Characterisation of the Host Immune Response in Cystic Fibrosis Mice

Palmer, James (2007). Characterisation of the Host Immune Response in Cystic Fibrosis Mice PhD Thesis, Institute for Molecular Bioscience , University of Queensland.

       
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Author Palmer, James
Thesis Title Characterisation of the Host Immune Response in Cystic Fibrosis Mice
School, Centre or Institute Institute for Molecular Bioscience
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Professor Brandon Wainwright
Abstract/Summary Lung disease is the major cause of mortality in cystic fibrosis patients. Lack of functional cystic fibrosis conductance transmembrane conductance regulator (CFTR) in airway epithelia alters the environment of the lung, predisposing patients to chronic infections predominated by the opportunistic pathogen Pseudomonas aeruginosa. Such infections cause an exaggerated host inflammatory response characterised by a sustained neutrophilic influx which ultimately destroys the lung tissue resulting in respiratory failure. In addition to the inability for CF patients to effectively clear airway infections evidence exists suggesting an innate defect in inflammatory signalling pathways due to a lack of CFTR, priming the CF lung for a heightened state of inflammation even in the absence of detectable infection. In order to develop effective strategies for dealing with CF lung disease a greater understanding is required of the dysregulation in the molecular signalling pathways that leads to the uncontrolled state of inflammation in the CF lung. The G551D CF mouse previously generated in our laboratory exhibits characteristic CF phenotypes including altered chloride conduction in the airways, reduced ability for bacterial clearance and a dysregulated lung inflammatory response upon bacterial challenge. The work presented in this study further characterises the response in these animals upon stimulation with an intratracheal dose of bacterial lipopolysaccharide (LPS). This treatment regime was sufficient to initiate an acute inflammatory response in the lungs of both WT and CF mice, from which animals of both genotypes routinely recovered. G551D animals consistently lost a greater amount of weight than their WT counterparts and exhibited enhanced recruitment of inflammatory cells, predominantly neutrophils, to the lungs. To profile transcriptional responses occurring in the lung after exposure to inflammatory stimulus both CF and WT animals were treated with LPS and sacrificed six hours post-treatment. Six hours marks a point where no discernable difference existed between the two genotypes, as measured by cellular infiltrate and cytokine levels in the airways. cDNA and oligonucleotide microarrays were utilised to determine relative differences in transcriptional activity between CF and WT mice. A series of comparisons was performed to incorporate potential differences in gene expression levels between the genotypes in the absence of stimulus. A robust transcriptional response was detected in both WT and CF animals in response to LPS. The majority of genes were similarly regulated in both genotypes, leaving overall levels of transcript comparable after stimulus. Similarly, few differences were detected between CF and WT mice not exposed to LPS. Analysis of microarrays directly comparing transcript levels in the lungs of WT and CF mice exposed to LPS revealed a number of genes expressed at lower levels in the CF samples which shared the common feature of being known targets of interferon signalling. Independent validation of a series of molecules integral to interferon beta signalling confirmed genes downstream of interferon beta, namely Stat1, Irf7 and Tap2, were consistently expressed at lower levels in the CF lung compared to WT after exposure to LPS. While all three of these genes were transcriptionally upregulated by LPS in both genotypes the response was diminished in the CF lung. Whether or not this perturbation in signalling stems from a deficiency in production of interferon beta itself or is a result of impaired downstream events remains to be elucidated. Nevertheless, with increasing evidence promoting interferon beta as an important molecule in inflammatory regulation the data presented in this thesis suggests this as a worthy point of intervention in managing the persistent deleterious effects associated with CF lung disease.

 
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Created: Fri, 21 Nov 2008, 16:21:49 EST