In vivo examination of viral exacerbations of airways inflammation function mucus hypersecretion and mucociliary clearance

Lead Research Organisation: Cardiff University
Department Name: Welsh School of Pharmacy


Inhaled microbes are likely drivers of lung inflammation and function and understanding airway responses to microbial challenge will provide insights to identify novel drugs. In vivo modelling of the airway responses is therefore critical. Lipopolysaccharide (LPS) is a major component of gram negative bacterial membranes, whose inhalation mimics bacterial inhalation by interacting with epithelial toll-like receptors (TLR-4) resulting in induction of NF-kappaB and transcription of proinflammatory genes and cytokines. We showed that single LPS inhalations by guinea-pigs cause inflammatory cell influx to the lungs and immediate airways hyperreactivity (AHR). After repeated LPS exposure, there was persistent AHR and goblet cell hyperplasia. Parainfluenza3 virus (PIV3) inoculation alone caused airways inflammation and AHR but no goblet cell hyperplasia indicating differential responses to microbial challenges. We hypothesise that combining LPS and PIV3 will induce heightened or differential responses. Lung function, AHR, inflammatory cell influx and goblet cell histology in guinea-pigs will identify any modified microbial responses in vivo. Influenza virus (H1N1 strains WSN33 and PR8/34) alone and with acute or chronic LPS challenge will be examined as well as measles (wildtype and vaccine virus) (mWTFB, mEdmonston) and poly[I:C]. Lung histology after these treatments will determine gross histology (haematoxylin and eosin), lung remodelling from collagen staining (Gomori's stain) and goblet cell mucin (Alcian blue/periodic acid Schiff stain). TLR-4 are implicated in innate immune responses to infection and inflammation after LPS, but not inflammation from influenza infection. Invasion of airways epithelial cells by viruses like influenza, release proinflammatory and antiviral products including IL-8, IL-6 and IFN-beta and we will measure NF-kappaB levels as an index of TLR-4 receptor activity and BAL fluid IL-8 as a marker of viral infection. These assays will depend upon sufficient cross-reactivity between reagents for guinea-pig and human. Studies will determine whether exacerbations are associated with changes in viral load using culture of live virus from lung tissue and lavage fluid, supported by real time PCR to detect viral nucleic acids. Corticosteroid use in viral inflammatory exacerbations is counter-intuitive since they should reduce immune responses and worsen viral infection. However, steroids have the advantage of being anti-inflammatory. The effects of injected dexamethasone and inhaled budesonide will be examined against functional and histological responses to LPS/virus and against viral load to assess any pro- or antiviral effects. Do steroids affect viral entry to epithelial cells and subsequent replication, and if so, what is the mechanism? A non-steroidal anti-inflammatory (NSAI) COX inhibitor, indomethacin, on viral infection and inflammation will also be examined. Mucociliary clearance will be examined from airway function and gamma scintigraphy. Inhaled mucus secretagogues (UTP, histamine) cause prolonged reduction in airways conductance, recovery of which will be an index of mucociliary clearance. Does UTP affect mucociliary clearance as well as mucus secretion? Drug interventions will be examined after inducing mucus secretion. Gamma scintigraphy will monitor clearance of radiolabelled 99mTc-Sn colloid particles instilled into anaesthetized guinea-pig trachea. Mucociliary clearance will be measured following UTP inhalation in guinea-pigs receiving LPS alone and superimposed virus. At Novartis, primary cultures of human and guinea-pig epithelial cells will enable the student to examine in vitro viral effects on cytokine production to relate to in vivo studies. Do prior exposure to IL-13, bacterial products (LPS) or oxidant stressors alter viral responses? Repeated LPS exposure desensitizes toll-like receptors. Steroids and NSAIs will be examined on viral entry into cells and subsequent replication.


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