Publication Date

2019

Document Type

Dissertation

Committee Members

Katherine J.D.A. Excoffon, Ph.D. (Advisor); Jeffrey B. Travers, M.D., Ph.D. (Committee Member); Dawn P. Wooley, Ph.D. (Committee Member); Dan R. Halm, Ph.D. (Committee Member); Hongmei Ren, Ph.D. (Committee Member)

Degree Name

Doctor of Philosophy (PhD)

Abstract

Human adenoviruses (AdV) are double-stranded DNA viruses that can cause a range of diseases. While AdV respiratory infections are often mild and self-limited, severe manifestations such as fulminant pneumonia and acute respiratory distress syndrome (ARDS) are not uncommon. In order to identify potential modalities to treat AdV infections and potentially enhance the effectiveness of AdV vector delivery, a thorough mechanistic understanding of how AdV enters host cells is needed. Previous studies have revealed polarized epithelia, one of the primary targets for AdV respiratory infections, are highly resistant to AdV entry from the apical (luminal) surface. However, upon exposure to interleukin 8 (IL-8), the 8-exon encoded isoform of the Coxsackie and Adenovirus receptor (CAR^(Ex8)) localizes to the apical surface of airway epithelial cells where it is able to mediate AdV entry. Furthermore, neutrophils, or polymorphonuclear leukocytes (PMN), that migrate to the epithelium as a result of the IL-8 stimulus have been shown to further enhance AdV infection of the epithelium through an uncharacterized mechanism. I hypothesized that neutrophilic factors alter epithelial physiology in such a way that renders them more susceptible to AdV infection and that a combination of enhanced apical CAR^(Ex8) expression and PMN factor signaling drastically enhances epithelial susceptibility to AdV5 infection. In order to address this hypothesis, a variety of pharmacological inhibitors were tested for their ability to influence AdV entry in epithelial cells that had been exposed to PMN or various PMN factors. Furthermore, CRISPR/Cas9 techniques were used to develop a CAR^(Ex8)-knockdown epithelial model system to address the importance of CAR^(Ex8) in AdV entry in the presence and absence of neutrophilic factors. Using these methods, I demonstrate that neutrophil elastase (NE), a PMN serine protease with diverse functions, is a major neutrophilic factor that drives PMN-mediated enhancement of epithelial AdV5 infection. Furthermore, I show that NE activates autophagic flux in epithelial cells and that this process is indirectly related to PMN enhancement of epithelial AdV5 transduction. Finally, using newly emerged CRISPR/Cas9 techniques, three epithelial model systems were generated and data suggest that NE mediated enhancement of epithelial AdV5 transduction is largely independent of CAR^(Ex8) expression. Taken together, this dissertation shows data that implicates NE as a key regulator of AdV5 infection of the airway epithelium and discusses the development of new epithelial cell lines with altered CAR^(Ex8) expression that may be useful for future studies of epithelial AdV entry. Future research should focus on establishing the role of PMN and NE in AdV5 entry in vivo.

Page Count

167

Department or Program

Biomedical Sciences

Year Degree Awarded

2019

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