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Title: Investigating the role of Tenascin-C and extracellular vesicles in human rhinovirus induced exacerbations of asthma
Author: Mills, Jake
ISNI:       0000 0004 7655 3082
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Introduction, Aims and Hypothesis: Viral infections are the cause of 75% of all asthma exacerbations, with human rhinovirus (RV) the most common trigger, but what happens during this process is not well understood. Tenascin-C (TN-C) is a large extracellular matrix protein that is present in small quantities in the airway of healthy individuals, but in high quantities in asthma sufferers. TN-C has been demonstrated to drive inflammation in diseases such as rheumatoid arthritis, but the inflammatory potential of TN-C in asthma has yet to be investigated. Furthermore, a subset of extracellular vesicles (EVs), known as exosomes, that are 50-120 nm in size, can contain TN-C and play role in airway inflammation. This study aimed to characterise the relationship between RV infection of airway epithelial cells (AECs) and TN-C expression and exosome release. It was hypothesised that RV infection of AECs promoted the release of TN-C and exosomes, leading to increased inflammatory cytokine and chemokine release in the airway and potentially contributing towards virally-induced exacerbations of asthma. Methods: WT mice were treated with the viral mimic poly(I:C) and bronchoalveolar lavage fluid analysed for TN-C by western blotting. AECs from asthmatic and non-asthmatic donors were also stimulated with poly(I:C) or infected with RV and assayed for TN-C expression and release by qPCR and western blotting. Exosomes were then isolated by differential ultracentrifugation and analysed for TN-C expression by western blot. Finally, recombinant purified TN-C, and exosomes isolated with and without TN-C siRNA pre-treatment, were used to stimulate AECs and monocyte derived macrophages (MDMs). Cytokine and chemokine release was then measured by enzymelinked immunosorbent assay (ELISA). Results: It was determined that TN-C mRNA expression and cell-associated TN-C expression could be modulated in response to RV infection, ultimately leading to the significant release of TN-C from the cell. This pathway was demonstrated to be TLR3-dependent and independent of TLR7 and cell cytotoxicity. TN-C release following RV infection was more pronounced in asthmatics, potentially revealing why TN-C is expressed in higher quantities in the asthmatic airway. The study also revealed that viral TLR3-dependent stimulation induced significant exosomal release and TN-C was associated with these exosomes, with expression correlating with exosome number. RV-dependent TN-C release induced large inflammatory CXCL8 release from MDMs and moderate CXCL8 release from AECs. Also of note, exosomes from RV-infected AECs promoted significant inflammatory and anti-viral cytokine / chemokine release from AECs, whilst exosomes from non-virally cells did not, and both types of exosomes induced cytokine release from MDMs. The role of TN-C in RV-induced exosomal inflammation, however, is yet to be elucidated. Conclusion: The results in this study reveal a pathway by which RV infection promotes the release of TN-C and exosomes that have the ability to induce inflammatory cytokine release in the airway. Further investigation is required to determine if TN-C or exosomes are viable therapeutic targets to modulate RV-induced asthma exacerbations in the future.
Supervisor: Parker, Lisa ; Midwood, Kim ; Sabroe, Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available