Mouse Models of Cystic Fibrosis

Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 www.elsevier.com/locate/jcf

Mouse models of cystic ﬁbrosis: Phenotypic analysis and research applications

Martina Wilke a,1, Ruvalic M. Buijs-Offerman b, Jamil Aarbiou b,2, William H. Colledge c, David N. Sheppard d, Lhousseine Touqui e, Alice Bot a, Huub Jorna a, Hugo R. de Jonge a, Bob J. Scholte b,*

a Erasmus MC, Biochemistry Department, 3000 CA Rotterdam, The Netherlands b Erasmus MC, Cell Biology Department, 3000 CA Rotterdam, The Netherlands c Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK d University of Bristol, School of Physiology & Pharmacology, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK e Unité de Défense Innée et Inﬂammation, Unité Inserm U.874, Institut Pasteur, 75015 Paris, France

Abstract

Genetically modified mice have been studied for more than fifteen years as models of cystic fibrosis (CF). The large amount of experimental data generated illuminates the complex multi-organ pathology of CF and raises new questions relevant to human disease. CF mice have also been used to test experimental therapies prior to clinical trials. This review recapitulates the major phenotypic traits of CF mice and highlights important new findings including aberrant alveolar macrophages, bone and cartilage abnormalities and abnormal bioactive lipid metabolism. Novel data are presented on the intestinal and nasal physiology of F508del-CFTR CF mice backcrossed onto different genetic backgrounds. Caveats, and sources of variability including age, gender and animal husbandry, are discussed. Interspecies differences limit comparison of lung pathology in CF mice to the human disease. The recent development of genetically modified pigs and ferrets heralds the application of more advanced animal models to CF research and drug development. © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Keywords: Cystic ﬁbrosis; Animal models; CFTR mice; Lung disease; Intestinal disease; F508del-CFTR

1. Introduction Because access to relevant patient tissues is limited, genetically modified mice are widely used to study CF pathology The life expectancy of cystic fibrosis (CF) patients in- and experimental therapeutics. These models have proven to creased from around 20 to 40 years over the past two decades, be very useful, but they have important limitations, due to mainly due to improved nutrition and treatment of chronic obvious physiological differences between humans and mice lung disease. However, there is still an urgent unmet need to [1–3]. Recently, pig [4,5] and ferret [6] cystic fibrosis trans- improve the lifespan and quality of life of CF patients. The membrane conductance regulator (CFTR) knockout animals complex multi-organ pathophysiology of secretory epithelia were reported, opening an exciting new field. Both models in CF is not yet completely understood, and a treatment show dominant intestinal disease [6,7], with severe perinatal that sufficiently corrects the primary defect remains to be lethality. There is also evidence for reduced clearance of developed. bacterial lung infection in a limited number of long-lived CF pigs [8]. In both models, F508del-CFTR mutants are being * Corresponding author: B.J. Scholte, PhD, Erasmus MC, Cell Biology developed, which will facilitate the testing of novel therapeu- Department, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Tel.: +31 tic strategies for CF. The challenge for the time to come will 10 7043205; fax: +31 10 7044743. E-mail address: [email protected] (B.J. Scholte) be to manage intestinal disease in CF pigs and ferrets to allow 1 Present address: Erasmus MC, Clinical Genetics Department. better understanding of early lung disease, which is still an 2 Present address: Galapagos NV, Leiden. underexplored area of CF pathology. While we can expect

1569-1993/$ - see front matter © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S153 great progress from these models, their application is limited The most recent and important addition to the CF mouse by the considerable practical and financial issues involved. family is a conditional (“loxed”) Cftr deletion that can be This paper will focus on CF mouse models, presenting an created by cell specific expression of the cre recombinase [12]. update on new developments and novel data, in particular on This genetically modified mouse will allow the contribution the properties of the mutant strains made available to the CF of different cell lineages to CF pathology to be elucidated. community through the EuroCareCF project. Although not a CFTR mouse model, genetically modified mice overexpressing the β subunit of the epithelial Na+ 2. CF mutant mice, portrait of an unruly family channel (ENaC) are important because they possess a lung phenotype resembling that of CF [13–16]. For further infor- Shortly after the discovery of the CFTR gene, several mation about this mouse model and its applications, see Zhou groups set out to generate mouse strains with mutations in the et al. [17]. Cftr locus using the homologous recombination technology and mouse embryonic stem cells pioneered by Mario Capec- 3. Mouse models, sources of variation and practical chi, Martin Evans and Oliver Smithies who jointly received considerations the Nobel Prize for these innovations in 2007. Table 1 lists the genetically modified mouse strains produced by different CF mutant mice are generally much less fertile than normal groups. Details of mouse strain nomenclature and availability mice, which makes it difficult to breed them from homozy- can be found at the Mouse Genome Informatics website gous mutant pairs. This is partly due to frequent obstruction (http://www.informatics.jax.org/). Table 2 summarizes briefly of the male vas deferens [18,19]. However, homozygous fe- the major phenotypic traits of CF mice in comparison with the males are also poor breeders. Occasionally mice can be bred human disease. successfully from homozygous pairs. However, there is risk Several loss of function, or “knock-out” models were made of “genetic drift”, the selection of compensating mutations in by interrupting the mouse Cftr coding region with a selectable either the background or the Cftr allele, which affect pheno- marker. The Cftrtm1Hgu shows low level of residual activity type in an unpredictable way. Breeding CF mutant mice from of wild-type CFTR and lower mortality, compared to the heterozygous animals is therefore recommended strongly. Im- other two frequently used knockout strains Cftrtm1Cam and portantly, this breeding strategy yields age- and sex-matched Cftrtm1Unc (Table 1). normal littermates, which are mandatory control for most In addition, common CF causing mutations were intro- experiments. Breeding requires considerable investment in duced into the mouse CFTR amino acid sequence. Three DNA genotyping of all neonates by ear or tail clips, the strains with the F508del mutation in the mouse Cftr gene identification of individual animals by chip, toe- or ear-clips were generated, two of which carry an intronic insertion that and a database to track individual animals. Genotyping is may interfere with gene transcription (Table 1). By contrast, primarily required to identify heterozygous breeders, because the Cftrtm1Eur allele was made by the two step “hit and homozygous mutants are easily recognized by their typical run” recombination procedure, which removes the selectable white incisor teeth [20]. marker. Therefore, this allele only contains the F508del mu- There is consensus about the most prominent phenotypic tation and has normal transcription rates in all tissues [9,10]. abnormalities of different Cftr mutant mice. Yet, close com- All three strains have been successfully used in phenotypic parison of published data from different laboratories shows studies. The F508del mutation in the mouse Cftr context considerable variation and even controversy about the nature causes severely reduced amounts of fully glycosylated CFTR and extent of lung disease. One obvious source of variation protein (band C) similar to the human form, and a phenotype is the different Cftr mutations, some of which are associated comparable to CFTR loss of function mutants [9]. In cultured with residual CFTR function (Table 1). Furthermore, Cftr gallbladder epithelial cells from F508del mutant mice, the alleles have been bred onto different genetic backgrounds, number of active CFTR Cl− channels observed in patch- which may cause subtle differences in phenotype, presumably clamp experiments is increased by incubating cells at low because of the expression of different “modifier genes”. Com- temperature [9], suggesting a similar temperature-dependent plete genomes of several laboratory mouse strains are now processing defect to the F508del mutation in human CFTR. available (for further information, see Sanger mouse genome Moreover, the G551D mutation which disrupts channel gating project, http://www.sanger.ac.uk/resources/mouse/genomes/). has the typical CF phenotype caused by loss of function This resource will provide a new and powerful tool to ana- mutants [11]. The less severe phenotype of the F508del- lyze phenotypic differences between CFTR mutant strains. It and G551D-CFTR models when compared to CFTR knock- will also facilitate comparative studies of modifier genes and outs, particularly lower perinatal mortality due to intestinal potential therapeutic targets in the human population. obstruction, has been attributed to residual CFTR activity. Animal husbandry is a critical issue, especially when in- To overcome the limitations imposed by the severe in- vestigating innate adaptive responses (e.g. lung inflammation), testinal disease presented by many knockout strains, “gut which are by their very nature designed to reflect external corrected” hybrid strains were created using a transgene ex- conditions. This can cause differences between strains, con- pressing CFTR from a promoter specific for the intestinal tribute to inter-individual variation, and lead to differences epithelium (Table 1). in results between laboratories. Unfortunately, there is no S154 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

Table 1 CFTR mutant mice

Mouse Mutation Cftr mRNA Genetic Survival to Body wt Contact References background maturity Null mutations Cftr tm1Unc S489X Ex10 R Not detectable* C57Bl/6 <5% 10–25% reduction BH Koller/Jax Labs [113,158] Cftr tm1Cam R487X Ex10 R Not detectable 129S6/Sv/Ev <5% 20% reduction WH Colledge [159] Cftr tm1Hsc M1X Ex1 R Not detectable CD1 x 129 25% Delayed LC Tsui [160] Cftr tm3Bay Ex2 R Not detectable C57Bl/6 x 129 40% Reduced AT Beaudet [161] Cftr tm3Uth Y122X Ex4 R Not detectable C57Bl/6 25% 25–50% reduction M Capecchi/PB Davis [113,162] Hypomorphic mutations Cftr tm1Hgu ** Ex10 I 10% of wt MF1 x 129 90% No reduction J Dorin [113] Cftr tm1Bay Ex3 I <2% wt C57Bl/6 x 129 40% 70% reduction AL Beaudet [163] F508del mutations Cftr tm2Cam F508del R 30% of wt 129S6/Sv/Ev <5% 10–20% reduction WH Colledge [164] Cftr tm1Kth F508del R Low in intestine C57Bl/6 x 129 40% 10–50% reduction KR Thomas/Jax labs [165] Cftr tm1Eur F508del (H&R) Normal levels FVB/129; FVB 90% 10–20% reduction BJ Scholte [9] C57Bl/6 Other point mutations Cftr tm2Hgu G551D R 50% of wt CD1/129 65% 30–50% reduction J Dorin [11] Cftr tm3Hgu G480C (H&R) Normal levels C57Bl/6/129 Normal No reduction J Dorin [166] Cftr tm2Uth R117H R 5–20% of wt C57/Bl6 95% 10–25% reduction M Capecchi/PB Davis [113,162] Transgenes Mouse Transgene Promoter Expression Phenotype References Tg(FABPCFTR) CFTR Rat intestinal fatty acid Intestinal villus epithelia Rescue of CF intestinal pathology [167] binding protein Tg(CCSPScnn1b) Scnn1b Clara cell secretory Airway surface epithelia Na+ hyperabsorption [13] protein (CCSP) Reduced airway surface ﬂuid volume Mucus accumulation, CF-like lung disease; 40% survival.

Legend: Mutations are introduced by homologous recombination in embryo stem cells. Key: R, replacement; I, insertion; (H&R), Hit & Run; * a splice variant was detected [168]. Transgenes are created by injection into mouse oocytes. General rules on mouse gene and strain nomenclature can be found at the Mouse Genome Informatics guidelines website. ** The strain Cftr tm1Hgu is also referred to as Cftr TgH(neoim)Hgu due to the unique nature of this recombinant which produces low level normal CFTR due to a splice variant introduced by the recombination event. global standard protocol for animal husbandry. Laboratories when designing experiments and analyzing data. Finally, age therefore differ in the level of pathogen exposure. The Rot- is another important variable. Its importance is demonstrated terdam animal facility maintains a FELASA+ pathogen-free by the progressive nature of lung disease in several CF mouse standard using sterilized ventilated cages supplied with steril- strains. Together, these considerations place severe demands ized, acidified water. Separation of animals causes stress and on the size and selection of experimental groups necessary to is avoided whenever possible; nesting material (e.g. sterile generate reproducible data and statistical power. paper tissue) is always provided. Strict light and dark regimes are maintained; nursing females are not disturbed until ten 4. CF mouse phenotype days after birth to reduce postnatal mortality. CF homozygous mutant animals do not travel very well and often die or The major phenotypic abnormalities of CF mutant mice suffer excessive weight loss during long distance transport. are briefly summarized in Table 2. This brief overview does Therefore, animals are moved at least two weeks in advance not do justice to all the available literature. of experiments to allow them to adapt. Under these condi- In the mutant strains investigated, pancreatic pathology tions we can maintain colonies of Cftrtm1Eur F508del and is not a prominent feature, although it was reported in a Cftrtm1Cam knockout mice in different genetic backgrounds C57Bl/6 Cftr knockout backcross [19]. Although pancreatic on normal chow at less than 20% mortality for homozygous insufficiency correlates with severe pathology, it is not seen mutants. in all CF patients. Similarly, male infertility caused by Gender is another source of variation that should not be obstruction or absence of the vas deferens is frequent in underestimated. Male and female mice differ with respect to CF mice depending on the strain and genetic background many physiological responses relevant to CF studies. This [18,19]. However, this pathology appears less severe than in is well illustrated in a recent study of a mouse model of humans where absence of the vas deferens is observed in asthma [21]. Gender differences should be taken into account all male CF patients. Apparently, factors other than CFTR M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S155

Table 2 Comparative CF pathology in human and mouse CF mutants

Human pathology [27,169] Tissues affected in Mouse pathology [1,2] CFTR mutant Recurrent bacterial lung infections, [170] Lung No CF type obstructive lung disease. Pro: [19,105,114, chronic inflammation, airway Inflammation and tissue remodeling in aging 117,171,172] remodeling, irreversible loss of animals. Con: [173] function Hyper-inflammation reduced clearance upon challenge with bacteria or LPS. Hyposecretion, viscous mucus, [174,175] Submucosal gland Nasal and tracheal glands present, reduced [81,82] plugging (nasal, bronchial) cAMP-induced secretion in bronchial glands of mutant mice Bacterial biofilm, viscous/purulent [176,177] Trachea, bronchial Reduced mucociliary clearance (contested). Pro [84] mucus, defective mucociliary epithelium (ciliated/ Electrophysiology influenced by CaCC Con: [83] clearance, goblet cell hyperplasia, goblet) (TMEM16A). inflammation, tissue remodeling Distal airways involved in early [95] Broncheoli (distal Spontaneous progressive remodeling and [19,102,172,178] disease, CFTR expression in Clara epithelium, Clara cells) inflammation (strain- and age-dependent) cells No known pathology, CFTR Alveoli (type I/II) Cftr expression in type II cells, abnormal fluid [97,99]. expression in cultured type II cells secretion in mutant mice. Meconium ileus, chronic [31,179, Intestine Lethal mucus plugging, lipid and bile acid [1,2,25,60,181, malabsorption, reduced body 180] reabsorption defect, reduced body weight, failure to 182] weight, failure to thrive (pancreas thrive. Goblet cell hyperplasia in ileum, mucus deficiency), typical accumulation in intestinal crypts, meconium ileus electrophysiology. equivalent, chronic inflammation, abnormal electrophyiology. Frequent biliary disease, [43,183– Liver/Biliary duct No morbidity, progressive liver disease in aging CF [19,186,187] progressive cirrhosis 185] KO mice biliary duct damage in KO mice upon induced colitis No frequent pathology [188] Kidney Functional CFTR in proximal tubule. In CF mice no [41,42,189,190] overt pathology, abnormalities in Na+ and K+ transport, defective endocytosis. Increased frequency of bile stones Gallbladder No cAMP-induced Cl− or water secretion [45,191] Pancreatitis, atrophy, digestive [192,193] Pancreas Normal function and ductal fluid secretion, [19,194] enzyme secretion deficiency, occasional duct plugging in Cftr tm1Unc mice diabetes, abnormal bicarbonate secretion and mucous plugging of ducts No pathology Tooth Abnormal enamel (white incisor) [20] Osteopenia, osteoporosis [73,74] Bone Abnormal bone density. [67–69] No pathology, reduced flow rate [195] Salivary gland Adrenergic fluid secretion defective [38] High NaCl in sweat (defective [169] Sweat gland Foot pad glands only, no report of deficiency. [196] reabsorption duct) Absent (CBAVD, male infertility Vas deferens Frequent plugging or absent (male infertility) [18,19] 100%) Frequent obstruction Oviduct Frequent obstruction (female infertility) No pathology reported Cornea Defective chloride permeability, enhanced amiloride [197–199] response, attenuated uptake of Pseudomonas Smooth muscle cell-related Smooth muscle cell CFTR expression, altered function and innervation, [47–50,200] pathology in lung and diaphragm degeneration in CF KO mice suspected but not firmly established No pathology reported Mast cells CFTR expression, enhanced presence in CF KO [59,60] intestine, no evidence for cell autonomous abnormality No pathology reported Macrophages CFTR expression, reduced capacity to kill [51,55–57] Pseudomonas, abnormal proinflammatory cytokine secretion S156 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 deficiency determine disease severity, in this case probably distinct hyperplasia of goblet cells is observed, comparable to the expression of alternative pathways for Cl− transport. published data for the original outbred strain (FVB/129) [10]. Consistent with this observation, the expression of the goblet 4.1. Intestinal disease in CF mouse models cell marker Clca3/Gob5 is increased 3-fold in the intestine of CF mutant mice [32] and individual goblet cells appear larger Similar to humans, intestinal disease is prominent in all than normal, possessing “plumes” of mucous material. Mucus CF mutant mice. Depending on the strain and laboratory plugs are often seen in the crypts and lumen. conditions many mutant mice may die of intestinal blockage A recent study in Cftrtm1Kth F508del (C57Bl/6) mice around the time of weaning. This is a stochastic effect at- highlights the crucial role of CFTR dependent bicarbonate tributed to abnormal fluid and mucus secretion (meconium secretion in PGE2-induced intestinal mucus secretion and hy- ileus equivalent). To reduce the frequency of intestinal ob- dration [33,34]. In the proposed model, bicarbonate secretion struction, mutant mice are often fed a liquid diet (Peptamen™) by CFTR in enterocytes is required for the proper expansion [22]. Because this drastic dietary intervention may affect the of tightly packed mucus-calcium ion complexes, which are phenotype of both control and mutant mice, the use of a secreted from vesicular granules by neighbouring goblet cells. mild laxative (Poly Ethylene Glycol; PEG) in combination In the absence of sufficient CFTR activity, the mucus does not with normal chow is recommended as an alternative [23]. unfold properly, which might play an important role in CF In the Rotterdam facility, Cftrtm1Eur and Cftrtm1Cam strains pathology. This indirect model for intestinal mucus secretion (FVB/n and C57Bl/6) survive well into adulthood (10–30 was invoked because CFTR expression in goblet cells has weeks, 80–90%) on normal chow based on animal fat (Hope not been demonstrated directly, whereas enterocytes show Farm™ SRMA 2131, Abfarm, Woerden, the Netherlands) relatively high levels of CFTR expression. If it can be shown combined with 6% (w/v) PEG in the drinking water for the that CFTR-mediated bicarbonate secretion plays a similar knockout mice. Male and female mutant mice of all strains role in mucus release in human airway epithelia, this would show a mild, but significant (10% average), reduction of body have important implications for our understanding of CF lung weight compared to normal age- and sex-matched littermates. disease. Although there is no evidence of an essential lipid deficiency The mechanism causing the apparent goblet cell hyperpla- [24], mutant mice display reduced lipid reabsorption, which is sia in the ileum of CF mutant mice has not been established. most pronounced in the Cftrtm1Cam strain [25]. Interestingly, However, abnormalities of mucus composition in Cftrtm1Unc the length and weight of the small intestine is larger in mutant knockout mice [35], suggest an effect on goblet cell differen- Cftrtm1Eur C57Bl/6 compared to wild-type (10%, N = 18, tiation and function, possibly by an adaptive process. P < 0.01, Wilke et al., unpublished data [26]). Immunohistochemistry using the R3195 anti-CFTR anti- In the mouse and human intestine, different proximal body (CR Marino, University of Tennessee) shows CFTR to distal regions with distinct morphological and functional expression on the apical membrane of enterocytes in villi characteristics can be distinguished. In the duodenum, bile and crypts of wild-type C57Bl/6 mice (Fig. 2). This signal from the gallbladder and digestive enzyme secretions from the is strongly reduced in Cftrtm1Eur F508del mutant mice and pancreas are delivered through a common duct and mixed with absent in Cftrtm1Cam knockout mice. Similar results were the acidic products of the stomach. In the jejunum and ileum, reported using the original Cftrtm1Eur 129/FVB strain [9] regulated fluid and mucus secretion maintain the optimal and the Cftr partial knockout Cftrtm1Hgu (also designated milieu required for lipid, sugar and amino-acid absorption. CftrTgh(neoim)Hgu) [36]. Consistent with these data, electro- The colon, including the caecum, is involved in mucus and physiological analyses demonstrate the absence or severe bicarbonate secretion, and net ion and fluid reabsorption, reduction of CFTR function in CF mutant mice. For exam- compacting the intestinal content to faeces. In addition to ple, Fig. 3 shows that in three different genetic backcrosses proximal to distal gradients along the length of the intestine, of the Cftrtm1Eur F508del allele forskolin-induced (cAMP- the epithelium is organized vertically by invaginations called dependent) short-circuit current (Isc),ameasureofCFTR- crypts and finger-like protrusions called villi. Stem cells at the mediated transepithelial ion transport, is reduced by more bottom of the crypts continuously produce new epithelial cells than 80%. Figure 3 also reveals that the carbachol response that differentiate to form the different cell types, lining crypts (Ca2+-dependent) is reduced, which in ileal epithelia reflects and villi. the activity of Ca2+-activated K+ channels in the basolateral CFTR is expressed mainly in enterocytes where it is membrane that facilitate CFTR-mediated Cl− secretion. In involved in chloride and bicarbonate secretion. The role of CFTR knockout mice of the same genetic background, both CFTR in intestinal ion transport [27,28] and in the small the forskolin and carbachol responses are completely lacking, intestine [29,30] has been reviewed. The ion transport prop- suggesting that the residual Isc response in intestinal epithelial erties of the colon, including the role of CFTR were reviewed of Cftrtm1Eur F508del mice reflects residual CFTR activity by Kunzelmann and Mall [31]. Here, we present general and (De Jonge & Bot, unpublished). novel data relevant to this review on CF mouse models. As for human CFTR, the intracellular transport defect Figure 1 demonstrates that the jejunum, ileum and colon and severely reduced activity of F508del-CFTR in Cftrtm1Eur of backcrossed Cftrtm1Eur C57Bl/6 and FVB/n mice exhibit homozygous mice is rescued by low temperature incubation a normal structure of crypts and villi. In the ileum a [9]. Full restoration of wild-type CFTR protein processing M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S157

Fig. 1. Goblet cells in CF mutant mouse intestine. Comparison of representative paraffin sections from mouse jejunum, ileum and colon stained with Alcian blue to identify goblet cells. Wild-type C57Bl/6 (A) and FVB (C) are compared with their Cftr tm1Eur C57Bl/6 F508del (B) and Cftr tm1Eur FVB F508del (D) homozygous mutant littermates (12–16 weeks). Ileum derived from both mutant strains (B and D) show significant hypertrophy of goblet cells. No differences in jejunum and colon between wild-type and mutant mice are observed (original magnification 20×). and channel activity was observed in stripped ileal mucosa restore function to Cftrtm1Eur F508del mutants in vivo.This from Cftrtm1Eur homozygous mice after 12 hours incubation assay offers the advantage of detecting very low levels of at 26°C [37] (Wilke, de Jonge, in preparation). Based on residual function in mutant mice (Fig. 4). Moreover, it can be this observation, small molecules that rescue the cell surface performed repetitively in the same mouse, serving as a highly expression of mutant CFTR (termed CFTR correctors) are sensitive indicator of CFTR function [39]. Other aspects of now being tested in Cftrtm1Eur homozygous mice. the physiology of the salivary acini and glands in normal and CF mutant mice were described recently by Catalan et al. 4.2. Salivary glands [40]. The authors’ data demonstrate that submandibular duct cells co-express CFTR and ENaC and that CFTR is required Best and Quinton [38] first demonstrated that β-adrenergic for effective reabsorption of NaCl [40]. (cAMP)-stimulated fluid secretion by salivary glands is a non- invasive and highly discriminative assay of CFTR function. 4.3. Kidney In this assay, atropine is first injected to prevent the stimulation of secretion by cholinergic agonists before isoproterenol CFTR is expressed in the proximal tubules of the mouse is injected to activate β-adrenergic-stimulated fluid secretion. kidney, localized to the apical and endosomal compartments Subsequently, Noël et al. [39] demonstrated that this assay can of epithelial cells. Though no overt kidney pathology is be used to evaluate the efficacy with which small molecules present, abnormalities in urinary protein secretion were ob- S158 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

Fig. 2. Immunohistological analysis of CFTR expression in the ileum. CFTR protein was stained with the R3195 anti-Cftr antibody on paraffin sections. Intense CFTR staining (brown) is observed in the ileum of FVB/n wild-type mice at the apical border of crypt and villus epithelial cells (A). Staining in Cftr tm1Eur F508del FVB mutant littermates is much less intense and more diffuse (B). Tissue of the Cftr tm1Cam FVB knockout mouse serves as negative control (C). This figure is representative for all backcrossed strains described in the present paper (original magnification 20×).

Fig. 3. Short-circuit measurements (Isc) of muscle-stripped excised ileum. Muscle stripped mucosa from ileum was analysed in an Ussing chamber as described in [77]. Bars represent average initial short-circuit current (Isc) or the change of Isc after sequential addition of the cAMP agonist forskolin (forsk), 2+ tm1Eur the Ca agonist carbachol and glucose. Isc measurements were performed on ileum derived from the Cftr F508del strain in three different congenic backgrounds (F13) A: FVB/n (F13) B: C57Bl/6 (F13) C: 129/sv (F12) comparing homozygous mutant (dd, open bars) with homozygous normal (++, black bars). In all homozygous mutant Cftr tm1Eur F508del mice, forskolin responses are significantly lower than normal (P < 0.001), but residual activity is detected. Similar results are observed with carbachol. Glucose is added to test a CFTR-independent function and to evaluate tissue viability. All animals were adult males and females in the range of 13–30 weeks. Data presented are means ± SEM (n = number of mice, two pieces of ileum per mouse, FVB +/+, n =9; FVB dd, n = 10; C57Bl/6 +/+, n = 10, C57Bl/6 d/d, n = 15; 129sv +/+, n = 11, 129sv d/d, n =8). served in Cftrtm1Cam knockout mice, suggesting a role for Moreover, by lowering the phospholipid-to-bile salt ratio, CFTR in membrane trafficking and brush border matura- chronic cholate administration increases the cytotoxicity of tion. To a lesser extent these abnormalities were also seen bile produced by CF mice. By contrast, either acute or chronic in Cftrtm2Cam and Cftrtm1Eur F508del mice, where they are ursodeoxycholic acid (UDCA) administration induces a bile attributed to partial processing of the mutant protein in this salt-specific choleresis in vivo in mice that is independent tissue [41,42]. of functional CFTR [44]. These observations may help to explain the beneficial effects of oral UDCA therapy in 4.4. Liver cholangiopathies including CFLD.

CF associated liver disease (CFLD) is an increasing 4.5. The gallbladder clinical problem, affecting more than 50% of adult CF patients. CFTR is expressed in cholangiocytes, where it is In mice, the gallbladder is an accessible and relatively presumably involved in ﬂuid homeostasis in the biliary duct simple epithelial tissue with both secretory and absorptive system [43]. In CF mice, no obvious liver pathology is properties. We have previously shown that CFTR-dependent tm1Cam detected. Chronic administration of cholate increases weakly forskolin-induced Isc (Isc forsk)isabsentinCftr bile production in CF mice when compared with control mice. C57Bl/6/129 knockout mice, but not normal mice [45,46]. We M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S159

inhibition of reabsorption and absence of fluid secretion is also observed in Cftrtm1Eur 129/FVB F508del mice (Fig. 5). This indicates that CFTR activity is insufficient for net fluid secretion in this CF mutant mouse. Gallbladder epithelial cells contain a large number of secretory granules containing mucins. Peters et al. [46] demonstrated that basal and ATP- stimulated mucus release from cultured gallbladder epithelial cells of Cftrtm1Eur 129/FVB mice are quantitatively normal. However, the potential role of CFTR-mediated bicarbonate secretion in mucus release in this model system and the physical properties of the secreted mucus remain to be investigated.

Fig. 4. Isoproterenol-stimulated salivary gland secretion. Salivary gland 4.6. Smooth muscle cells and CFTR function secretion was measured in anesthetized mice for 21 minutes as described in [38]. Mice were pretreated with atropine to block cross-stimulation of CFTR is expressed in smooth muscle cells and skeletal cholinergic-stimulated secretion. Isoproterenol was used in the presence muscle myotubes, colocalised with the sarcoplasmic reticu- of atropine to stimulate adrenergic secretion. There was no significant difference between the maximum secretion of FVB wild-type (WT) male (n lum [47–50]. Recently Divangahi et al. [47] demonstrated that 2+ = 6) and female mice (n = 4). The CF mutant homozygous Cftr tm1Eur FVB CFTR deficient myotubes display increased Ca signaling F508del (DF/DF) littermates (DF/DF: male: n = 123, female: n = 63) and upon depolarisation and excessive NF-κB translocation in re- tm1Cam homozygous Cftr FVB knockout mice (−/−: male: n = 7, female: n = sponse to inflammatory stimuli. Furthermore, degeneration of < 11) of either sex had a very low response compare to wild-type (P 0.001). diaphragmatic muscle was observed selectively in Cftrtm1Unc The Cftr tm1Eur F508del mice do not differ from the Cftr tm1Cam. mice challenged with Pseudomonas aeruginosa, which chron- ically infects the lungs of CF patients [47]. The authors further showed that unstimulated gallbladders from normal suggest therefore that reduced muscle strength, particularly mice reabsorb fluid to concentrate bile, whereas they secrete of the diaphragm, as frequently observed in CF patients, fluid when stimulated with forskolin [45]. By contrast, in may contribute to CF lung disease. Because smooth muscle gallbladders from Cftrtm1Cam knockout mice forskolin abol- cells of the airways and arteries express functional CFTR ished fluid reabsorption without stimulating fluid secretion, [48–50], elements of the CF lung phenotype (e.g. wheezing, demonstrating that fluid secretion is CFTR-dependent [45]. hyperreactivity and progressive tissue remodeling) might also tm1Eur In Cftr 129/FVB F508del mice, forskolin-elicited Isc result from the dysfunction of smooth muscle. responses were reduced by 80% [9]. Because Cftrtm1Cam FVB mice also show low, but detectable, forskolin-elicited Isc 4.7. The cellular immune system is abnormal in CF mutant responses through a CFTR-independent pathway, the residual mice forskolin-induced Isc is not mediated by F508del-CFTR (De Jonge, Bot unpublished data). Here, we present previously Several authors have reported abnormal behaviour of unpublished data demonstrating that the forskolin-induced alveolar and peritoneal macrophages in CF mutant mice. Di et al. [51] demonstrated defective killing of bacteria after uptake, possibly due to dysfunctional CFTR-dependent acidification of endosomes/phagosomes [52], though this explanation is contested [53,54]. Leal and co-authors showed that peritoneal and alveolar macrophages from Cftrtm1Eur mice display a pro- inflammatory bias after stimulation in culture, characterized by enhanced secretion of IL1β, but reduced secretion of IL10 [55,56]. Similarly, Bruscia et al. [57] reported abnormal pro- inflammatory cytokine secretion by lung and bone marrow- derived macrophages in CF mutant mice after exposure to lipopolysaccharide from Pseudomonas aeruginosa (PA LPS). These data strongly suggest that macrophages from CFTR mutant mice display defects that might, at least in part, explain the pro-inflammatory lung phenotype in CF mutant Fig. 5. Fluid transport in mouse gallbladder. Net fluid transport rates (Jv) mice. Other cells of the myeloid lineage (e.g. neutrophils in isolated gallbladders of the 129/FVB Cftr tm1Eur F508del strain [10] were [58]; mast cells [59,60] and dendritic cells [61]) are now measured as described in [45]. Negative values show initial transport of fluid under investigation in CF patients and mouse models. If it from the lumen of the bladder to the bath. After addition of the cAMP can be demonstrated that such defects are prominent in CF agonist forskolin, Jv changes to positive values in normal mice, i.e. net patients and cell-autonomous, not adaptive, in origin, this fluid secretion towards the lumen. By contrast, in homozygous Cftr tm1Eur F508del littermates transport towards the bath is inhibited and there is no net would support the conclusion that CF is a disease of the secretion into the lumen. immune system. S160 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

4.8. Abnormal bone and cartilage formation in CF mutant per part of the skull is covered with olfactory epithelium mice (OE). The OE consists of a multilayer of neuronal cells projecting sensory cilia from the surface, covered with a Bonvin et al. [62] first showed that rings of tracheal carti- layer of specialized epithelial cells called sustentacular cells lage in CF mutant mice (Cftrtm1Unc knockout and Cftrtm1Eur (Fig. 6A and B). This tissue expresses CFTR and ENaC and F508del (129/FVB)) are frequently incomplete at birth. Be- is thought to contribute to fluid and ion homeostasis across cause, neonatal CFTR knockout ferrets [6] and pigs [4,5] also the OE [76]. Under the OE and the NAE, there are numerous exhibit abnormal tracheal cartilage, this developmental abnor- submucosal glands embedded in connective tissue. CF mutant mality in CFTR deficient animals is the first to be verified in mice display an age-dependent progressive degeneration of multiple species. An explanation for this phenomenon is not the olfactory neuronal layer that is evident in adult mutant readily available. Interestingly, mouse chondrocytes, express mice from the age of 16–24 weeks. This was first reported functional CFTR [63], suggesting a cell autonomous defect for Cftrtm1Unc knockout and CftrTgHm1 G551D mutant mice in cartilage formation. However, a response to local and sys- on different genetic backgrounds [75,76]. Furthermore, an temic inflammation cannot be excluded. Of note, a similar, but increase of NAE thickness and apparent hyperplasia of mucus equally unexplained, phenotype was reported in TMEM16A producing cells is observed in CftrTgHm1 G551D mutants mutant mice [64–66], which lack the Ca2+-activated Cl− [75]. At age 14–20 weeks, Cftrtm1Eur FVB mice did not show channel (see above, section 4.12). histological evidence of OE degeneration in our facility (Fig. Reduced bone density in CFTR mutant mice was described 6A, B), indicating that this abnormality is not only age-, but initially by Dif et al. [67] and confirmed by others [68,69]. also mouse strain-dependent. It is currently unknown whether this defect is caused by When the OE and underlying tissue is removed from a cell autonomous CFTR-related defect of bone forming the nasal cavity, it can be inserted into a small aperture osteoblasts or bone degrading osteoclasts. Alternatively, this Ussing chamber for ion transport studies [77]. In all five CF defect might be secondary to systemic inflammation and mutant strains tested, we observed a significantly increased abnormal bioactive lipid metabolism (ceramides, vitamin D). (P < 0.01) Isc response to amiloride (Isc amil) (Fig. 7). This Low amounts of CFTR protein are expressed in mouse includes the Cftrtm1Eur F508del mutation on different genetic and human osteoblasts and odontoblasts, together with low backgrounds and the Cftrtm1Cam knockout allele on mixed levels of CFTR mRNA [70]. The location of CFTR in these background (C57Bl/6-129) and FVB. An enhanced Isc amil cells was mainly intracellular, suggesting that CFTR might and PDamil is also observed in human CF airway epithelia, function as an anion transporter in intracellular organelles. supporting the hypothesis that CF airway disease results, at The dysfunction of the ClC transporter ClC-7 in osteopetrosis least in part, from ENaC hyperactivity. Consequently, ENaC [71,72] raises the possibility that CFTR dysfunction in is a primary therapeutic target in CF mutant mice [15,78]. organelle membranes of bone cells might underlie the changes In excised nasal OE from Cftrtm1Eur F508del mice, Cftr in bone metabolism in CFTR-deficient mice and CF patients, mRNA expression is normal (Scholte et al., data not shown), leading to osteopenia. whereas CFTR protein expression is much reduced (Fig. 6A The clinical relevance of these data remains to be estab- and B) and in Cftrtm1Cam knockout mice this signal is absent lished. To date, no cartilage abnormalities have been reported (not shown). The absence of olfactory marker proteins from in CF patients. However, osteopenia and osteoporosis are CFTR-expressing cells argues that CFTR is not expressed serious problems in CF patients [73,74]. Taken together, the in ciliated sensory neurons, but in a subset of microvillous data suggest that animal model studies of CF-related bone secondary chemosensory cells (named brush cells), where it disease might lead to novel treatments for CF patients. is co-localized with ENaC (De Jonge et al, unpublished data). Whether this cell type represents a cell specialized for fluid 4.9. Nasal airways of CF mutant mice and electrolyte transport or plays a role in CFTR-dependent regeneration of the olfactory system [79,80] remains to be The nasal epithelium is an important focus of CF mouse established. model research, because of its similarities in structure and Forskolin responses observed in different mouse strains are function with the human bronchial airway epithelium. Indeed, complex parameters that cannot, at face value, be attributed early studies of the nasal epithelium demonstrated electro- to CFTR activity. In Cftrtm1Cam C57Bl/6-129 knockout mice, physiological abnormalities consistent with CFTR dysfunc- we observe a forskolin response comparable to normal tion in CF mutant mice (i.e. abrogated cAMP-stimulated Cl− littermates. However, in Cftrtm1Cam FVB mutant mice this conductance and enhanced amiloride-sensitive Na+ currents response is virtually absent (Fig. 7). These data suggest that in microperfusion studies, Table 2). a cAMP-stimulated Cl− conductance other than CFTR is More recent studies draw attention to the complex ar- present in the OE of Cftrtm1Cam C57Bl/6-129 mice, but is chitecture of the mouse nasal epithelium and the effects of absent from Cftrtm1Cam FVB mice. Studies are underway to CFTR dysfunction on this tissue [75,76]. The mouse nasal identify this cAMP-stimulated Cl− conductance. cavity is a septated cartilage and bone structure covered with In all Cftrtm1Eur F508del backcrossed strains the nasal nasal airway epithelium (NAE), mainly ciliated and mucus forskolin-stimulated Isc in mutant mice is comparable to producing (goblet) cells. However, the cavity lining the up- normal littermates. Because this includes the FVB strain M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S161

Fig. 6. CFTR immunostaining in mouse olfactory epithelium (OE) and trachea. In wild-type FVB (FVB WT) mice punctuated CFTR staining in brush cells of the olfactory epithelium (OE) (A) and diffuse CFTR staining in ciliated cells of the trachea (C) is observed. In homozygous mutant littermates (FVB F508del Cftr tm1Eur), the CFTR staining in brush cells of the OE (B) and ciliated cells of the trachea (D) and is much reduced. The CFTR signal is completely absent in sections from Cftr tm1Cam FVB knockout mice, stained in parallel (not shown). that does not show a response with the Cftrtm1Cam knockout the intracellular calcium and cAMP signaling pathways, allele, one possible explanation is that the nasal forskolin- respectively. The cAMP-stimulated component is lacking stimulated Isc reflects residual CFTR activity resulting from in tracheal glands from CF mutant mice, and is therefore partial processing of the mutant protein [9]. However, the thought to be CFTR-dependent [81,82]. The contribution nasal forskolin-stimulated Isc does not influence the enhanced of CFTR-mediated secretion by submucosal glands to CF amiloride response in this mutant strain (Fig. 7). Together mouse pathology is unclear, but this experimental paradigm with the immunohistology data (Fig. 6A, B), this argues that constitutes a valid model to study CFTR function and test there is not a linear relationship between the nasal forskolin- drugs targeting CFTR. stimulated Isc and the number of active CFTR channels. It is important to note that the submucosal glands of the nasal cavity, and their contribution to its CF phenotype, have 4.10. Submucosal glands in CF mutant mice not yet been studied. It is conceivable, that nasal submucosal cells have properties different from tracheal glands, since they Mice have composite serous and mucous submucosal have developed in a different cellular context. glands, not only in the nasal cavity, but also in the upper part of the trachea. This is relevant, because abnormal 4.11. CFTR expression in the mouse trachea submucosal glands are thought to play an important role in the development of CF airway disease, contributing to The cellular architecture of the pseudostratified columnar fluid hypo-secretion and the abnormal consistency of mucus mouse tracheal epithelium is similar to that of the nasal in the proximal airways [81,82]. Secretion of fluid from airways. It is dominated by CFTR-expressing ciliated cells submucosal glands is dependent on two complementary (Fig. 6C and D), interspersed with infrequent mucus secreting combinations of transport systems, in particular basolateral neuroendocrine and Clara cells. Mucociliary clearance and K+ channels and apical CFTR Cl− channels, regulated by epithelial ion transport are not significantly affected in tracheal S162 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

Fig. 7. Summary of short-circuit current (Isc) measurements in nasal epithelium. Short-circuit current responses (Isc) to amiloride (10 μM) and forskolin (10 μM) were measured in isolated nasal epithelium in an Ussing chamber as described in [77]. Bioelectric characteristics of 12–18 weeks old normal (nn) and homozygous mutant (dd) mice from the different backcrossed Cftr tm1Eur F508del strains (FVB, FVB/129, C57Bl/6, 129sv) are compared. Homozygous Cftr tm1Cam knockout mice were analysed in FVB (FVB/n -/-), and the original Cftr tm1Cam strain (C57Bl/6-129 -/-), the latter compared to their normal (nn) littermates. The number of individual animals measured per experimental group is given in parenthesis. The amiloride response is significantly (P < 0.01) increased in all Cftr mutant strains compared to their wild-type littermates, except for the 129sv backcross. The forskolin response does not differ significantly between the mutant strains and the wild-type mice in this assay, with the exception of the FVB Cftr tm1Cam knockout mouse strain (P < 0.001). epithelia from CFTR knockout mice [83], but see [84]. al., not shown). Immunohistological detection of CFTR ex- Interestingly, in tracheal epithelia the Ca2+-activated Cl− pression in the lung, confirmed by using CFTR knockout mice channel (CaCC) is the dominant apical membrane Cl− as a negative control, is not feasible with current methods. channel, CFTR activity is only observed after maximal Mouse bronchi possess ciliated and mucus producing cells in stimulation of CaCC [85]. a declining proximal to distal distribution, whereas airways in the mouse lung are dominated by Clara cells, constituting 4.12. CaCC: the dark horse in CF pathology 60–80% of the total epithelial cell population. Clara cells are non-ciliated secretory cells that (i) display phenotypic adapta- CaCC is expressed in the apical membrane of epithelia tion and proliferative capacity during injury and inflammation, affected by CF, where it operates in parallel with CFTR, but (ii) are capable of adopting a mucus secretory phenotype [90– is regulated by a different intracellular signaling pathway. 92] and (iii) express functional CFTR [93,94]. Clara cells are As a result, CaCC is a potentially important element in CF also prominent in human bronchioles, which, small in size, pathology. The recent demonstration that the TMEM16A gene but large in number, comprise a major portion of the airway encodes a CaCC [86–88] will allow the molecular behaviour surface in the human lung. Like airways in the mouse lung, of CaCC in different secretory epithelia to be elucidated. human bronchioles lack cartilage and submucosal glands. Interestingly, TMEM16A knockout mice exhibit phenotypic Of note, bronchioles play an important role in the initiation abnormalities consistent with a role in fluid and electrolyte of CF lung disease. For example, Tiddens et al. [95] and homeostasis [64–66]. It is therefore likely that the phenotype Stick et al. [96] observed severe distal airway disease in at of both CF mutant mice and CF patients is, at least in part, least 50% of CF patients before the age of 5 years, including dependent on TMEM16A expression and function. To what patients free of bacterial colonization. Notwithstanding impor- extent CaCC affects the phenotypic abnormalities observed in tant functional differences between human and murine airway different organs of CF mice, as well as in CF patients, remains epithelia, murine nasal airway epithelia is therefore struc- to be established. turally and functionally related to human bronchial airways, whereas murine bronchi are similar to human bronchioles. 4.13. CFTR expression in the mouse lung In alveolar epithelia, CFTR is expressed in type II epithelial cells [97], where it is involved in cAMP-stimulated fluid In mouse lung, Cftr mRNA expression is readily detected reabsorption [98]. Using an ingenious optical method, Lindert by RT-PCR and in situ hybridization [89], but expression et al. [99] demonstrated that basal fluid secretion in the levels are 10-fold lower than in nasal epithelium (Scholte et alveolar space was absent in Cftrtm1Unc homozygous mutant M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S163 mouse lung and sensitive to the CFTR inhibitor CFTRinh-172 in normal mice.

5. Lung pathology in CF mutant mice

CF lung pathology has been studied intensively using CF mutant mice and has been thoroughly reviewed [1–3]. Here we present a summary of the literature and some relevant recent data. Two issues are prominent in this field. First, to what extent can we compare lung pathology between humans and mice? Second, which parameters in a mouse model provide relevant information about CF pathology? In view of the major differences in size, cellular archi- Fig. 8. Lung inflammatory score is increased in Cftr tm1Eur FVB mutant mice. Paraffin sections were made from paraformaldehyde inflated lungs of tecture, physiology, host-pathogen interactions, lifestyle and tm1Eur lifespan, it is not surprising that CF mice do not reproduce FVB Cftr F508del mice, homozygous normal (+/+, n = 5) and mutant (dd, n = 6), age-matched (16–19 weeks comparable numbers of males and exactly the lung disease typical of CF patients. However, there females) littermates. The animals were kept in the Erasmus MC animal is considerable heterogeneity in the severity and progression facility, pathogen-free (FELASA+), in sterilized ventilated cages, provided of CF lung disease in humans. Another caveat is that we know ad lib with normal chow and sterilized acidified water. Five micron sections surprisingly little about the early development of CF lung were stained with hematoxylin/eosin and scored blind by an independent disease from direct observations of human airways in situ, pathologist, for oedema, haemorrhages, recruitment of poly-morphonuclear cells, macrophages, lymphocytes and plasma cells, lesions of alveolitis and especially the distal airways [95]. It is important to take these bronchitis, area of focal or diffuse consolidation, necrosis and metaplasia of considerations into account when comparing lung pathology pneumonocytes. The scores in each category are expressed and averaged on in mouse models and CF patients. a scale of zero (no abnormalities) through one (mild inflammation) to five Several groups reported an inflammatory lung phenotype (severe, destruction of the lung with lesions of alveolitis, severe bronchitis, associated with progressive tissue remodeling in different with necrosis and large areas of consolidation extended to all lobules). The results show mild, but significant inflammation in CF mutant mice compared unchallenged CF mutant mice [55,100–103]. The severity and to normal, under these conditions. presentation of this phenotype, most prominent in a C57Bl/6 backcross of the Cftrtm1Unc mouse [19], appears to be strain- and age-dependent. As discussed above, this phenotype will 5.1. A challenged mouse counts for two be influenced by animal husbandry. Here, we show that the inflammatory score based on quan- Waiting for CF mice to develop lung disease under stan- titative histology is moderately, but significantly increased in dard laboratory conditions is a tedious, ineffective method. adult (16–19 weeks) unchallenged Cftrtm1Eur F508del (FVB) Therefore, CF mutant mice of various strains have been mice kept in ventilated cages under pathogen-free conditions subjected to bacterial challenge protocols, resulting in a (Fig. 8). Inflammation, enhanced levels of the macrophage at- substantial, albeit bewildering, body of data. tractant CCL2/MCP-1 and increased numbers of macrophages CF mice were infected with Pseudomonas aeruginosa were reported in lung lavage fluid of a different colony of (PA), because this opportunistic pathogen causes life threaten- Cftrtm1Eur F508del (129/FVB) mice, which were reduced ing chronic infections in CF patients. At first sight this seems by the macrolide azythromycin [55]. Importantly, this in- a rather naïve approach, since host pathogen interactions are flammatory phenotype is not related to detectable pathogen very complex, differ in mice and humans and are certainly colonization, suggesting that CFTR deficiency by itself is not dependent on CFTR activity alone. Moreover, PA adapts sufficient to generate a pro-inflammatory environment in the rapidly to different environments, from hospital taps and mouse lung. At present it has not been established whether shower heads to patient lungs, forming mucoid variants and this inflammatory lung phenotype is exclusively attributed to biofilms. Nevertheless, several authors report enhanced mor- the abnormal behaviour of cells from the myeloid lineage, in tality and reduced clearance of acute PA infection in loss of particular macrophages [57,104], or whether abnormal proin- function CF mutants [105–108] and G551D mice [109]. What flammatory signaling in CFTR-deficient epithelial cells plays causes mortality in acute PA infection models is not obvious, a role [104]. However, it should be noted that these animals and should probably be distinguished from the intensity and are not kept in a sterile environment. Exposure to fecal resolution of inflammation. Interestingly, epithelial, but not bacteria, dust and other allergens are expected to be more macrophage, specific Cftr gene complementation is necessary challenging to CF mutant animals than to normal littermates. and sufficient to protect G551D CF mice from PA mortality Therefore, it might be misleading to call the inflammatory [110]. lung phenotype “spontaneous”. Moreover, animal husbandry, While on the whole data from acute PA infection experi- genetic background and gender likely affect the data, which ments are satisfactory, several concerns need to be addressed. explains why reports from different laboratories differ with It proved difficult to establish a true chronic infection with respect to the severity and presentation of the inflammatory clinical PA isolates. Mice, including CF mice, either rapidly lung phenotype. clear an intranasal or intra-tracheal dose of PA or they die S164 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 quickly depending on the dose and method of delivery. Dif- [122] reported that Cftrtm1Unc mice with different genetic ferent clinical isolates and delivery methods have therefore backgrounds did not exhibit goblet cell hyperplasia com- been tested [111,112]. Virulent PA strains embedded in agar pared to normal controls, and both groups of mice responded beads elicit a prolonged inflammatory reaction, simulating similarly to ovalbumin challenge. More recently, Touqui and chronic infection. Interestingly, responses to this challenge colleagues observed increased numbers of mucus producing are more severe in CF mutant mice [113], including the gut- cells and MUC5AC antigen in lungs of homozygous mu- corrected version of the Cftrtm1Unc knockout mouse [114]. tant C57Bl/6 Cftrtm1Unc mice [123]. Of note, the authors In the latter model, azithromycin reduced inflammation and demonstrated that this response was mediated by abnormal improved bacterial clearance after a challenge with alginate activation of cytosolic phospholipase A2 and could be reduced embedded PA [115]. Though this is an elegant way to create by a specific inhibitor of this enzyme [123]. Similar results a prolonged inflammatory challenge, it does not necessarily were obtained in Cftrtm1Eur mice (Scholte et al., unpublished reflect accurately the response of human CF epithelia to PA results). infection or how biofilms are formed and maintained in CF In summary, unchallenged CF mutant mice present a mod- lungs. est, but significant, increase in the number of mucus producing In a different approach, Pier and coauthors showed that cells in the proximal airways and mucus hypersecretion in delivery of PA in drinking water results in low level chronic lavage. However, overt airway plugging and air trapping such colonization of CF mouse airways, but not those of controls as that reported in CF patients and recently in CF ferrets and [116,117]. In this model, the inability to clear infection pigs is not observed. Because cells that stain with standard was linked to abnormal IL-1β signaling in epithelial cells goblet cell markers are confined to the proximal airways in the [118]. However, this model lacked the intense inflammatory mouse lung, careful analysis of sections is required. In view responses of mice infected with high doses of PA. of the known susceptibility of this parameter to environmental Intra-tracheal lipopolysaccharide (LPS) isolated from bac- and genetic factors care must be taken to provide properly teria can also be used to induce transient lung inflammation. controlled conditions. Similar to bacterial infection, this response is characterized The molecular biology of this response remains to be by activation of alveolar macrophages, cytokine production, investigated. On the basis of available data it is most likely a massive neutrophil influx, but little or no mortality. The an adaptive response of Clara cells to the pro-inflammatory responses seen in these models are primarily triggered by Toll environment created by CFTR deficiency. Whether a similar like receptors (TLR) of the innate immune system. In addition response occurs in human distal airways is not known. to TLR2 and TLR3 responses to LPS, bacterial flagellin is Finally, newborn mice overexpressing the β subunit of recognized by TLR5 and can be used to provoke an inflam- ENaC exhibit a considerably stronger version of the mucus matory response [119,120]. In CF mutant mice responses to hypersecretion phenotype (for further information see the LPS challenge are stronger than in normal mice [56,57]. accompanying article [17]).

5.2. Mucus production in CF mutant mouse lung 6. Bioactive lipids and CF lung disease

Mucus plugging of the airways is an important aspect of CF CFTR mutant mice display distinct abnormalities in the lung disease. This has been attributed to abnormal hydration metabolism of several lipids that are known to play a of CF mucus by the surface epithelium hyperabsorbing Na+ role in intra- and extracellular signaling of inflammation [78] and alternatively to a lack of bicarbonate secreting and apoptosis. Freedman et al. [124,125] first reported that cells, which prevents normal mucus hydration and secretion Cftrtm1Unc C57Bl/6 knockout mice show an increased ratio [33,34,121]. Furthermore, goblet cell hyperplasia and mucus of arachidonic acid (AA) to docosahexaenoic acid (DHA), hyper-secretion induced by inflammation are likely to play a which was reversed by oral DHA. Importantly, this finding role. correlated with a normalization of several phenotypic param- In the CF mouse nasal cavity, goblet cell hyperplasia eters in CF mutant mice [126]. More recently, Radzioch and does not correlate with infection [75]. In distal airways of colleagues confirmed the abnormal AA/DHA ratio in adult the normal mouse lung, we observe no goblet cells using Cftrtm1Unc C57Bl/6 CF mutant mice [101] and CF patients PAS/Alcian Blue, Muc5AC, and Clca3/Gob5 stains. However, [127]. Furthermore, these authors reported reduced levels of in proximal airways of the lung, second and third bifurcation major ceramide species in plasma and tissue samples in both bronchi, we do observe areas containing PAS/Alcian Blue and species, which were reversed by treatment with the vita- Clca3/Gob-5 positive cells. min A analogue fenretinide (N-(4-hydroxyphenyl)retinamide; Kent et al. [101] observed increased PAS staining together 4-HPR) [127]. In CF mutant mice, fenretinide reduced the with other lung alterations in unchallenged congenic C57Bl/6 sensitivity to intranasal challenge with PA [103] and reduced Cftrm1Unc mice. In an independent study, Durie et al. [19] bone abnormalities in CF knockout mice [128]. Whether confirmed progressive lung disease and airway accumula- fenretinide can reduce the development of progressive CF tion of mucus in the same mutant strain. Cftrtm1Hgu mice lung disease in patients remains to be established. Also the demonstrate mucus accumulation compared to controls after molecular mechanism explaining the relationship between this challenge with bacteria [107]. By contrast, Cressman et al. aspect of the CF phenotype and CFTR deficiency remains M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S165 to be determined. It is hypothesized that both oral DHA 7.1. CFTR activators and fenretinide act by reducing pro-inflammatory signaling through the PPAR pathway. In this context, Harmon et al. The mutant strains bearing F508del and G551D were made [129] recently demonstrated that Cftrtm1Unc (B6.129P2) ho- specifically to test compounds that aim to correct defec- mozygous knockout mice (males and females, four weeks tive processing and trafficking (CFTR correctors) or defective old) have attenuated PPARγ activity due to reduced levels of channel gating (CFTR potentiators). A caveat is that these mu- PPAR agonists, including 15-keto-PGE2. The authors further tations are introduced into the mouse CFTR sequence, which showed that the PPARγ agonist rosiglitazone, used to treat differs from the human sequence. Therefore, the properties of diabetes, corrected inflammatory and intestinal pathology in the mutant protein, including its interaction with therapeutic the mouse model [129]. In accordance with these observa- drugs, might differ from the human form. Indeed, the murine tions, we have reported previously that expression of the CFTR Cl− channel exhibits significant differences in its bio- 15-keto-PGE2 precursor, PGF2α and Cbr2 (lung carbonyl physical properties and regulation compared to human CFTR. reductase), the enzyme responsible for PGF2α production in These differences include (i) a smaller single-channel conduc- airway epithelial cells, are reduced in BALF of homozygous tance, (ii) a markedly different pattern of channel gating and tm1Eur Cftr lungs [130]. How these findings relate to defective (iii) insensitivity to AMP-PNP and pyrophosphate (PPi), two CFTR function is not yet fully understood. Borot et al. [131] agents that potentiate robustly the activity of human CFTR presented evidence suggesting that CFTR can form a com- [136–138]. Consistent with these data, a number of CFTR plex with cytosolic phospholipase A2 through the S100A10 potentiators (e.g. VRT-532, NPPB-AM and VX-770), which adaptor protein, which might explain the hyperactivity of augment strongly human CFTR channel gating are without cellular phospholipase A2 in CF mutant mice and its effect on effect on murine CFTR [139,140]. Moreover, Scott-Ward et mucin secretion [123]. We hypothesize that CFTR dysfunc- al. [141] demonstrated that transfer of both nucleotide-binding tion affects the enzymatic pathways involved, either directly domains (NBDs) of murine CFTR to human CFTR endowed or indirectly, through an adaptive process. the human CFTR Cl− channel with the gating behaviour and In a different CF mouse model carrying the Cftrtm1Hgu pharmacological properties of the murine CFTR Cl− chan- partially active knockout allele (Table 1), Gulbins and col- nel. These data suggest that human-murine CFTR chimeras leagues reported an age-dependent accumulation of ceramide might be employed to identify the binding sites of CFTR species in lung tissue, which correlates with progressive lung potentiators. inflammation and sensitivity to PA challenge [102]. Reduction of acid sphingomyelinase activity (ASM), either by mutation 7.2. CFTR correctors (Smpd−/−) or using the ASM inhibitor amitryptilin normal- ized ceramide levels and reduced lung pathology in these CF Because CFTR correctors target the cellular protein pro- mutant mice [102]. Further studies confirmed and extended cessing and trafficking machinery, they are less dependent these data in the mouse model [132]; initial experiments in on a direct interaction with the CFTR protein. As a re- CF patients demonstrated that amitriptylin is safe and might sult, a number of CFTR correctors have been successfully be effective in reducing CF disease [133]. At this time, tested in the F508del mouse model (De Jonge et al., in the apparent contradictory changes in ceramide levels in CF preparation). For example, miglustat, currently under eval- mutant mice between the Radzioch and Gulbins groups have uation as a therapy for Gauchers disease, shows limited, not been resolved using verified experimental protocols. It is but significant, efficacy in the Cftrtm1Eur mouse model by possible that differences in experimental protocols may cause reducing the amiloride response in the nasal epithelium a bias towards distinct subsets of ceramide species. [142] and increasing F508del CFTR processing efficiency in intestinal epithelium [143]. Moreover, intraperitoneal instil- 7. Pumping the pipeline, testing experimental therapies in lation of sildenafil and vardenafil, related phosphodiesterase mouse models 5 inhibitors, activated nasal Cl− conductance in Cftrtm1Eur F508del mice, but not Cftrtm1Cam null mice with the same A key application of CF mutant mice is to test experimental genetic background [144]. In a recent study, Robert et al. therapeutics. Elsewhere in this issue the development of [145] showed that glafenine, an off-patent drug with analgesic pharmacotherapy [134] and gene therapy for CF [135] are properties, improved the trafficking of human F508del-CFTR reviewed. New antagonists of ENaC have been developed and in BHK and CFBE41o− cells to 40% of normal values. are being tested in a mouse model of ENaC hyperactivity In isolated intestinal epithelia from Cftrtm1Eur mice, glafe- [17]. Compounds affecting bioactive lipid metabolism and nine increased the Isc response to forskolin plus genistein, downstream transcription factor activity were discussed in and it partially restored salivary secretion in vivo in mutant the previous section. The cloning of the CaCC TMEM16A mice, without causing adverse effects [145]. These results also presents new opportunities for intervention. As discussed illustrate that mutant CFTR mouse strains can play a signif- below, several experimental therapeutics have been tested in icant role in testing experimental drugs that are identified in CF mouse models. high-throughput screening programs [134]. S166 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

7.3. Anti-inﬂammatory drugs 9. Conclusion and perspective

In addition to drugs targeting the bioactive lipid pathways We can conclude that despite their obvious limitations, (see above, section 6), anti-inflammatory drugs can be investi- Cftr mutant mouse models have delivered. First, a better gated successfully in mouse models. The macrolide antibiotic understanding of CF pathology beyond clinical and cell azithromycin has anti-inflammatory action and reduced the culture studies. Second, novel and promising therapeutics hyper inflammatory condition in Cftrtm1Eur F508del mutant have been identified, which are now being tested in these mice [55], apparently through an action at the level of alve- models. Large animal models will certainly offer exciting new olar macrophages [146], rather than at the level of epithelial dimensions in this field. However, the versatility and relative cells [104]. Similar results were reported for the Cftrtm1Unc- affordability of mouse strains in genetic and physiological TgN(FABPCFTR) gut corrected CFTR null mouse model studies will ensure their continued use. challenged with a clinical PA isolate [115]. The synthetic triterpenoid CDDO reduced the hyper inflammatory pheno- Acknowledgements type of a R117H mutant mouse model, presumably through inhibition of the NF-κB pro-inflammatory pathway [147]. We thank EuroCareCF (LSHM-CT-2005-018932), the Dutch CF Foundation NCFS (R.B-O.) and the Dutch Maag 8. Modifier genes and new therapeutic targets Lever Darm Stichting MLDS (M.W.), for financial support. We gratefully acknowledge the expert assistance of Michel Not only environmental factors but also genetic elements Huerre (Institut Pasteur, Paris, France) in the quantitative determine CF pathology. First, the many different mutations analysis of histological data. in the CFTR gene found in CF patients give rise to variable levels of residual activity [27,148]. A different category of Conflict of interest genetic variations, often referred to as “modifier genes”, are polymorphisms in genes that affect CFTR function or All authors report no conflict of interest pathways involved in CF pathology [149–151]. Identification of modifier genes not only improves the CF diagnosis, but References also identifies novel therapeutic targets. CF mouse models and the toolbox allowing genetic experiments in this species [1] Grubb BR, Boucher RC. Pathophysiology of gene-targeted mouse offer unique opportunities to study modifier genes and their models for cystic fibrosis. Physiol Rev 1999;79(1 Suppl):S193–214. [2] Scholte BJ, Davidson DJ, Wilke M, De Jonge HR. Animal models of potential therapeutic effects. First, phenotypic differences cystic fibrosis. 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