co-Chair - Keywords:
- Asthma
Exosome
Extracellular Vesicle
Airway Inflammation
Inflammation
Steroid
T cell
Neutrophil
IL-8
Epithelial Cell
microRNA
miRNA
Severe Asthma
Allergen
Dust Mite
Glucocorticoid
EV
miR-155
miR-146a
Biomarker
proinflammatory - Abstract:
- Asthma is a heterogeneous clinical syndrome that encompasses a remarkably diverse spectrum of chronic lung diseases involving episodic airflow obstruction, bronchial hyperresponsiveness and airway inflammation. Rather than a single disease entity, asthma consists of disease variants or phenotypes that possess divergent immunologic, physiologic, and pathologic features, including varied responses to treatment. One such phenotype, severe asthma, represents a subset of asthmatics who fail to respond to standard therapy with glucocorticoids and remain poorly controlled despite adherence to medication and escalations in clinical management. Representing only 10-15% of asthma, severe asthmatics are a minority of patients who consume a majority of asthma-related healthcare resources and suffer greater morbidity and mortality compared to other asthma subtypes. The exact biologic pathways or ‘endotypes’ responsible for this vulnerable and difficult-to-treat cluster of patients are still undefined due to the group’s overwhelming heterogeneity. We aim to fill this unmet research area through the investigation of inflammatory pathways in severe asthma. Asthma heterogeneity at a population level is the consequence of heterogeneity in cell-to-cell interactions at the molecular level. Assorted cell populations in the lung form diverse networks that collaboratively communicate to initiate and maintain chronic inflammation in various asthma endotypes. Chief among these relationships, airway epithelial cells and T lymphocytes engage in inflammatory dialogue with one another. Their methods for communicating and the content of their messages have not been fully unraveled; although, understanding of these pathways is anticipated to give way to crucial advances in targeted therapeutics for severe, treatment-refractory asthma. In this dissertation, we address the critical need to decipher communications and disentangle intracellular conversations between members of the airway lining and the immune system. Novel cell-to-cell signaling mediators, extracellular vesicles (EVs) have been shown to participate in cellular crosstalk and play roles in immune regulation. We aimed to elucidate the role of extracellular vesicles in severe asthmatic inflammation and hypothesized that EVs would serve as proinflammatory directives from T lymphocytes to airway epithelial cells. Utilizing both peripheral blood mononuclear cells and peripheral CD4+ T lymphocytes of human asthmatics, we studied T-cell derived extracellular vesicle production and function. EVs were produced in response to activating inflammatory stimuli, including dust mite allergen exposure and TCR engagement, and were shed in greater quantities from asthmatic cells compared to healthy controls. Qualitatively, the phenotype of EVs secreted from activated asthmatic T cells mirrored the inflammatory stress of their parental cell and also transmitted the inflammatory phenotype to airway epithelial cells in vitro. EVs upregulated epithelial expression of neutrophilic chemoattractants like CXCL8 (aka IL-8), a pathway associated with severe asthma. Together the data suggest that T-cell derived EVs are generated as a pathogenic swarm of proinflammatory cues capable of altering airway immune function and sustaining chronic inflammation through leukocyte recruitment. More in-depth analysis and profiling of severe asthmatic T cell EVs, uncovered miRNA cargo within extracellular vesicles. Like messages in a bottle, microRNA missives are able to be shuttled between cells, sharing genetic instructions for inflammatory regulation between T lymphocytes and airway epithelium. A proinflammatory miRNA, miR-155 was found in greater abundance within severe asthmatic EVs, while anti-inflammatory molecule and endogenous opponent, miR-146a was found in reduced quantities. The composition of severe asthmatic EVs and the delicate balance between immunoregulatory miRNAs, miR-155 and miR-146a, within EVs likely contributes to the proinflammatory nature of particles and status of inflammation in airway epithelium. We posit that low concentrations of EV-encased miR-146a may result in negligible transfer of anti-inflammatory molecules to airway epithelial cells in severe asthma As a result, we investigated miRNA-based approaches to enhance anti-inflammatory action and boost glucocorticoid functions in airway epithelial cells. We show that overexpression of miR-146a in airway epithelial cells reduces proinflammatory cytokines and when combined with glucocorticoids further suppresses inflammation. This work identifies a novel therapeutic target and innovative approach to overcome steroid resistance in severe asthmatics through exogenous administration of miR-146a. It would be valuable in future studies to investigate exosomal or EV-mediated delivery of miR-146a to airway epithelium. Alternatively, approaches to skew T cell EV cargo towards anti-inflammatory cargo (miR-146a) and away from proinflammatory stowaways (miR-155) may be a worthy line of future investigation to alter EV phenotypes and immune regulation in asthma. In conclusion, our work makes significant strides to characterize novel molecular messages coordinating diverse inflammatory processes in asthma. Our data show that T-cell derived extracellular vesicles and their associated miRNAs participate in cell-to-cell conversations that ultimately coordinate inflammatory responses in the airway. T-cell EVs command inflammatory changes in their target airway cells, inducing neutrophilic chemoattractants required to recruit additional leukocytes to sustain chronic and severe unchecked asthmatic inflammation. Finally, we detect increased numbers of extracellular vesicles and unique extracellular vesicle miRNA signatures for severe asthma. We report that both measures are promising biomarker candidates for the clinical management of severe asthma. Together, these studies elucidate the contributions of extracellular vesicles and miRNAs to a neutrophil-predominant inflammatory endotype associated with severe asthma.