CF mouse model

The cause of cystic fibrosis may be simply explained by a number of mutations in the gene for CFTR leading to ion channel dysfunction. As detailed previously, this leads to depletion of ASL volume, impaired mucus clearance, and bacterial infection. Our understanding of the disease would predict that the simple deletion of CFTR would produce similar effects in mice, but this is not the case. Unlike in humans, the removal of CFTR activity does not lead to spontaneous lung disease, even in older mice raised

outside the sterile environments commonly used in animal facilities. This is

found consistently in CFTR-/- models and may be due to the fact that CFTR is

not expressed in the lower airways of mice. Consequently, its removal will not have major pathological effects on the peripheral lung. In contrast, CFTR

is common in the murine gastrointestinal tract and the CFTR-/- model has

been found to closely mimic the intestinal pathophysiology of human patients (7).

Defective Cl- secretion is not the only ionic characteristic of CF. The lung

epithelium (and indeed other organ systems) is characterised by increased

Na+ absorption which, in combination with chloride dysregulation, leads to

depletion of water on the airway surface (250). To this end, an alternative

mouse model has been developed that is characterised by Na+

hyperabsorption as opposed to reduced Cl- secretion. It is based on the “low-

volume” hypothesis i.e. that CFTR also regulates the epithelial sodium channel (ENaC), with dysfunction of the former leading to reduced NaCl concentrations in the ASL and a resulting reduction in osmotic force for water transport to the lumen. This is in contrast to the alternative “high-salt”

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NaCl concentrations in the ASL and the inhibition of HDP activity, exacerbating bacterial infection. While evidence exists to support both hypotheses, the fact that ASL volume has been demonstrated to be lower in CF supports the “low-volume” hypothesis. Figure 6.1 demonstrates how

defective Cl- and Na+ transport reduce the height of the ASL and impair

mucociliary clearance. If the “high-salt” hypothesis was correct, the increased NaCl concentrations would induce an accompanying movement of water across the lumen and increase ASL depth. This increase in ASL depth, as seen in the disease pseudohypoaldosteronism, would increase mucociliary clearance, in contrast to the situation in CF, and one would expect it would act as an increased barrier to bacterial adherence rather than facilitate it (251). The “low-volume” hypothesis also predicts that increasing the salt concentration of the ASL would induce water entry into the lumen, increase the ASL depth, and improve clearance. Supporting this is the fact that hypertonic saline has been repeatedly shown to improve mucociliary clearance and lung function in CF (73).

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Figure 6.1: A demonstration of the effects of CFTR and ENaC on

mucociliary clearance (MCC) based on the “low-volume” hypothesis. Under normal conditions the balance of secretory and absorptive processes

ensures that the ASL is an appropriate height (~7µm), compatible with

effective MCC. In CF, defective Cl- clearance and inappropriate Na+

absorption leads to a dehydrated, compressed ASL (~3-4µm) that collapses

the cilia, prevents MCC, and promotes bacterial adherence. Taken from (251).

Mice have been generated that overexpress the β-ENaC subunit (encoded

by the Scnn1b gene) which results in increased Na+ absorption, reduced

airway surface volume, mucus obstruction of the lungs, neutrophilic inflammation, and, crucially, reduced clearance of a bacterial challenge compared to wild-type mice. The pulmonary disease phenotype of these mice is similar to the human disease (252). These have also been crossed

with a murine NE (mNE)-/- mouse line (48) to examine the effects of mNE in a

CF phenotype. The genetic deletion of mNE has been demonstrated in one study to reduce neutrophil recruitment. In addition, large quantities of mNE

were found to be bound to the membranes of β-ENaC neutrophils, although

free mNE was not detected in BAL fluid, potentially due to inhibition by a robust anti-protease defence. The results suggest that membrane-bound NE plays a large role in tissue degeneration in the mouse model (253). A similar observation has been made with MMP-12 levels in these mice (70). The lack of free enzyme has the potential to pose an issue for testing NE-activated

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pro-HDPs, but in mice from the same background it has been shown in some studies active NE is expressed in BAL fluid after nasal instillation of P.

aeruginosa or LPS (254). Overall, there are mixed reports on whether P. aeruginosa infection in these mice can induce substantial free NE activity (255, 256).

Other CF in vivo models have been developed, using different species whose lung pathology more closely resembles human CF in many respects. CF is the first human disease to have two non-rodent knock-out models, pigs

and ferrets. Unlike in CFTR-/- mice, the lungs of both CFTR-/- and

CFTR∆F508/∆F508 pigs are characterised by lung infection, inflammation, and

remodelling in the first months of life (257). The similar lung pathology allows important observations to be made that are precluded in CF patients, e.g. whether inflammation precedes infection, with the pig model demonstrating no significant inflammation a few hours after birth. A barrier to using this model however, beyond the obvious increased costs with raising enough

pigs for statistically significant data, is that 100% of CFTR-/- pigs require early

intestinal surgery for meconium ileus, i.e. intestinal blockage, comparable to the surgery to clear intestinal blockages that is sometimes required in CF patients (258).

Ferret CF models have also been developed. They are an attractive species for modelling lung disease because they reproduce relatively quickly and share many features of lung biology with humans, such as a similar

distribution of submucosal glands. It has been shown that CFTR-/- ferrets are

extremely susceptible to lung infection early in life. This model, however, is also complicated by the fact that ferrets are obligate carnivores (unlike mice and pigs) which requires special considerations for their nutritional needs. Intestinal pathology is again an issue, with 75% having meconium ileus. This pathology of the ferret model is severe, resembling human CF in many respects (259). But while their susceptibility to infection and gastrointestinal pathology makes them attractive for establishing the link between CFTR and disease, it also means many CF ferrets die before reaching maturity (260). In addition, the necessity of antibiotic treatment from birth complicates

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Considering all the options, the β-ENaC mouse model was the most

attractive in terms of breeding speed, precedents set with other HDP

treatments, parallels with human pathology, and economic considerations for the study.

As the activity of pro-WMR is enzyme-dependent, an important consideration for the study was the potential differences between human NE and mNE, which could preclude the use of a mouse CF model. A study examining some of the differences in substrate affinity demonstrated that mNE had a

higher Kcat/Km, i.e. catalytic efficiency, for the substrate Suc-AAA-pNa, nearly

twice that of the human enzyme. From this, one would expect the AAAG linker of pro-WMR to be at least as labile to the mouse enzyme as to the human (168). Human NE and mNE share 69% sequence homology, in comparison to the 50% similarity between human NE and PR-3. However, there are subtle differences between the binding sites of both NE enzymes, such as the ability of mNE to bind acidic residues at P2’ (261). To ensure that any antibacterial activity was due to the active peptide and not the intact pro-HDP, the lability to purified mNE would need to be quantified.

While generation of a respirable aerosol of pro-WMR was demonstrated in Results Chapter Three, the differences in breathing parameters between mice and humans means that delivery to the mouse lung via nebuliser is not used here. Discounting the differences in lung morphology which are a caveat when using in vivo models (262), the difference in breathing

frequency is an order of magnitude greater. Previously, 18 breaths/min has been used as a parameter of human CF in model lungs (240) while the

measured frequency of β-ENaC mice, though reduced from wildtype, is 172

breaths/min (263). Most rodents are primarily nose-breathers and, in comparison to humans, lung deposition is lower (dogs and primates have closer deposition) (264). These differences could potentially compromise the delivery of HDPs via nebulisation and, as a result, intratracheal instillation was used as an alternative delivery method. This method is commonly used for delivering HDPs to rodent lungs (248, 265).

167 6.2 Results

6.2.1 Murine NE cleaves pro-WMR but is less active than the human