Parallels between humans and pigs in regards to anatomy, physiology, biochemistry, size, life span, and genetics make pigs a suitable candidate for modelling a range of human diseases. Porcine lungs share several anatomical and histological features with human lungs, including similar tracheobronchial tree structure, and abundance of airway submucosal glands [
90]. Pigs have previously been used to model pulmonary diseases involving inflammation and infection such as chronic bronchitis [
91]. Furthermore, as human CF lung disease progresses over the lifetime of an individual, the longevity of pigs allows for long-term investigations of lung disease pathogenesis and assessment of therapeutics, which is not possible in rodent or ferret models due to their relatively short lifespan or disease severity, respectively [
90]. The pig CFTR amino acid sequence is also 95% conserved with human, and the electrophysiological properties of porcine airway epithelium and submucosal glands resemble those observed in humans [
36,
92].
Adeno-associated virus-mediated gene targeting of CFTR was used to generate the first CF pig models containing either a null allele (CFTR
−/−) or the common Phe508del mutation (CFTR
ΔF508/ΔF508) [
6,
93]. Crossbreeding of CFTR
+/− and CFTR
+/ΔF508 heterozygotes was also performed to produce a third model, denoted CFTR
-/ΔF508 [
94]. Shortly thereafter, Klymiuk et al. generated another knockout CF pig using an alternative approach that involved sequential targeting of CFTR using bacterial artificial chromosome (BAC) vectors [
7]. Following breeding of the first CF pigs, it was realised that maintenance of the animals requires intensive and costly husbandry due to the severe gastrointestinal phenotype. Newborn CF pigs have a 100% penetrance of meconium ileus, with piglets requiring surgery soon after birth to relieve the obstruction. CF pigs also demonstrate pancreatic insufficiency and other gut-related issues requiring treatments including enzyme replacement therapy to aid digestion, oral vitamin supplements to prevent malnutrition, oral proton pump inhibitors or H2 blockers to control gastric acid, and laxatives to prevent bowel obstruction [
93]. Using the same approach employed to create the gut-corrected CF mouse and ferret models, a transgenic CFTR
−/− pig was generated to express porcine CFTR in the intestines thereby overcoming the need for surgical correction of meconium ileus at birth [
95].
Airway disease in CF pig models
CFTR
−/−, CFTR
ΔF508/ΔF508 and CFTR
-/ΔF508 pig strains (hereafter collectively referred to as CF pigs) spontaneously develop key features of human CF lung disease within months of birth. These manifestations include airway inflammation, infection, tissue remodelling, mucus accumulation, and obstruction [
94]. As in human CF populations, the severity of the lung phenotype varies between animals, with individual lobes also demonstrating heterogeneity [
93]. Examination of neonatal CF pig airways shows no evidence of inflammation, comparable to newborn humans with CF [
94,
96]. BAL profiles also indicate no difference in leukocyte and interleukin 8 (IL-8) concentrations between newborn CF and non-CF pigs. Over time however, inflammation ensues, ranging from mild to severe leukocytic infiltration, with severe cases occasionally exhibiting ulceration and abscess formation of the airway wall, and destruction of submucosal glands. Shortly after birth, CF pig lungs demonstrate defective bacteria eradication with newborn CF pigs challenged with
Staphylococcus aureus failing to eradicate the bacteria as effectively as wild-type pigs [
94]. This defective bacterial killing has been attributed to reduced airway surface pH in the CF pig and subsequent diminishing of the ASL antimicrobial function [
78].
CF pigs demonstrate heterogeneous airway remodelling, with some cases showing evidence of goblet cell hyperplasia, airway wall thickening, and rarely, distended submucosal glands [
94]. As observed clinically in infants with CF [
97,
98], CF pigs present with airway obstruction comprising of atelectasis, hyperinflation, air trapping, and pneumonia [
99]. Purulent material appears to obstruct the trachea and bronchi, often containing bacteria, neutrophils, and macrophages [
94]. Tracheal abnormalities are also observed in CF pigs including a triangular rather than circular shaped trachea [
7], smaller lumen area and circumference, irregular cartilage rings, and altered smooth muscle [
93,
100]. Similar tracheal malformations have been noted in CF mouse models [
43], CF rats [
4], and infants with CF [
100]. Interestingly, newborn CF pigs do not demonstrate reduced PCL depth in the trachea [
101], but they do exhibit impaired MCT [
102].
A range of ion transport measures performed on the nasal, tracheal and bronchial epithelia of CF pigs using tissues, cultures and in vivo approaches reveals electrophysiological defects consistent with loss of CFTR activity [
101]. These abnormalities tend to be more pronounced in CFTR
−/− airway epithelia when compared to CFTR
ΔF508/ΔF508 pigs, most likely due to the CFTR
ΔF508/ΔF508 epithelia retaining some residual CFTR function [
93]. Characteristic of CF, CFTR-mediated chloride transport is substantially reduced in the nasal, tracheal, and bronchial epithelium of CF pigs when compared to wild-type. CF pig nasal epithelia also exhibits a CF-like response to amiloride perfusion, but the trachea does not. Further investigation into this phenomenon revealed that the amiloride response observed in the CF pig nasal epithelia was due to reduced CFTR-mediated chloride conductance rather than sodium hyperabsorption [
101]. Excised tracheal tissue from CF pigs also demonstrates diminished bicarbonate conductance, which is consistent with the human CF phenotype [
93,
101].
Given that CF pigs capture many features of human CF airway disease, they have been useful for several research applications including investigations of lung disease pathogenesis [
94], studying electrolyte transport defects [
101], and exploring mechanisms involved in CFTR-Phe508del biosynthesis and misprocessing [
92]. CF pigs have also provided a useful platform for exploring the origins of inflammation and infection within CF airways [
94], identifying the role of ASL acidification in lung disease development [
78], and trialling viral-mediated airway gene therapy approaches [
103,
104]. In the future, CF pigs may also be useful for the long-term testing of therapeutics such as CFTR modulators, and for assessing airway disease prevention strategies [
105].