Alejandro A. Pezzulo

Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn Pigs

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis (CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.

Reduced Airway Surface pH Impairs Bacterial Killing in the Porcine Cystic Fibrosis Lung

Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO3− transport. Without CFTR, airway epithelial HCO3− secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.

Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth.

The lungs of just-born piglets with cystic fibrosis fail to efficiently eliminate bacteria, suggesting that lung problems in cystic fibrosis patients may be secondary to impaired antibacterial defense mechanisms. A Matter of Life and Breath The CafePress and Zazzle Web sites and most yoga-wear boutiques sport an array of teeshirts, bumper stickers, and water bottles prepared to offer simple advice to those living a harried life: “Just breathe.” Not so simple for a cystic fibrosis (CF) patient. Very early on, physicians recognized that difficulty breathing was the most ominous of the mosaic of symptoms that characterize this syndrome. Indeed, lung disease is the main cause of death in cystic fibrosis patients, but the lack of an animal model that mirrors the CF lung pathology seen in people has slowed translational cystic fibrosis research. Now, Stoltz et al. report findings in cystic fibrosis pigs that survive long enough to develop human-like lung disease. At the heart of this recessive genetic disease is the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride-ion channel. CF-causing mutations in the CFTR gene give rise to an aberrant channel that is defective in its ability to transport ions and water across cell membranes, resulting in a dizzying array of defects in the pancreas, intestines, reproductive system, liver, and lungs. It has been hypothesized that the impaired channel causes cells that line body cavities and passageways to become coated with thick mucus. In such an environment, bacteria thrive, leading to the chronic infections characteristic of this disease. However, the precise mechanisms by which CFTR mutations manifest as the complex phenotypes that constitute CF remain unclear, particularly with respect to the inflamed and infected airways of the CF lung. Despite substantial research efforts, scientists have been unable to achieve two crucial goals,to mold an animal model that mimics human CF lung disease and to pinpoint the trigger of CF lung pathology in pristine airways. Stoltz et al. tackled both of these obstacles by producing genetically modified CF pigs and analyzing their airways from birth to 6 months of age. Their studies revealed a spontaneously arising human-like lung disease that developed over time and had the CF hallmarks: multibacterial infections, inflammation, and mucus buildup. Although the lungs of the newborn CF piglets were not yet inflamed, they were less likely to be sterile and less able to eliminate bacteria that had been introduced into their lungs, relative to wild-type animals. Together, these findings suggest that bacterial infiltration spurs the pattern of lung inflammation and pathogenesis associated with CF. Having a clearer conception of CF lung disease can help clinicians devise preventive treatments that can be initiated early in the lives of CF patients. Such interventions may let CF suffers live and breath more fully. Lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). Understanding the pathogenesis of this disease has been hindered, however, by the lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with mutated CFTR genes. We now report that, within months of birth, CF pigs spontaneously developed hallmark features of CF lung disease, including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting that the lungs of CF pigs have a host defense defect against a wide spectrum of bacteria. In humans, the temporal and causal relations between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation but were less often sterile than controls. Moreover, after introduction of bacteria into their lungs, pigs with CF failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Our finding that pigs with CF have a host defense defect against bacteria within hours of birth provides an opportunity to further investigate CF pathogenesis and to test therapeutic and preventive strategies that could be deployed before secondary consequences develop.

Loss of Anion Transport without Increased Sodium Absorption Characterizes Newborn Porcine Cystic Fibrosis Airway Epithelia

Defective transepithelial electrolyte transport is thought to initiate cystic fibrosis (CF) lung disease. Yet, how loss of CFTR affects electrolyte transport remains uncertain. CFTR⁻(/)⁻ pigs spontaneously develop lung disease resembling human CF. At birth, their airways exhibit a bacterial host defense defect, but are not inflamed. Therefore, we studied ion transport in newborn nasal and tracheal/bronchial epithelia in tissues, cultures, and in vivo. CFTR⁻(/)⁻ epithelia showed markedly reduced Cl⁻ and HCO₃⁻ transport. However, in contrast to a widely held view, lack of CFTR did not increase transepithelial Na(+) or liquid absorption or reduce periciliary liquid depth. Like human CF, CFTR⁻(/)⁻ pigs showed increased amiloride-sensitive voltage and current, but lack of apical Cl⁻ conductance caused the change, not increased Na(+) transport. These results indicate that CFTR provides the predominant transcellular pathway for Cl⁻ and HCO₃⁻ in porcine airway epithelia, and reduced anion permeability may initiate CF airway disease.

The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Produces Abnormalities in Tracheal Development in Neonatal Pigs and Young Children

RATIONALE Although airway abnormalities are common in patients with cystic fibrosis (CF), it is unknown whether they are all secondary to postnatal infection and inflammation, which characterize the disease. OBJECTIVES To learn whether loss of the cystic fibrosis transmembrane conductance regulator (CFTR) might affect major airways early in life, before the onset of inflammation and infection. METHODS We studied newborn CFTR⁻(/)⁻ pig trachea, using computed tomography (CT) scans, pathology, and morphometry. We retrospectively analyzed trachea CT scans in young children with CF and also previously published data of infants with CF. MEASUREMENTS AND MAIN RESULTS We discovered three abnormalities in the porcine CF trachea. First, the trachea and mainstem bronchi had a uniformly small caliber and cross-sections of trachea were less circular than in controls. Second, trachealis smooth muscle had an altered bundle orientation and increased transcripts in a smooth muscle gene set. Third, submucosal gland units occurred with similar frequency in the mucosa of CF and control airways, but CF submucosal glands were hypoplastic and had global reductions in tissue-specific transcripts. To learn whether any of these changes occurred in young patients with CF, we examined CT scans from children 2 years of age and younger, and found that CF tracheas were less circular in cross-section, but lacked differences in lumen area. However, analysis of previously published morphometric data showed reduced tracheal lumen area in neonates with CF. CONCLUSIONS Our findings in newborn CF pigs and young patients with CF suggest that airway changes begin during fetal life and may contribute to CF pathogenesis and clinical disease during postnatal life.

The ΔF508 Mutation Causes CFTR Misprocessing and Cystic Fibrosis–Like Disease in Pigs

A common mutation in human cystic fibrosis, CFTR-ΔF508, results in misprocessed CFTR and a cystic fibrosis–like clinical phenotype in pigs. Four Legs Good, Two Legs Bad In Animal Farm, George Orwell describes a pasture in which the pigs lead an animal revolt, resulting eventually in the porcine dwellers becoming indistinguishable from the human ones against whom they revolted. Scientists similarly wish for pigs to model humans, although as large animal models of human disease, not despotic rulers. Ostedgaard et al. extended this idea to cystic fibrosis (CF), generating pigs that carry the most common human CF mutation, Δ508. CF is a devastating genetic disease characterized by difficulty breathing, progressive disability, persistent infections, and, often, early death. CF is caused by a mutation in the gene that encodes the CF transmembrane conductance regulator (CFTR), which is an anion channel that modulates the components of sweat, digestive juices, and mucus. The most common mutation in CF patients results in an altered version of CFTR, CFTR-Δ508, which is found in 1 of 25 people of Caucasian descent. CF is difficult to study in human patients, and mouse models do not accurately reflect the human disease. Pigs may provide a better model of CF because they have more similar anatomy, biochemistry, physiology, size, and genetics to humans than mice. Thus, the authors generated a pig model of CF with the CFTR-Δ508 mutation. Similar to pigs that completely lack expression of CFTR, the CFTR-Δ508 pigs developed CF symptoms that mimicked those in human patients. In these animals, much of the CFTR-Δ508 protein was misprocessed; specifically, it was retained in the endoplasmic reticulum and rapidly degraded. However, pigs with CFTR-Δ508 retained small amounts of CFTR conductance (~6%), although this level of function was not sufficient to prevent disease. This new model may help to determine which levels of CFTR are sufficient for function and, therefore, guide future therapeutic strategies. After all, all animal models are equal, but some are more equal than others. 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 CFTRΔF508/ΔF508 pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTRΔF508/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 CFTRΔF508/ΔF508 epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTRΔF508/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.

Pathology of Gastrointestinal Organs in a Porcine Model of Cystic Fibrosis

Cystic fibrosis (CF), which is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), is characterized by multiorgan pathology that begins early in life. To better understand the initial stages of disease, we studied the gastrointestinal pathology of CFTR-/- pigs. By studying newborns, we avoided secondary changes attributable to environmental interactions, infection, or disease progression. Lesions resembling those in humans with CF were detected in intestine, pancreas, liver, gallbladder, and cystic duct. These organs had four common features. First, disease was accelerated compared with that in humans, which could provide a strategy to discover modifying factors. Second, affected organs showed variable hyperplastic, metaplastic, and connective tissue changes, indicating that remodeling was a dynamic component of fetal life. Third, cellular inflammation was often mild to moderate and not always present, which raises new questions as to the role of cellular inflammation in early disease pathogenesis. Fourth, epithelial mucus-producing cells were often increased, producing a striking accumulation of mucus with a layered appearance and resilient structure. Thus, mucus cell hyperplasia and mucus accumulation play prominent roles in early disease. Our findings also have implications for CF lung disease, and they lay the foundation for a better understanding of CF pathogenesis.

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth

People with cystic fibrosis (CF) exhibit growth defects. That observation has been attributed, in part, to decreased insulin-like growth factor 1 (IGF1) levels, and the reduction has been blamed on malnutrition and pulmonary inflammation. However, patients with CF already have a reduced weight at birth, a manifestation not likely secondary to poor nutrition or inflammation. We found that, like humans, CF pigs were smaller than non-CF littermates and had lower IGF1 levels. To better understand the basis of IGF1 reduction, we studied newborn pigs and found low IGF1 levels within 12 h of birth. Moreover, humerus length and bone mineral content were decreased, consistent with less IGF1 activity in utero. These findings led us to test newborn humans with CF, and we found that they also had reduced IGF1 levels. Discovering lower IGF1 levels in newborn pigs and humans indicates that the decrease is not solely a consequence of malnutrition or pulmonary inflammation and that loss of cystic fibrosis transmembrane conductance regulator function has a more direct effect. Consistent with this hypothesis, we discovered reduced growth hormone release in organotypic pituitary slice cultures of newborn CF pigs. These findings may explain the long-standing observation that CF newborns are smaller than non-CF babies and why some patients with good clinical status fail to reach their growth potential. The results also suggest that measuring IGF1 levels might be of value as a biomarker to predict disease severity or the response to therapeutics. Finally, they raise the possibility that IGF1 supplementation beginning in infancy might be beneficial in CF.