Pseudomonas infection and mucociliary and absorptive clearance in the cystic fibrosis lung

Discussion

ASL dehydration is a key defect of cystic fibrosis lung disease that is directly linked to CFTR dysfunction. Dehydrated ASL is thought to cause defective MCC, linking CFTR dysfunction to the opportunistic bacterial infections often associated with cystic fibrosis lung disease. The key finding of our study is that defective MCC is present only in cystic fibrosis subjects with a previous history of P. aeruginosa infection. Noncolonised subjects demonstrated MCC rates similar to healthy controls. In addition, cystic fibrosis subjects were found to have consistently increased ABS, which may be associated with ASL dehydration. Our studies also indicated potential confounding effects for both MCC and ABS measurements with high central lung aerosol deposition.

Our first goal was to determine the repeatability of the ABS and MCC measurements. Using values for the standard deviation of the intrasubject measurement differences for subjects with similar pulmonary function on both testing days (ABS σ=5.2 and MCC σ=7.7) we can consider power. For comparison purposes, the inhalation of 7% hypertonic saline caused a 9.8%·80 min⁻¹ decrease in ABS and an 18.8%·80 min⁻¹ increase in MCC versus the inhalation of isotonic saline [12]. For a crossover study using parametric statistics to estimate power, a group size of 11 is needed to demonstrate a 5%·80 min⁻¹ change in ABS through a paired t-test, assuming that σ=5.2, Δ=5, α=0.05 and β=0.2. Group sizes of seven and 21 are needed to demonstrate 10%·80 min⁻¹ and 5%·80 min⁻¹ changes in MCC, respectively, assuming σ=7.7, Δ=10, α=0.05 and β=0.2 [15]. These group sizes are very attractive and feasible for proof-of-concept studies with ASL-modulating drugs.

Aerosol deposition pattern has been previously shown to affect MCC measurements [9], and most current techniques use controlled breathing to promote consistency between studies and deliver aerosols primarily to the large airways [8]. However, obstructive lung disease may cause extremely high rates of central lung aerosol deposition in some subjects [16]. Excessive deposition in the highly ciliated proximal airways will result in very high MCC measurements that make accurate measurement of ABS impossible as the In-DTPA probe clears through MCC too rapidly to absorb. Here, in order to determine the specific patient and disease factors affecting baseline MCC/ABS, we utilised an analysis group with a central deposition percentage <50%. This is not intended to be a general limit to be applied to other studies. In particular, crossover studies involving only MCC measurement, where deposition is well matched between study days, may allow for the evaluation of therapeutic effect even with high central deposition percentages.

We generated a combined baseline dataset of MCC/ABS measurements that included measurements from several recent and previous studies [12]. Previously reported trends were confirmed in this larger group. Specifically, baseline ABS is significantly increased in cystic fibrosis subjects versus healthy controls and baseline MCC is not detectably lower in cystic fibrosis. When analysis was restricted to subjects with optimal deposition (central deposition <50%, 37 out of 51 measurements), MCC differences between cystic fibrosis and control subjects approached significance and the significance of ABS differences increased.

The effect of P. aeruginosa colonisation on MCC was previously considered by Laube et al. [14]. In that study, MCC with included cough clearance was depressed in children with P. aeruginosa colonisation when compared to those without. Controversy has existed about whether cystic fibrosis subjects demonstrate baseline deficits in 60–90 min measurements of MCC, with some studies demonstrating no differences versus healthy controls [6, 12] and others demonstrating clear deficits [17, 18]. These differences are probably related to P. aeruginosa status, since the latter group of studies enrolled only PA⁺ subjects. (This was also the case for several other studies utilising MCC to evaluate therapeutics [7, 19].) One of these studies did identify two PA⁻ subjects as having normal clearance, but did not include them in the final analysis [18]. Some studies have noted an MCC defect in cystic fibrosis using techniques designed to assess peripheral lung MCC, such as 24-h measurements [6]. Shorter measurement periods, including those used here, are dominated by large airway clearance. Our results indicate that PA⁻ cystic fibrosis subjects have MCC rates very similar to healthy controls while PA⁺ cystic fibrosis subjects have MCC rates that are significantly decreased versus controls. MCC differences between the PA⁺ and PA⁻ cystic fibrosis groups were not statistically significant, but trends indicated a likely association between MCC depression and recent P. aeruginosa infection.

MCC may be depressed by P. aeruginosa through multiple mechanisms. Ciliary beat frequency can be reduced after exposure to P. aeruginosa [20, 21], potentially mediated by virulence factors such as pyocyanin [22] and rhamnolipids [23]. Previous studies have demonstrated that mucus production, composition and rheology could be affected by P. aeruginosa [24], and pyocyanin in particular is associated with goblet cell hyperplasia and mucus hypersecretion [25]. As a host response to pathogens in healthy airways, CFTR facilitates ASL secretion, increasing the transportability of mucus. The failure of this mechanism in cystic fibrosis, compounded by infection-driven increases in mucus production, could defeat otherwise normal rates of MCC in the cystic fibrosis lung. Acute impairments of clearance may extend beyond P. aeruginosa. We can hypothesise that other bacterial or viral infections may depress MCC in cystic fibrosis leading to vulnerability to secondary infections, including P. aeruginosa or other cystic fibrosis pathogens. In support of this, previous studies have associated initial P. aeruginosa infection with respiratory syncytial virus and other upper respiratory infections [26, 27]. However, the cause-and-effect relationship between P. aeruginosa and depressed clearance cannot be clearly determined from our data. Depressed MCC could be the direct cause of P. aeruginosa infection, in line with classic descriptions of cystic fibrosis lung pathophysiology. Loss of this host defence mechanism would certainly make subjects more vulnerable to infection. Another possibility is that airway conditions, such as mucus plugging and associated local hypoxia, could independently predispose subjects to both depressed MCC and P. aeruginosa infection.

In longitudinal studies of MCC/ABS in paediatric subjects, there was no indication that ABS or MCC predicted clinical outcomes over a 2-year interval. Infection status and age may have confounded our ability to determine these relationships. The role of P. aeruginosa infection has already been described and trends within the paediatric group matched those of the larger analysis group in this regard. Ultimately the small sample size and the use of only a single follow-up measurement probably decreased our ability to detect any subtle effects with these measurements.

Many of the limitations of the current studies are associated with the inherent variability of the aerosol-based techniques applied. We have confirmed the previously described potential for confounding based on changes in aerosol deposition pattern [9]. In addition, our techniques rely on two-dimensional planar imaging of an inherently complex three-dimensional organ that contains large and small airways and alveoli. Our aerosol delivery techniques are intended to target deposition primarily to large airways, but some alveolar contribution to outcomes is likely. Limitations to consider with regard to our analysis of baseline relationships between MCC/ABS and disease state include the use of multiple measurements from some subjects. Our primary analysis group included 37 measurements from 24 subjects obtained through several different protocols that utilised identical techniques. The time between measurements varied from a minimum of ∼30 days to ≥2 years. Since each of these studies was associated with a unique set of variables describing patient condition and ABS/MCC, we opted to utilise all measurements. The study was also limited by having only a small group of control subjects for comparison.

In summary, measurements of MCC and ABS demonstrated good repeatability. The primary experimental factor affecting the measurements is the extent of central deposition. Excluding subjects with high central deposition appeared to increase the statistical significance of observed trends. The primary patient factor associated with depressed MCC is P. aeruginosa infection. PA⁻ cystic fibrosis subjects had MCC rates similar to healthy controls while PA⁺ cystic fibrosis subjects had depressed MCC, apparently more so with recent infection. Neither ABS nor MCC predicted the clinical course of disease in a small number of paediatric cystic fibrosis subjects though a 2-year follow-up. Multiple longitudinal measurements may provide more comprehensive information on disease evolution and the factors leading to initial P. aeruginosa colonisation. We hypothesise that defects in ASL secretion in the cystic fibrosis airway result in clearance that is not depressed at baseline, but rather is vulnerable to depression by factors such as infection that in turn might lead to cycles of secondary infection and disease progression.