Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets

Abstract

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

Figure 1.

Cystic fibrosis (CF) ferrets have impaired growth rates. (A) Representative pictures of CF and non-CF control ferrets between 15 days and 3 months of age. (B) Average weights of CF and non-CF control ferrets between birth and 3 months of age. Values past 5 days of age were significantly different between genotypes by two-way Student’s t test (P < 0.05). (C) Body mass index (BMI) of CF and non-CF control ferrets between birth and 3 months of age. *Values were significantly different between genotypes by two-way Student’s t test (P < 0.05). ^†The window in which pancreatic enzyme supplementation was initiated. The mechanism for the decrease in BMI of non-CF controls is unclear, although this does coincide with transition to nipple feeding and pancreatic enzyme supplementation.

Figure 2.

Early treatment of suspected lung infection is key to rearing older CF ferrets. The weights of three CF and non-CF matched pairs (pairs reared on the same jill) were taken every 6 hours. (A) Total weight gain profiles of CF (blue bars) and non-CF (red bars) animals for each pair. (B) The rolling average of weight gain over a 6-hour period was calculated by averaging five measurements over a 24-hour period, and is plotted for three CF (blue bars) and non-CF (red bars) pairs. A decline in this rolling average was indicative of an early lung infection (yellow-shaded regions), and antibiotics were instituted at the positions marked by an arrowhead. Graph of the absolute 6-hour weight gain were not as informative as the 6-hour rolling average in predicting this decline, due to greater fluctuations in weights. Several other features of clinical management were useful from the rolling average 6-hour weight gains. For example, gut obstruction (†) could easily be seen as a very rapid spike in the value. When this was observed, animals were gavaged with Golytely or the percentage was increased in Elecare gavages. This was typically followed by a significant decline in the following average weight gain as the obstruction was passed in the feces. In addition, fluctuations in weight gain observed in both CF and non-CF animals (*) were typically associated with jill performance and lactation. (C) The ratio of the rolling average 6-hour weight gain between non-CF and CF animals most clearly shows the decline in weight of CF kits associated with lung infection by referencing this weight change with a non-CF littermate.

Figure 3.

Gross abnormalities in the CF ferret lung. Lungs from three CF ferrets and one non-CF ferret ranging from 3 to 8 months of age are shown. (A–C) Mucus obstruction of airways in a CF animal. Inset in (A) shows mucus accumulation in the trachea, (B) shows air-trapping (arrows) in a lobe, and (C) shows mucus accumulation in an intralobar airway. (D and E) Airway mucus from this CF animal contained numerous neutrophils, bacterial colonies (E, arrow), and neutrophil extracellular traps. (F and G) A second example of a CF lung with (F) mucus accumulation in the trachea and (G) infection with hemorrhage (*) in various lobes demonstrating interstitial pneumonia. (H) A third example of a CF lung with hemorrhage and cranial bronchopneumonia (*). (I) Gross image of a control non-CF lung. Scale bars, 100 μm (D), 25 μm (E).

Figure 4.

Histopathology in the CF ferret lung. Lungs from four CF animals ranging from 3–8 months of age are shown. (A–C) Proximal airway mucus obstruction in a CF animal demonstrating complete occlusion (B) and partial occlusion (C) as compared with the non-CF control (A). Insets in (A) and (B) are higher-power images of the surface airway epithelium. (D and E) Distal airway occlusion in a CF (E) as compared with non-CF (D) animal. (F–G) Submucosal gland plugging with mucus (F and G) and expansion of bronchial-associated lymphoid tissue (G) in a proximal airway of a CF animal. (H and I) Distal airway occlusion in two different CF animals with inflammatory cell debris in the lumen. (J and K) Accumulation of inflammatory cells in the lumen of a distal airway (J) and submucosal glands (K) extending into alveoli from a CF animal. The four independent CF animals are grouped in panels as follows: (B, C, and E–G), (H), (I), (J and K). Images in (A–C) are periodic acid-Schiff stains and (D–K) are hematoxylin and eosin stains. Scale bars, 1 mm (A–C), 200 μm (H), 100 μm (D–G, J), 50 μm (I and K). *Air-trapping in CF lung (B).

Figure 5.

CF animals have impaired airway mucociliary clearance (MCC) and age-dependent increases in epithelial Na⁺ channel (ENaC) activity. (A) Time-lapse fluorescent photomicrographs of the tracheal MCC assay. The origin of fluorescent bead placement is marked by the arrows, and the distal and proximal ends of each tracheal segment are on the left and right of each photomicrograph, respectively. (B) Quantified MCC rates for seven CF and non-CF matched pairs at 3–8 months of age. *CF animal that was killed due to a rectal prolapse with more mild lung disease. ^†A pair in which the CF animal was found dead in the cage at roughly 3 hours postmortem; MCC on the non-CF animal in this pair was performed at 3 hours after killing to control postmortem influences on MCC. Differences between MCC rates between genotypes were determined using a paired two-way Student’s t test with P value given in the figure. (C) Fold difference (± SEM) in MCC rates between non-CF and CF animals (n = 7). (D) Ussing chamber short-circuit current analysis (I_SC) of tracheal tissue from CF and non-CF animals older than 3 months of age. I_SC was measured after the sequential addition of amiloride (Amil), 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDS), 1-methyl-3-isobutylxanthine/forskolin (IF), N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH101; GlyH), and bumetanide (Bumet). Quantification of the change in I_SC for each of the indicated drugs is shown (mean ± SEM from n = 7 animals of each genotype). At least two independent tissue samples were evaluated for each animal and the average ∆I_SC for each animal/condition used to calculate the SEM. Significant differences between genotypes by two-tailed Student’s t test are marked (**P < 0.005, *P < 0.05). On average, amiloride-sensitive I_SC was not significantly different between genotypes (P = 0.0654). However, there was a significant age-dependent increase in amiloride-sensitive currents in CF, but not in non-CF, animals (CF, P = 0.0009; non-CF, P = 0.7637 [by Spearman correlation]; see Figure E3). (E) Bacterial titers of lung homogenates from three non-CF and 11 CF animals. (F) Quantification of bacteria taxa found in lung homogenates from 10 CF animals using matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) fingerprinting. Only genera are shown; for complete genus and species, see Figure E4A.

Figure 6.

Overlap in bacteria found in the CF ferret lung and intestine. The types of bacteria observed in both the lung and intestine of seven CF animals were evaluated by MALDI-TOF MS and 16S sequencing. (A) Schematic representation of graphs for each of the seven animals. Bacteria found in the small intestine and colon are shown in the outer circle, whereas bacteria found in the lung lysates are shown in the inner circle. The animal identification number is in the center of the circles. (B–F) Results of bacteria identified in seven independent CF animals. *Bacteria found in both the intestinal and lung samples of the same animal; ^#bacteria found in both the lung and intestinal samples of at least two animals; ^†bacteria found in the lung and intestinal sample of only one of the seven CF ferrets. Each CF ferret had at least one unique bacterial strain found in both the lung and intestine.