- Title
- Investigation of the pathogenesis of influenza infection in asthma and COPD; potential therapeutic interventions
- Creator
- Dua, Kamal
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- People with chronic lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD) are more susceptible to influenza A virus (IAV) infections that subsequently exacerbate these diseases, leading to more severe symptoms with increased risk of mortality. However, the immune mechanisms driving the increased susceptibility of these patients to IAV infection and consequent exacerbations are largely unknown. Current prevention options are primarily vaccinations that have serious limitations including long manufacturing processes, vaccine mismatches and inability to target all virus strains that also undergo frequent mutations and recombination. Current anti-IAV treatments are only effective if given within the first 48 h of infection. This is normally before symptoms appear and are severe enough to present to a clinic or hospital. Thus, there is an urgent need to develop novel effective therapies for the management of IAV infections, particularly in patients with underlying respiratory diseases. The primary objective of this PhD was to determine the mechanisms responsible for increased susceptibility to IAV infection in asthma and COPD, and understand how infection causes exacerbations. To accomplish the aims of the study, we used established and developed novel mouse models of IAV infection in experimental asthma (Ovalbumin (Ova)- and house dust mite (HDM)-induced) and COPD. Firstly, we found that Ova- and HDM-induced allergic airways disease (AAD) increased susceptibility to IAV infection as evident by increased viral titre. In addition, the infection exacerbated AAD with severe histopathology, increased eosinophils, mucus hypersecretion, impaired antiviral responses, increased collagen deposition around small airways, small airways epithelial thickening and airway hyper-responsiveness (AHR). Also, an allergen rechallenge in infected mice with AAD demonstrated an ongoing remodelling characterised by excessive mucus and collagen production that are resistant to dexamethasone treatment. Secondly, to confirm whether production of IL-13, an important Th2 cytokine, is a likely mechanism driving infection and exacerbating disease, we assessed the levels of IL-13 and its receptor, IL-13Rα1, in Ova-induced AAD lung tissues. Non-infected allergic mice had AAD features that were associated with increased protein levels of IL-13 compared to non-allergic controls. IAV infection did not induce IL-13 in non-AAD mice, but further increased IL-13 levels in AAD. IHC determined the localisation of IL-13Rα1 in lung tissues and showed its increased expression in airway epithelium. We then used IL-13-reporter mice to show that AAD with or without IAV infection induced IL-13 production predominantly from IL-13+CD4+ T-cells and to a lesser extent from IL-13+ ILC2 cells but IAV infection alone induced high level of IL-13 production by IL-13+ NKT cells. Treatment of IAV infected mice with recombinant (r)IL-13 induced severe histopathology, increased eosinophils, mucus hypersecretion and AHR. Further, we identified the involvement of microRNA (miR)-21 in IL-13/IL-13Rα1-induced susceptibility to IAV infection in experimental AAD. We found significantly increased miR-21 expression in allergic mice, which was further increased after IAV infection. ISH determined an increased localisation of miR-21 expression to the airway epithelium. Also, rIL-13 treatment showed increased miR-21 expression which was further increased after IAV infection. Similar observations were found in HDM-induced AAD. To further assess the role of IL-13 in susceptibility to IAV in AAD, we used mice deficient in IL-13 (IL-13-/- mice). A significant decrease in viral titre, lung tissue inflammation and transpulmonary resistance was observed in infected IL-13-/- compared to infected WT mice. Infected IL-13-/- mice also demonstrated reduced miR-21 expression compared with infected WT mice. These observations clearly indicate that the absence of IL-13 during IAV infection improved disease features. Using IL-13-specific monoclonal antibody (Anti-IL-13), we showed that inhibition of IL-13 signalling protected mice against infection and reduced the severity of IAV infection-induced exacerbations of AAD. This was shown by reduced viral titres, tissue inflammation, eosinophil counts, mucus secreting cells (MSCs) and transpulmonary resistance, IL-13 and IL-13Rα1 mRNA expression in Anti-IL-13-treated groups compared to isotype-treated controls. Further, anti-IL13-treated infected non-allergic mice also had significantly reduced miR-21 expression compared to controls, whilst similar levels of expression were observed in both anti-IL-13-treated and isotype-treated infected mice with AAD. We also assessed the effects of corticosteroid (dexamethasone) on IAV infection and exacerbations of AAD. We found that the disease features in Ova-induced AAD mice with IAV infection were steroid-insensitive. miR-21 may promote PI3K activity and we found that anti-miR-21 (Ant-21) significantly reduced viral titres, tissue inflammation, numbers of MSCs and tissue eosinophils in AAD with IAV infection. However, Ant-21 treatment did not suppress AHR in Ova-induced AAD with IAV infection. PI3K inhibition using a pan-inhibitor (LY294002) had similar effects and reduced viral titres and features of asthma. The association of IL-13 with COPD is not well understood. We demonstrated that IAV infection in experimental COPD increased the expression of IL-13Rα1 in the airway epithelium of COPD mice compared to non-infected normal air-exposed mice. Surprisingly, infected normal air-exposed mice and infected mice with had further substantial increases in IL-13Rα1 levels in the airway epithelium. To further identify the mechanisms involved downstream of IL-13Rα1, miR-21 expression was measured in the lung sections and observed to be increased in response to IAV infection in experimental COPD. We also observed a significant increase in the numbers of MSCs in infected mice with COPD compared to COPD alone. We found that inhibiting IL-13 using Anti-IL-13 reduced viral replication and severity of IAV infection in experimental COPD. This reduction was accompanied by significantly reduced tissue inflammation, numbers of MSCs and miR-21 expression in infected mice with COPD. We next demonstrated that treatment with Ant-21 protected mice against IAV infection in experimental COPD as assessed by significant reductions in viral titre, tissue inflammation, numbers of MSCs and miR-21 expression. Thirdly, we demonstrated that targeting miR125 a and b with specific antagomirs reduced IAV-mediated inflammation and reversed immune signalling abnormalities in COPD. We also observed that COPD pBECs and mice with experimental COPD infected with IAV had higher levels of inflammatory cytokines, reduced antiviral responses and increased levels of miR-125 a and b. The study discovered that the mechanism underlying excessive inflammation and increased susceptibility to IAV infection in COPD was facilitated by a miR-125-mediated pathway that reduced A20 (negative regulator of NF-κB) and MAVS proteins. In conclusion, this is the first study to define the functional relevance of IAV infection-induced activation and maintenance of novel IL-13/IL-13Rα1/miR-21 and IL-13Rα1/miR-21 axes in AAD and COPD, respectively. Our data identified promising therapeutic interventions for increased susceptibility to IAV infection in AAD (anti-IL-13, Ant-21 and PI3K inhibitors) and COPD (anti-IL-13, Ant-21, Ant-125 a and b) which supressed various key features of the respective airway disease. While we show promising findings experimentally, we have yet to explore some of these findings in clinical settings. This would be the next critical step to translate these promising findings from bench to bedside. The models used and developed in this study have clear utility in identifying new and effective therapeutic interventions and are well suited for in vivo studies investigating modulation of immune responses in exacerbation-induced expression of various cytokines and corticosteroid insensitivity.
- Subject
- influenza; asthma; chronic obstructive pulmonary disease (COPD); microRNA; interleukin-13
- Identifier
- http://hdl.handle.net/1959.13/1385094
- Identifier
- uon:32170
- Rights
- Copyright 2018 Kamal Dua
- Language
- eng
- Full Text
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