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The regulation of inflammatory responses of airway epithelial cells and fibroblasts to rhinoviral infection

Ismail, Saila (2015) The regulation of inflammatory responses of airway epithelial cells and fibroblasts to rhinoviral infection. PhD thesis, University of Sheffield.

Final thesis S Ismail Sept2015(1).pdf
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Human rhinoviral (RV) infection is a major trigger of exacerbations of airway diseases such as asthma and COPD. Whilst many studies have shown that RV-infected airway epithelial cells secrete many proinflammatory cytokines that may exacerbate airway inflammation, limited studies have sought to investigate the potential role of airway fibroblasts in mediating RV-induced inflammation in airway diseases. Furthermore, the signalling pathways involved in the regulation of cytokine responses of structural airway cells to RV infection remain unclear, particularly with regard to signalling via PI3K, and the PI3K-dependent pathway, autophagy. The first aim of this thesis was to investigate the innate immune responses of human airway fibroblasts to RV infection, and to compare those responses with those of airway epithelial cells. It was found that RV infection induced differential responses in normal human airway fibroblasts and epithelial cells. In comparison to airway epithelial cells, the lack of viral-detecting PRRs TLR3, RIG-I, MDA5 and virally-induced transcription factors IRF1 and IRF7 in fibroblasts may be a potential explanation as to why the fibroblasts do not secrete the IFN-stimulated cytokines CCL5 and CXCL10 (indicating that the RV-infected fibroblasts do not produce IFNs), therefore providing no antiviral response to limit viral replication, with concomitant cell death. The work presented here demonstrated the permissiveness of lung fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) patients to RV infection. This study also showed the ability of IL-1β to enhance proinflammatory responses of RV-infected epithelial cells. Furthermore, it was demonstrated that in the presence of monocytes, RV and bacterial-derived LPS coinfections could act in synergy to augment the proinflammatory responses of airway tissue cells. The second aim of this thesis was to determine whether the PI3K-dependent pathway autophagy is involved in the detection of RV infection, and therefore regulates the RV-induced responses of airway epithelial cells. It was found that the PI3K pharmacological inhibitor 3-MA, typically used to inhibit autophagy, suppressed RV-induced cytokine production in airway epithelial cells. In contrast to the actions of 3-MA, specific targeting of the autophagy proteins Bec1, LC3, Atg7, or the autophagy-specific class III PI3K Vps34 by siRNA had very modest effects on RV-induced cytokine responses. Knockdown of autophagy proteins by siRNA also had minimal effects on RV replication. However, it was found that RV infection induced autophagy in the airway epithelial cells, although additional work is required to confirm this finding. Subsequent experiments performed using a panel of broad and class I-selective PI3K small-molecule inhibitors demonstrated functional redundancy of class I PI3K isoforms in modulating the RV-induced inflammation. The PI3K inhibitors 3-MA and LY294002 also remarkably reduced viral replication, suggesting that PI3Ks exert their roles in controlling RV infection via multiple mechanisms. Moreover, preliminary data suggests a potential role for mTOR in regulating the proinflammatory responses to RV infection. In conclusion, the work outlined in this thesis demonstrates two major findings: (i) a potential role for lung fibroblasts in mediating airway inflammation following RV infection and (ii) the involvement of PI3Ks and mTOR in induction of proinflammatory cytokines in response to RV infection, and that autophagy plays a limited role in the cytokine response to RV infection or control of RV replication.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.675046
Depositing User: Miss Saila Ismail
Date Deposited: 26 Nov 2015 16:40
Last Modified: 03 Oct 2016 12:19
URI: http://etheses.whiterose.ac.uk/id/eprint/10166

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