The ΔF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CFTR(ΔF508/ΔF508) pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTR(ΔF508/ΔF508) airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3',5'-monophosphate (cAMP) agonists were less potent at stimulating current in CFTR(Δ)(F508/)(Δ)(F508) epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTR(ΔF508/ΔF508) pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

Figure 1. Pathology of newborn CFTR^{ΔF508/ΔF508} pigs

A. Location (in cm) of meconium ileus obstruction in CFTR^{ΔF508/ΔF508} (n=10) and CFTR^−/− (n=9) pigs. B. CFTR^{ΔF508/ΔF508}ileum distal to the obstruction had a small caliber and was heterogeneously filled with mucocellular debris (arrows). Ileal Peyer’s patches (asterisks) appeared similar in CFTR^+/+ and CFTR^{ΔF508/ΔF508} pigs. Bars=725 top and 145 μm bottom. C. Pancreas from CFTR^{ΔF508/ΔF508} pigs had increased connective tissue (asterisks) and destruction compared to CFTR^+/+. Histopathological changes in CFTR^{ΔF508/ΔF508} pancreas were slightly less severe than in CFTR^−/−. HE stain. Bar=457 μm. D. Lobular parenchyma in CFTR^{ΔF508/ΔF508} pigs (n=17) was reduced compared to CFTR^+/+ (n=9, * P<0.001) and greater than in CFTR^−/− (n=19, # P<0.05, Dunn’s post-test). Data from CFTR^+/+ and CFTR^−/− pigs were previously published (24). E. Liver from newborn CFTR^{ΔF508/ΔF508}and CFTR^−/− pigs showed portal areas that were focally expanded (arrows) by inflammation, duct proliferation and connective tissue. HE stain. Bar=46 μm. F. CFTR^{ΔF508/ΔF508} pigs had microgallbladder variably filled by mucus and bile. HE stain, bars=928 μm (+/+) and 463 μm (ΔF508/ΔF508). G. Lung from newborn CFTR^{ΔF508/ΔF508} pigs lacked mucus accumulation or inflammatory changes. HE stain. Bar=93 μm. H–J. Bronchoalveolar lavage liquid analyses from newborn pigs, including total cell counts (H), neutrophil percentages (I), and IL-8 concentrations (J) revealed no statistically significant differences between genotypes. CFTR^+/+ (n=5) combined with CFTR^+/ΔF508 (n=4); CFTR^{ΔF508/ΔF508} (n=11).

Figure 2. Morphometry of newborn CFTR^{ΔF508/ΔF508} trachea

A. Cross section of trachea. MT stain. Bars=1 mm. Images from CFTR^+/+ and CFTR^−/− are from reference (29). B–E. Tracheal morphometry in CFTR^+/+ (n=20), CFTR^{ΔF508/ΔF508} (n=19), and CFTR^−/− (n=18) newborn pigs. * indicates different from CFTR^+/+ and # indicates different from CFTR^−/− (* P<0.05 vs. CFTR^+/+, and # P<0.05 vs. CFTR^−/−, 1-way ANOVA with Bonferroni’s post test). B. Tracheal lumen cross-sectional area. C. Tracheal circumference. D. Submucosal gland area normalized to tracheal lumen circumference. E. Smooth muscle area normalized to tracheal lumen area.

Figure 3. Disease progression in pigs ~2-weeks of age and older

A. Pancreas from a 77-day old CFTR^{ΔF508/ΔF508} pig and 69-day old CFTR^+/+ pig for comparison. Islands of degenerative, fibrotic and inflamed CFTR^{ΔF508/ΔF508} pancreas were surrounded by abundant adipose tissue (asterisk). HE stain. Bar=75 μm. B–D. Porcine liver. MT stain. Bars=570 top and 57 μm bottom. B. Liver from a 136-day old CFTR^+/+ pig. C. Diffuse zone 1 steatosis (black arrows) in a 77-day old CFTR^{ΔF508/ΔF508} pig. D. A 62-day old CFTR^{ΔF508/ΔF508} pig had focal to bridging expansion (black arrows) of triads by fibrosis, duct proliferation and inflammation. E–M. Histopathological evaluation of CFTR^{ΔF508/ΔF508} lungs. E–H are HE stain and I–M are PAS stain. E. Lung from a 69-day old CFTR^+/+ pig; changes like those in panels F–H were not observed in wild-type pigs. F–H. Lung from 13-day old CFTR^{ΔF508/ΔF508} pig. F. Lungs showed mucopurulent airway obstruction (arrow) and adjacent atelectasis (asterisks). Bar=757 μm. G. Affected airway lumens often contained a heterogeneous mixture of mucopurulent debris obstructing the airway (arrows) and adjacent atelectasis (asterisks). Bar = 378 μm. H. Airways sometimes showed nominal inflammatory changes in the wall (asterisks) adjacent to luminal neutrophils (arrows) suggesting the dispersion of the luminal mucocellular debris from more severely affected airways. Bar=38 μm. I–J. Lung from 87-day old CFTR^{ΔF508/ΔF508} pig. I. Some airways showed focal airway mucus obstruction (arrow). Bar=162 μm. J. The surface epithelium showed focal goblet cell hyperplasia (black arrows) and inflammation in the airway wall around submucosal glands (arrowheads). Bar=81 μm. Insets: magnified PAS-stained images of airway epithelia of 4.5-mo CFTR^+/+ (top) and 87-day CFTR^{ΔF508/ΔF508} (bottom) pigs. K–L. Lung from 62-day old CFTR^{ΔF508/ΔF508} pig. K. Lungs showed mucopurulent inflammation associated with focal dilated submucosal glands and ducts (arrow). Bar=40 μm. L. Airway lumens showed mucopurulent material in lumen (arrow) with epithelial proliferation and wall inflammation. Bar=40 μm. M. Lung from 77-day old pig showed lesions included complete lobular atelectasis (arrows), although in this image airway obstruction was not present. Bar=378 μm.

Figure 4. mRNA and protein expression in intestine and airway

A. Quantitative RT-PCR of CFTR mRNA in CFTR^{ΔF508/ΔF508} and CFTR^+/+ pigs. Data are from triplicate assays repeated on multiple days. For each tissue, amounts of CFTR mRNA were normalized to β-actin mRNA. These normalized values were then expressed relative to that in wild-type duodenum. Data are mean±SE from intestinal tissues from 6 CFTR^+/+ and 6 CFTR^{ΔF508/ΔF508}piglets, and from cultured nasal epithelia from 1 CFTR^+/+ piglet (n=3) and 1 CFTR^{ΔF508/ΔF508}piglet (n=4). B. Northern blot analysis of duodenal CFTR mRNA, indicated by arrow. C. Immunoprecipitated and in vitro phosphorylated CFTR isolated from intestine. “rec” (lanes 1,8,13) indicates recombinant protein. Lanes 2–7, proximal intestine. CFTR^+/+ and CFTR^−/− 500 μg and CFTR^{ΔF508/ΔF508} 750 μg. Lanes marked with * show enhanced exposure. Lanes 6 and 7 are same as lanes 4 and 5. Lanes 9–12, distal intestine. CFTR^+/+ 200 μg and CFTR^{ΔF508/ΔF508} 1000 μg. Lanes 11 and 12 are same as 9 and 10. D. Immunoprecipitated and in vitro phosphorylated CFTR isolated from airway epithelia. Recombinant protein, lanes 1,6,7. Lanes 2–5, trachea; CFTR^+/+ 623 μg and CFTR^{ΔF508/ΔF508} 1208 μg. Lanes 4 and 5 are same as lanes 2 and 3. Lanes 8–10, cultured bronchial epithelia; each lane 750 μg.

Figure 5

Immunocytochemical localization of CFTR in intestinal and airway epithelia of newborn pigs. Data are stacks of confocal images, except as noted. Scale bars=10 μm. A. Sections of intestine from newborn pigs. Third panel (asterisk) shows an electronically enhanced image of second panel. CFTR is green, ZO-1 is red, and nuclei are blue. Nonspecific staining was occasionally found in lumen of some CFTR^{ΔF508/ΔF508}and CFTR^−/− crypts in areas of extensive mucus. B. Sections of trachea. C. Images of cultured airway epithelia. Top panels are enface images, and bottom panels are single vertical sections. Images from CFTR^ΔF508ΔF508 epithelia are electronically enhanced (*) to show CFTR. Cell size heterogeneity was observed with all genotypes.

Figure 6

Electrophysiological properties of freshly excised porcine tracheal epithelia. Data are from CFTR^+/+(23 tissues, 23 pigs), CFTR^{ΔF508/ΔF508} (19 tissues, 17 pigs), and CFTR^−/−(16 tissues, 14 pigs) epithelia. Data from CFTR^−/− and most CFTR ^+/+ pigs were previously reported(31). * indicates CFTR^{ΔF508/ΔF508} differs from CFTR^+/+, # indicates CFTR^−/− differs from CFTR ^+/+, and † indicates CFTR^{ΔF508/ΔF508} differs from CFTR^−/−, all at P<0.017 by unpaired t test with Welch’s correction. A). Transepithelial voltage (Vt) and response to sequential apical addition of 100 μM amiloride, 100 μM DIDS, 10 μM forskolin and 100 μM IBMX, and 100 μM GlyH-101. B). ΔVt_amil indicates change in Vt with addition of amiloride. C). ΔVt_cAMP indicates change in Vt with addition of forskolin and IBMX. D). ΔVt_GlyH indicates change in Vt with addition of GlyH-101. E–H). Short-circuit current (Isc) measurements corresponding to Vt measurements in panels A–D. I–L). Transepithelial conductance (Gt) measurements corresponding to Vt measurements in panels A–D. Changes in Vt, Isc, and Gt with DIDS were small and did not differ by genotype.

Figure 7. HCO₃⁻ transport, apical Cl⁻ currents, and effect of increasing cAMP-dependent stimulation

A–B. Changes in Isc and Gt in tracheal epithelia bathed in Cl⁻-free solution containing 25 mM HCO₃⁻. Change in Isc (ΔIsc) (A) and Gt (ΔGt) (B). stimulated by forskolin (10 μM) and IBMX (100 μM) (F&I) and inhibited by GlyH-101 (100 μM, apical). * indicates P < 0.05, unpaired t- test. N=7 CFTR^+/+and 7 CFTR^{ΔF508/ΔF508}. C–E Changes in Cl⁻ current after permeabilization of basolateral membrane. N=7 CFTR^+/+and 7 CFTR^{ΔF508/ΔF508}. C). Current traces in response to indicated agents in CFTR^+/+, CFTR^{ΔF508/ΔF508}, and CFTR^−/− epithelia. Concentrations are those indicated in Fig. 6 legend; nystatin was 0.36 mg/ml. D). Change in current in response to nystatin plus forskolin and IBMX (ΔI_Nystatin+F&I). E). Change in current in response to GlyH-101 (ΔI_GlyH). F). Examples of Isc current traces following addition of increasing forskolin and IBMX concentrations. For concentrations, see panel G. G). Changes in Isc with increasing forskolin and IBMX concentrations. N=7 CFTR^+/+and 6 CFTR^{ΔF508/ΔF508}. H). Examples of Isc current traces following addition of increasing 8-cpt-cAMP concentrations. For concentrations, see panel I. I). Changes in Isc with increasing 8-cpt-cAMP concentrations. N=6 CFTR^+/+and 7 CFTR^{ΔF508/ΔF508}.