1 s2.0-s1569199311600209-main

Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

www.elsevier.com/locate/jcf

Mouse models of cystic fibrosis: Phenotypic analysis andresearch applications

Martina Wilkea,1, Ruvalic M. Buijs-Offermanb, Jamil Aarbioub,2, William H. Colledgec,David N. Sheppardd, Lhousseine Touqui e, Alice Bot a, Huub Jornaa, Hugo R. de Jongea,

Bob J. Scholte b,*aErasmus MC, Biochemistry Department, 3000 CA Rotterdam, The NetherlandsbErasmus MC, Cell Biology Department, 3000 CA Rotterdam, The Netherlands

cPhysiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UKdUniversity of Bristol, School of Physiology & Pharmacology, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK

eUnité 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 ofexperimental data generated illuminates the complex multi-organ pathology of CF and raises new questions relevant to human disease. CFmice have also been used to test experimental therapies prior to clinical trials. This review recapitulates the major phenotypic traits of CF miceand highlights important new findings including aberrant alveolar macrophages, bone and cartilage abnormalities and abnormal bioactive lipidmetabolism. Novel data are presented on the intestinal and nasal physiology of F508del-CFTR CF mice backcrossed onto different geneticbackgrounds. Caveats, and sources of variability including age, gender and animal husbandry, are discussed. Interspecies differences limitcomparison of lung pathology in CF mice to the human disease. The recent development of genetically modified pigs and ferrets heralds theapplication 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

The life expectancy of cystic fibrosis (CF) patients in-creased from around 20 to 40 years over the past two decades,mainly due to improved nutrition and treatment of chroniclung disease. However, there is still an urgent unmet need toimprove the lifespan and quality of life of CF patients. Thecomplex multi-organ pathophysiology of secretory epitheliain CF is not yet completely understood, and a treatmentthat sufficiently corrects the primary defect remains to bedeveloped.

* Corresponding author: B.J. Scholte, PhD, Erasmus MC, Cell BiologyDepartment, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Tel.: +3110 7043205; fax: +31 10 7044743.

E-mail address: [email protected] (B.J. Scholte)1 Present address: Erasmus MC, Clinical Genetics Department.2 Present address: Galapagos NV, Leiden.

1569-1993/$ - see front matter © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Because access to relevant patient tissues is limited, genet-ically modified mice are widely used to study CF pathologyand experimental therapeutics. These models have proven tobe very useful, but they have important limitations, due toobvious physiological differences between humans and mice[1–3]. Recently, pig [4,5] and ferret [6] cystic fibrosis trans-membrane conductance regulator (CFTR) knockout animalswere reported, opening an exciting new field. Both modelsshow dominant intestinal disease [6,7], with severe perinatallethality. There is also evidence for reduced clearance ofbacterial lung infection in a limited number of long-lived CFpigs [8]. In both models, F508del-CFTR mutants are beingdeveloped, which will facilitate the testing of novel therapeu-tic strategies for CF. The challenge for the time to come willbe to manage intestinal disease in CF pigs and ferrets to allowbetter understanding of early lung disease, which is still anunderexplored area of CF pathology. While we can expect

M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171 S153

great progress from these models, their application is limitedby the considerable practical and financial issues involved.This paper will focus on CF mouse models, presenting an

update on new developments and novel data, in particular onthe properties of the mutant strains made available to the CFcommunity through the EuroCareCF project.

2. CF mutant mice, portrait of an unruly family

Shortly after the discovery of the CFTR gene, severalgroups set out to generate mouse strains with mutations in theCftr locus using the homologous recombination technologyand mouse embryonic stem cells pioneered by Mario Capec-chi, Martin Evans and Oliver Smithies who jointly receivedthe Nobel Prize for these innovations in 2007. Table 1 liststhe genetically modified mouse strains produced by differentgroups. Details of mouse strain nomenclature and availabilitycan be found at the Mouse Genome Informatics website(http://www.informatics.jax.org/). Table 2 summarizes brieflythe major phenotypic traits of CF mice in comparison with thehuman disease.Several loss of function, or “knock-out” models were made

by interrupting the mouse Cftr coding region with a selectablemarker. The Cftr tm1Hgu shows low level of residual activityof wild-type CFTR and lower mortality, compared to theother two frequently used knockout strains Cftr tm1Cam andCftr tm1Unc (Table 1).In addition, common CF causing mutations were intro-

duced into the mouse CFTR amino acid sequence. Threestrains with the F508del mutation in the mouse Cftr genewere generated, two of which carry an intronic insertion thatmay interfere with gene transcription (Table 1). By contrast,the Cftr tm1Eur allele was made by the two step “hit andrun” recombination procedure, which removes the selectablemarker. Therefore, this allele only contains the F508del mu-tation and has normal transcription rates in all tissues [9,10].All three strains have been successfully used in phenotypicstudies. The F508del mutation in the mouse Cftr contextcauses severely reduced amounts of fully glycosylated CFTRprotein (band C) similar to the human form, and a phenotypecomparable to CFTR loss of function mutants [9]. In culturedgallbladder epithelial cells from F508del mutant mice, thenumber of active CFTR Cl− channels observed in patch-clamp experiments is increased by incubating cells at lowtemperature [9], suggesting a similar temperature-dependentprocessing defect to the F508del mutation in human CFTR.Moreover, the G551D mutation which disrupts channel gatinghas the typical CF phenotype caused by loss of functionmutants [11]. The less severe phenotype of the F508del-and G551D-CFTR models when compared to CFTR knock-outs, particularly lower perinatal mortality due to intestinalobstruction, has been attributed to residual CFTR activity.To overcome the limitations imposed by the severe in-

testinal disease presented by many knockout strains, “gutcorrected” hybrid strains were created using a transgene ex-pressing CFTR from a promoter specific for the intestinalepithelium (Table 1).

The most recent and important addition to the CF mousefamily is a conditional (“loxed”) Cftr deletion that can becreated by cell specific expression of the cre recombinase [12].This genetically modified mouse will allow the contributionof different cell lineages to CF pathology to be elucidated.Although not a CFTR mouse model, genetically modi-

fied mice overexpressing the β subunit of the epithelial Na+

channel (ENaC) are important because they possess a lungphenotype resembling that of CF [13–16]. For further infor-mation about this mouse model and its applications, see Zhouet al. [17].

3. Mouse models, sources of variation and practicalconsiderations

CF mutant mice are generally much less fertile than normalmice, which makes it difficult to breed them from homozy-gous mutant pairs. This is partly due to frequent obstructionof the male vas deferens [18,19]. However, homozygous fe-males are also poor breeders. Occasionally mice can be bredsuccessfully from homozygous pairs. However, there is riskof “genetic drift”, the selection of compensating mutations ineither the background or the Cftr allele, which affect pheno-type in an unpredictable way. Breeding CF mutant mice fromheterozygous animals is therefore recommended strongly. Im-portantly, this breeding strategy yields age- and sex-matchednormal littermates, which are mandatory control for mostexperiments. Breeding requires considerable investment inDNA genotyping of all neonates by ear or tail clips, theidentification of individual animals by chip, toe- or ear-clipsand a database to track individual animals. Genotyping isprimarily required to identify heterozygous breeders, becausehomozygous mutants are easily recognized by their typicalwhite incisor teeth [20].There is consensus about the most prominent phenotypic

abnormalities of different Cftr mutant mice. Yet, close com-parison of published data from different laboratories showsconsiderable variation and even controversy about the natureand extent of lung disease. One obvious source of variationis the different Cftr mutations, some of which are associatedwith residual CFTR function (Table 1). Furthermore, Cftralleles have been bred onto different genetic backgrounds,which may cause subtle differences in phenotype, presumablybecause of the expression of different “modifier genes”. Com-plete genomes of several laboratory mouse strains are nowavailable (for further information, see Sanger mouse genomeproject, http://www.sanger.ac.uk/resources/mouse/genomes/).This resource will provide a new and powerful tool to ana-lyze phenotypic differences between CFTR mutant strains. Itwill also facilitate comparative studies of modifier genes andpotential therapeutic targets in the human population.Animal husbandry is a critical issue, especially when in-

vestigating innate adaptive responses (e.g. lung inflammation),which are by their very nature designed to reflect externalconditions. This can cause differences between strains, con-tribute to inter-individual variation, and lead to differencesin results between laboratories. Unfortunately, there is no

S154 M. Wilke et al. / Journal of Cystic Fibrosis Volume 10 Suppl 2 (2011) S152–S171

Table 1CFTR mutant mice

Mouse Mutation Cftr mRNA Genetic Survival to Body wt Contact Referencesbackground maturity

Null mutationsCftr 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 mutationsCftr 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 mutationsCftr 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 mutationsCftr 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 variantwas detected [168]. Transgenes are created by injection into mouse oocytes. General rules on mouse gene and strain nomenclature can be found at the MouseGenome 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 asplice variant introduced by the recombination event.

global standard protocol for animal husbandry. Laboratoriestherefore differ in the level of pathogen exposure. The Rot-terdam animal facility maintains a FELASA+ pathogen-freestandard using sterilized ventilated cages supplied with steril-ized, acidified water. Separation of animals causes stress andis avoided whenever possible; nesting material (e.g. sterilepaper tissue) is always provided. Strict light and dark regimesare maintained; nursing females are not disturbed until tendays after birth to reduce postnatal mortality. CF homozygousmutant animals do not travel very well and often die orsuffer excessive weight loss during long distance transport.Therefore, animals are moved at least two weeks in advanceof experiments to allow them to adapt. Under these condi-tions we can maintain colonies of Cftr tm1Eur F508del andCftr tm1Cam knockout mice in different genetic backgroundson normal chow at less than 20% mortality for homozygousmutants.Gender is another source of variation that should not be

underestimated. Male and female mice differ with respect tomany physiological responses relevant to CF studies. Thisis well illustrated in a recent study of a mouse model ofasthma [21]. Gender differences should be taken into account

when designing experiments and analyzing data. Finally, ageis another important variable. Its importance is demonstratedby the progressive nature of lung disease in several CF mousestrains. Together, these considerations place severe demandson the size and selection of experimental groups necessary togenerate reproducible data and statistical power.

4. CF mouse phenotype

The major phenotypic abnormalities of CF mutant miceare briefly summarized in Table 2. This brief overview doesnot do justice to all the available literature.In the mutant strains investigated, pancreatic pathology

is not a prominent feature, although it was reported in aC57Bl/6 Cftr knockout backcross [19]. Although pancreaticinsufficiency correlates with severe pathology, it is not seenin all CF patients. Similarly, male infertility caused byobstruction or absence of the vas deferens is frequent inCF mice depending on the strain and genetic background[18,19]. However, this pathology appears less severe than inhumans where absence of the vas deferens is observed inall male CF patients. Apparently, factors other than CFTR


Table 2Comparative CF pathology in human and mouse CF mutants

Human pathology [27,169] Tissues affected inCFTR mutant

Mouse pathology [1,2]

Recurrent bacterial lung infections,chronic inflammation, airwayremodeling, irreversible loss offunction

[170] Lung No CF type obstructive lung disease.Inflammation and tissue remodeling in aginganimals.Hyper-inflammation reduced clearance uponchallenge with bacteria or LPS.

Pro: [19,105,114,117,171,172]Con: [173]

Hyposecretion, viscous mucus,plugging

[174,175] Submucosal gland(nasal, bronchial)

Nasal and tracheal glands present, reducedcAMP-induced secretion in bronchial glands ofmutant mice

[81,82]

Bacterial biofilm, viscous/purulentmucus, defective mucociliaryclearance, goblet cell hyperplasia,inflammation, tissue remodeling

[176,177] Trachea, bronchialepithelium (ciliated/goblet)

Reduced mucociliary clearance (contested).Electrophysiology influenced by CaCC(TMEM16A).

Pro [84]Con: [83]

Distal airways involved in earlydisease, CFTR expression in Claracells

[95] Broncheoli (distalepithelium, Clara cells)

Spontaneous progressive remodeling andinflammation (strain- and age-dependent)

[19,102,172,178]

No known pathology, CFTRexpression in cultured type II cells

Alveoli (type I/II) Cftr expression in type II cells, abnormal fluidsecretion in mutant mice.

[97,99].

Meconium ileus, chronicmalabsorption, reduced bodyweight, failure to thrive (pancreasdeficiency), typicalelectrophysiology.

[31,179,180]

Intestine Lethal mucus plugging, lipid and bile acidreabsorption defect, reduced body weight, failure tothrive. Goblet cell hyperplasia in ileum, mucusaccumulation in intestinal crypts, meconium ileusequivalent, chronic inflammation, abnormalelectrophyiology.

[1,2,25,60,181,182]

Frequent biliary disease,progressive cirrhosis

[43,183–185]

Liver/Biliary duct No morbidity, progressive liver disease in aging CFKO mice biliary duct damage in KO mice uponinduced colitis

[19,186,187]

No frequent pathology [188] Kidney Functional CFTR in proximal tubule. In CF mice noovert pathology, abnormalities in Na+ and K+

transport, defective endocytosis.

[41,42,189,190]

Increased frequency of bile stones Gallbladder No cAMP-induced Cl− or water secretion [45,191]

Pancreatitis, atrophy, digestiveenzyme secretion deficiency,diabetes, abnormal bicarbonatesecretion and mucous plugging ofducts

[192,193] Pancreas Normal function and ductal fluid secretion,occasional duct plugging in Cftr tm1Unc mice

[19,194]

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 (defectivereabsorption duct)

[169] Sweat gland Foot pad glands only, no report of deficiency. [196]

Absent (CBAVD, male infertility100%)

Vas deferens Frequent plugging or absent (male infertility) [18,19]

Frequent obstruction Oviduct Frequent obstruction (female infertility)

No pathology reported Cornea Defective chloride permeability, enhanced amilorideresponse, attenuated uptake of Pseudomonas

[197–199]

Smooth muscle cell-relatedpathology in lung and diaphragmsuspected but not firmly established

Smooth muscle cell CFTR expression, altered function and innervation,degeneration in CF KO mice

[47–50,200]

No pathology reported Mast cells CFTR expression, enhanced presence in CF KOintestine, no evidence for cell autonomousabnormality

[59,60]

No pathology reported Macrophages CFTR expression, reduced capacity to killPseudomonas, abnormal proinflammatory cytokinesecretion

[51,55–57]


deficiency determine disease severity, in this case probablythe expression of alternative pathways for Cl− transport.

4.1. Intestinal disease in CF mouse models

Similar to humans, intestinal disease is prominent in allCF mutant mice. Depending on the strain and laboratoryconditions many mutant mice may die of intestinal blockagearound the time of weaning. This is a stochastic effect at-tributed to abnormal fluid and mucus secretion (meconiumileus equivalent). To reduce the frequency of intestinal ob-struction, mutant mice are often fed a liquid diet (Peptamen™)[22]. Because this drastic dietary intervention may affect thephenotype of both control and mutant mice, the use of amild laxative (Poly Ethylene Glycol; PEG) in combinationwith normal chow is recommended as an alternative [23].In the Rotterdam facility, Cftr tm1Eur and Cftr tm1Cam strains(FVB/n and C57Bl/6) survive well into adulthood (10–30weeks, 80–90%) on normal chow based on animal fat (HopeFarm™ SRMA 2131, Abfarm, Woerden, the Netherlands)combined with 6% (w/v) PEG in the drinking water for theknockout mice. Male and female mutant mice of all strainsshow a mild, but significant (10% average), reduction of bodyweight compared to normal age- and sex-matched littermates.Although there is no evidence of an essential lipid deficiency[24], mutant mice display reduced lipid reabsorption, which ismost pronounced in the Cftr tm1Cam strain [25]. Interestingly,the length and weight of the small intestine is larger in mutantCftr tm1Eur C57Bl/6 compared to wild-type (10%, N = 18,P < 0.01, Wilke et al., unpublished data [26]).In the mouse and human intestine, different proximal

to distal regions with distinct morphological and functionalcharacteristics can be distinguished. In the duodenum, bilefrom the gallbladder and digestive enzyme secretions from thepancreas are delivered through a common duct and mixed withthe acidic products of the stomach. In the jejunum and ileum,regulated fluid and mucus secretion maintain the optimalmilieu required for lipid, sugar and amino-acid absorption.The colon, including the caecum, is involved in mucus andbicarbonate secretion, and net ion and fluid reabsorption,compacting the intestinal content to faeces. In addition toproximal to distal gradients along the length of the intestine,the epithelium is organized vertically by invaginations calledcrypts and finger-like protrusions called villi. Stem cells at thebottom of the crypts continuously produce new epithelial cellsthat differentiate to form the different cell types, lining cryptsand villi.CFTR is expressed mainly in enterocytes where it is

involved in chloride and bicarbonate secretion. The role ofCFTR in intestinal ion transport [27,28] and in the smallintestine [29,30] has been reviewed. The ion transport prop-erties of the colon, including the role of CFTR were reviewedby Kunzelmann and Mall [31]. Here, we present general andnovel data relevant to this review on CF mouse models.Figure 1 demonstrates that the jejunum, ileum and colon

of backcrossed Cftrtm1Eur C57Bl/6 and FVB/n mice exhibita normal structure of crypts and villi. In the ileum a

distinct hyperplasia of goblet cells is observed, comparable topublished data for the original outbred strain (FVB/129) [10].Consistent with this observation, the expression of the gobletcell marker Clca3/Gob5 is increased 3-fold in the intestine ofCF mutant mice [32] and individual goblet cells appear largerthan normal, possessing “plumes” of mucous material. Mucusplugs are often seen in the crypts and lumen.A recent study in Cftrtm1Kth F508del (C57Bl/6) mice

highlights the crucial role of CFTR dependent bicarbonatesecretion in PGE2-induced intestinal mucus secretion and hy-dration [33,34]. In the proposed model, bicarbonate secretionby CFTR in enterocytes is required for the proper expansionof tightly packed mucus-calcium ion complexes, which aresecreted from vesicular granules by neighbouring goblet cells.In the absence of sufficient CFTR activity, the mucus does notunfold properly, which might play an important role in CFpathology. This indirect model for intestinal mucus secretionwas invoked because CFTR expression in goblet cells hasnot been demonstrated directly, whereas enterocytes showrelatively high levels of CFTR expression. If it can be shownthat CFTR-mediated bicarbonate secretion plays a similarrole in mucus release in human airway epithelia, this wouldhave important implications for our understanding of CF lungdisease.The mechanism causing the apparent goblet cell hyperpla-

sia in the ileum of CF mutant mice has not been established.However, abnormalities of mucus composition in Cftrtm1Unc

knockout mice [35], suggest an effect on goblet cell differen-tiation and function, possibly by an adaptive process.Immunohistochemistry using the R3195 anti-CFTR anti-

body (CR Marino, University of Tennessee) shows CFTRexpression on the apical membrane of enterocytes in villiand crypts of wild-type C57Bl/6 mice (Fig. 2). This signalis strongly reduced in Cftr tm1Eur F508del mutant mice andabsent in Cftr tm1Cam knockout mice. Similar results werereported using the original Cftr tm1Eur 129/FVB strain [9]and the Cftr partial knockout Cftr tm1Hgu (also designatedCftrTgh(neoim)Hgu) [36]. Consistent with these data, electro-physiological analyses demonstrate the absence or severereduction of CFTR function in CF mutant mice. For exam-ple, Fig. 3 shows that in three different genetic backcrossesof the Cftr tm1Eur F508del allele forskolin-induced (cAMP-dependent) short-circuit current (Isc), a measure of CFTR-mediated transepithelial ion transport, is reduced by morethan 80%. Figure 3 also reveals that the carbachol response(Ca2+-dependent) is reduced, which in ileal epithelia reflectsthe activity of Ca2+-activated K+ channels in the basolateralmembrane that facilitate CFTR-mediated Cl− secretion. InCFTR knockout mice of the same genetic background, boththe forskolin and carbachol responses are completely lacking,suggesting that the residual Isc response in intestinal epithelialof Cftr tm1Eur F508del mice reflects residual CFTR activity(De Jonge & Bot, unpublished).As for human CFTR, the intracellular transport defect

and severely reduced activity of F508del-CFTR in Cftr tm1Eur

homozygous mice is rescued by low temperature incubation[9]. Full restoration of wild-type CFTR protein processing


Fig. 1. Goblet cells in CF mutant mouse intestine. Comparison of representative paraffin sections from mouse jejunum, ileum and colon stained with Alcianblue 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 differencesin jejunum and colon between wild-type and mutant mice are observed (original magnification 20×).

and channel activity was observed in stripped ileal mucosafrom Cftr tm1Eur homozygous mice after 12 hours incubationat 26°C [37] (Wilke, de Jonge, in preparation). Based onthis observation, small molecules that rescue the cell surfaceexpression of mutant CFTR (termed CFTR correctors) arenow being tested in Cftr tm1Eur homozygous mice.

4.2. Salivary glands

Best and Quinton [38] first demonstrated that β-adrenergic(cAMP)-stimulated fluid secretion by salivary glands is a non-invasive and highly discriminative assay of CFTR function.In this assay, atropine is first injected to prevent the stimula-tion of secretion by cholinergic agonists before isoproterenolis injected to activate β-adrenergic-stimulated fluid secretion.Subsequently, Noël et al. [39] demonstrated that this assay canbe used to evaluate the efficacy with which small molecules

restore function to Cftr tm1Eur F508del mutants in vivo. Thisassay offers the advantage of detecting very low levels ofresidual function in mutant mice (Fig. 4). Moreover, it can beperformed repetitively in the same mouse, serving as a highlysensitive indicator of CFTR function [39]. Other aspects ofthe physiology of the salivary acini and glands in normal andCF mutant mice were described recently by Catalan et al.[40]. The authors’ data demonstrate that submandibular ductcells co-express CFTR and ENaC and that CFTR is requiredfor effective reabsorption of NaCl [40].

4.3. Kidney

CFTR is expressed in the proximal tubules of the mousekidney, localized to the apical and endosomal compartmentsof epithelial cells. Though no overt kidney pathology ispresent, abnormalities in urinary protein secretion were ob-


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 inCftr tm1Eur F508del FVB mutant littermates is much less intense and more diffuse (B). Tissue of the Cftr tm1Cam FVB knockout mouse serves as negativecontrol (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 asdescribed in [77]. Bars represent average initial short-circuit current (Isc) or the change of Isc after sequential addition of the cAMP agonist forskolin (forsk),the Ca2+ agonist carbachol and glucose. Isc measurements were performed on ileum derived from the Cftr tm1Eur F508del strain in three different congenicbackgrounds (F13) A: FVB/n (F13) B: C57Bl/6 (F13) C: 129/sv (F12) comparing homozygous mutant (dd, open bars) with homozygous normal (++, blackbars). 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 adultmales 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 Cftr tm1Cam knockout mice, suggesting a role forCFTR in membrane trafficking and brush border matura-tion. To a lesser extent these abnormalities were also seenin Cftr tm2Cam and Cftr tm1Eur F508del mice, where they areattributed to partial processing of the mutant protein in thistissue [41,42].

4.4. Liver

CF associated liver disease (CFLD) is an increasingclinical problem, affecting more than 50% of adult CFpatients. CFTR is expressed in cholangiocytes, where it ispresumably involved in fluid homeostasis in the biliary ductsystem [43]. In CF mice, no obvious liver pathology isdetected. Chronic administration of cholate increases weaklybile production in CF mice when compared with control mice.

Moreover, by lowering the phospholipid-to-bile salt ratio,chronic cholate administration increases the cytotoxicity ofbile produced by CF mice. By contrast, either acute or chronicursodeoxycholic acid (UDCA) administration induces a bilesalt-specific choleresis in vivo in mice that is independentof functional CFTR [44]. These observations may help toexplain the beneficial effects of oral UDCA therapy incholangiopathies including CFLD.

4.5. The gallbladder

In mice, the gallbladder is an accessible and relativelysimple epithelial tissue with both secretory and absorptiveproperties. We have previously shown that CFTR-dependentforskolin-induced Isc (�Isc forsk) is absent in Cftr tm1Cam

C57Bl/6/129 knockout mice, but not normal mice [45,46]. We


Fig. 4. Isoproterenol-stimulated salivary gland secretion. Salivary glandsecretion was measured in anesthetized mice for 21 minutes as describedin [38]. Mice were pretreated with atropine to block cross-stimulation ofcholinergic-stimulated secretion. Isoproterenol was used in the presenceof atropine to stimulate adrenergic secretion. There was no significantdifference between the maximum secretion of FVB wild-type (WT) male (n= 6) and female mice (n = 4). The CF mutant homozygous Cftr tm1Eur FVBF508del (DF/DF) littermates (DF/DF: male: n = 123, female: n = 63) andhomozygous Cftr tm1Cam FVB knockout mice (−/−: male: n = 7, female: n =11) of either sex had a very low response compare to wild-type (P < 0.001).The Cftr tm1Eur F508del mice do not differ from the Cftr tm1Cam .

further showed that unstimulated gallbladders from normalmice reabsorb fluid to concentrate bile, whereas they secretefluid when stimulated with forskolin [45]. By contrast, ingallbladders from Cftr tm1Cam knockout mice forskolin abol-ished fluid reabsorption without stimulating fluid secretion,demonstrating that fluid secretion is CFTR-dependent [45].In Cftr tm1Eur 129/FVB F508del mice, forskolin-elicited Iscresponses were reduced by 80% [9]. Because Cftr tm1Cam

FVB mice also show low, but detectable, forskolin-elicited Iscresponses through a CFTR-independent pathway, the residualforskolin-induced Isc is not mediated by F508del-CFTR (DeJonge, Bot unpublished data). Here, we present previouslyunpublished data demonstrating that the forskolin-induced

Fig. 5. Fluid transport in mouse gallbladder. Net fluid transport rates (Jv)in isolated gallbladders of the 129/FVB Cftr tm1Eur F508del strain [10] weremeasured as described in [45]. Negative values show initial transport of fluidfrom the lumen of the bladder to the bath. After addition of the cAMPagonist forskolin, Jv changes to positive values in normal mice, i.e. netfluid secretion towards the lumen. By contrast, in homozygous Cftr tm1Eur

F508del littermates transport towards the bath is inhibited and there is no netsecretion into the lumen.

inhibition of reabsorption and absence of fluid secretion isalso observed in Cftr tm1Eur 129/FVB F508del mice (Fig. 5).This indicates that CFTR activity is insufficient for net fluidsecretion in this CF mutant mouse. Gallbladder epithelialcells contain a large number of secretory granules containingmucins. Peters et al. [46] demonstrated that basal and ATP-stimulated mucus release from cultured gallbladder epithelialcells of Cftr tm1Eur 129/FVB mice are quantitatively normal.However, the potential role of CFTR-mediated bicarbonate se-cretion in mucus release in this model system and the physicalproperties of the secreted mucus remain to be investigated.

4.6. Smooth muscle cells and CFTR function

CFTR is expressed in smooth muscle cells and skeletalmuscle myotubes, colocalised with the sarcoplasmic reticu-lum [47–50]. Recently Divangahi et al. [47] demonstrated thatCFTR deficient myotubes display increased Ca2+ signalingupon depolarisation and excessive NF-κB translocation in re-sponse to inflammatory stimuli. Furthermore, degeneration ofdiaphragmatic muscle was observed selectively in Cftr tm1Unc

mice challenged with Pseudomonas aeruginosa, which chron-ically infects the lungs of CF patients [47]. The authorssuggest therefore that reduced muscle strength, particularlyof the diaphragm, as frequently observed in CF patients,may contribute to CF lung disease. Because smooth musclecells of the airways and arteries express functional CFTR[48–50], elements of the CF lung phenotype (e.g. wheezing,hyperreactivity and progressive tissue remodeling) might alsoresult from the dysfunction of smooth muscle.

4.7. The cellular immune system is abnormal in CF mutantmice

Several authors have reported abnormal behaviour ofalveolar and peritoneal macrophages in CF mutant mice. Di etal. [51] demonstrated defective killing of bacteria after uptake,possibly due to dysfunctional CFTR-dependent acidificationof endosomes/phagosomes [52], though this explanation iscontested [53,54]. Leal and co-authors showed that peritonealand alveolar macrophages from Cftr tm1Eur mice display a pro-inflammatory bias after stimulation in culture, characterizedby 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 tolipopolysaccharide from Pseudomonas aeruginosa (PA LPS).These data strongly suggest that macrophages from CFTRmutant mice display defects that might, at least in part,explain the pro-inflammatory lung phenotype in CF mutantmice. Other cells of the myeloid lineage (e.g. neutrophils[58]; mast cells [59,60] and dendritic cells [61]) are nowunder investigation in CF patients and mouse models. If itcan be demonstrated that such defects are prominent in CFpatients and cell-autonomous, not adaptive, in origin, thiswould support the conclusion that CF is a disease of theimmune system.


4.8. Abnormal bone and cartilage formation in CF mutantmice

Bonvin et al. [62] first showed that rings of tracheal carti-lage in CF mutant mice (Cftr tm1Unc knockout and Cftr tm1Eur

F508del (129/FVB)) are frequently incomplete at birth. Be-cause, neonatal CFTR knockout ferrets [6] and pigs [4,5] alsoexhibit abnormal tracheal cartilage, this developmental abnor-mality in CFTR deficient animals is the first to be verified inmultiple species. An explanation for this phenomenon is notreadily available. Interestingly, mouse chondrocytes, expressfunctional CFTR [63], suggesting a cell autonomous defectin cartilage formation. However, a response to local and sys-temic inflammation cannot be excluded. Of note, a similar, butequally unexplained, phenotype was reported in TMEM16Amutant mice [64–66], which lack the Ca2+-activated Cl−channel (see above, section 4.12).Reduced bone density in CFTR mutant mice was described

initially by Dif et al. [67] and confirmed by others [68,69].It is currently unknown whether this defect is caused bya cell autonomous CFTR-related defect of bone formingosteoblasts or bone degrading osteoclasts. Alternatively, thisdefect might be secondary to systemic inflammation andabnormal bioactive lipid metabolism (ceramides, vitamin D).Low amounts of CFTR protein are expressed in mouseand human osteoblasts and odontoblasts, together with lowlevels of CFTR mRNA [70]. The location of CFTR in thesecells was mainly intracellular, suggesting that CFTR mightfunction as an anion transporter in intracellular organelles.The dysfunction of the ClC transporter ClC-7 in osteopetrosis[71,72] raises the possibility that CFTR dysfunction inorganelle membranes of bone cells might underlie the changesin bone metabolism in CFTR-deficient mice and CF patients,leading to osteopenia.The clinical relevance of these data remains to be estab-

lished. To date, no cartilage abnormalities have been reportedin CF patients. However, osteopenia and osteoporosis areserious problems in CF patients [73,74]. Taken together, thedata suggest that animal model studies of CF-related bonedisease might lead to novel treatments for CF patients.

4.9. Nasal airways of CF mutant mice

The nasal epithelium is an important focus of CF mousemodel research, because of its similarities in structure andfunction with the human bronchial airway epithelium. Indeed,early studies of the nasal epithelium demonstrated electro-physiological abnormalities consistent with CFTR dysfunc-tion in CF mutant mice (i.e. abrogated cAMP-stimulated Cl−conductance and enhanced amiloride-sensitive Na+ currentsin microperfusion studies, Table 2).More recent studies draw attention to the complex ar-

chitecture of the mouse nasal epithelium and the effects ofCFTR dysfunction on this tissue [75,76]. The mouse nasalcavity is a septated cartilage and bone structure covered withnasal airway epithelium (NAE), mainly ciliated and mucusproducing (goblet) cells. However, the cavity lining the up-

per part of the skull is covered with olfactory epithelium(OE). The OE consists of a multilayer of neuronal cellsprojecting sensory cilia from the surface, covered with alayer of specialized epithelial cells called sustentacular cells(Fig. 6A and B). This tissue expresses CFTR and ENaC andis thought to contribute to fluid and ion homeostasis acrossthe OE [76]. Under the OE and the NAE, there are numeroussubmucosal glands embedded in connective tissue. CF mutantmice display an age-dependent progressive degeneration ofthe olfactory neuronal layer that is evident in adult mutantmice from the age of 16–24 weeks. This was first reportedfor Cftr tm1Unc knockout and CftrTgHm1 G551D mutant miceon different genetic backgrounds [75,76]. Furthermore, anincrease of NAE thickness and apparent hyperplasia of mucusproducing cells is observed in CftrTgHm1 G551D mutants[75]. At age 14–20 weeks, Cftr tm1Eur FVB mice did not showhistological evidence of OE degeneration in our facility (Fig.6A, B), indicating that this abnormality is not only age-, butalso mouse strain-dependent.When the OE and underlying tissue is removed from

the nasal cavity, it can be inserted into a small apertureUssing chamber for ion transport studies [77]. In all five CFmutant strains tested, we observed a significantly increased(P < 0.01) Isc response to amiloride (�Isc amil) (Fig. 7). Thisincludes the Cftr tm1Eur F508del mutation on different geneticbackgrounds and the Cftr tm1Cam knockout allele on mixedbackground (C57Bl/6-129) and FVB. An enhanced �Isc amiland �PDamil is also observed in human CF airway epithelia,supporting the hypothesis that CF airway disease results, atleast in part, from ENaC hyperactivity. Consequently, ENaCis a primary therapeutic target in CF mutant mice [15,78].In excised nasal OE from Cftr tm1Eur F508del mice, CftrmRNA expression is normal (Scholte et al., data not shown),whereas CFTR protein expression is much reduced (Fig. 6Aand B) and in Cftr tm1Cam knockout mice this signal is absent(not shown). The absence of olfactory marker proteins fromCFTR-expressing cells argues that CFTR is not expressedin ciliated sensory neurons, but in a subset of microvilloussecondary chemosensory cells (named brush cells), where itis co-localized with ENaC (De Jonge et al, unpublished data).Whether this cell type represents a cell specialized for fluidand electrolyte transport or plays a role in CFTR-dependentregeneration of the olfactory system [79,80] remains to beestablished.Forskolin responses observed in different mouse strains are

complex parameters that cannot, at face value, be attributedto CFTR activity. In Cftr tm1Cam C57Bl/6-129 knockout mice,we observe a forskolin response comparable to normallittermates. However, in Cftr tm1Cam FVB mutant mice thisresponse is virtually absent (Fig. 7). These data suggest thata cAMP-stimulated Cl− conductance other than CFTR ispresent in the OE of Cftr tm1Cam C57Bl/6-129 mice, but isabsent from Cftr tm1Cam FVB mice. Studies are underway toidentify this cAMP-stimulated Cl− conductance.In all Cftr tm1Eur F508del backcrossed strains the nasal

forskolin-stimulated Isc in mutant mice is comparable tonormal littermates. Because this includes the FVB strain


Fig. 6. CFTR immunostaining in mouse olfactory epithelium (OE) and trachea. In wild-type FVB (FVB WT) mice punctuated CFTR staining in brush cells ofthe olfactory epithelium (OE) (A) and diffuse CFTR staining in ciliated cells of the trachea (C) is observed. In homozygous mutant littermates (FVB F508delCftr 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 insections from Cftr tm1Cam FVB knockout mice, stained in parallel (not shown).

that does not show a response with the Cftr tm1Cam knockoutallele, one possible explanation is that the nasal forskolin-stimulated Isc reflects residual CFTR activity resulting frompartial processing of the mutant protein [9]. However, thenasal forskolin-stimulated Isc does not influence the enhancedamiloride response in this mutant strain (Fig. 7). Togetherwith the immunohistology data (Fig. 6A, B), this argues thatthere is not a linear relationship between the nasal forskolin-stimulated Isc and the number of active CFTR channels.

4.10. Submucosal glands in CF mutant mice

Mice have composite serous and mucous submucosalglands, not only in the nasal cavity, but also in the upperpart of the trachea. This is relevant, because abnormalsubmucosal glands are thought to play an important rolein the development of CF airway disease, contributing tofluid hypo-secretion and the abnormal consistency of mucusin the proximal airways [81,82]. Secretion of fluid fromsubmucosal glands is dependent on two complementarycombinations of transport systems, in particular basolateralK+ channels and apical CFTR Cl− channels, regulated by

the intracellular calcium and cAMP signaling pathways,respectively. The cAMP-stimulated component is lackingin tracheal glands from CF mutant mice, and is thereforethought to be CFTR-dependent [81,82]. The contributionof CFTR-mediated secretion by submucosal glands to CFmouse pathology is unclear, but this experimental paradigmconstitutes a valid model to study CFTR function and testdrugs targeting CFTR.It is important to note that the submucosal glands of the

nasal cavity, and their contribution to its CF phenotype, havenot yet been studied. It is conceivable, that nasal submucosalcells have properties different from tracheal glands, since theyhave developed in a different cellular context.

4.11. CFTR expression in the mouse trachea

The cellular architecture of the pseudostratified columnarmouse tracheal epithelium is similar to that of the nasalairways. It is dominated by CFTR-expressing ciliated cells(Fig. 6C and D), interspersed with infrequent mucus secretingneuroendocrine and Clara cells. Mucociliary clearance andepithelial ion transport are not significantly affected in tracheal


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. HomozygousCftr 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 significantlybetween 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].Interestingly, in tracheal epithelia the Ca2+-activated Cl−channel (CaCC) is the dominant apical membrane Cl−channel, CFTR activity is only observed after maximalstimulation of CaCC [85].

4.12. CaCC: the dark horse in CF pathology

CaCC is expressed in the apical membrane of epitheliaaffected by CF, where it operates in parallel with CFTR, butis regulated by a different intracellular signaling pathway.As a result, CaCC is a potentially important element in CFpathology. The recent demonstration that the TMEM16A geneencodes a CaCC [86–88] will allow the molecular behaviourof CaCC in different secretory epithelia to be elucidated.Interestingly, TMEM16A knockout mice exhibit phenotypicabnormalities consistent with a role in fluid and electrolytehomeostasis [64–66]. It is therefore likely that the phenotypeof both CF mutant mice and CF patients is, at least in part,dependent on TMEM16A expression and function. To whatextent CaCC affects the phenotypic abnormalities observed indifferent organs of CF mice, as well as in CF patients, remainsto be established.

4.13. CFTR expression in the mouse lung

In mouse lung, Cftr mRNA expression is readily detectedby RT-PCR and in situ hybridization [89], but expressionlevels are 10-fold lower than in nasal epithelium (Scholte et

al., not shown). Immunohistological detection of CFTR ex-pression in the lung, confirmed by using CFTR knockout miceas a negative control, is not feasible with current methods.Mouse bronchi possess ciliated and mucus producing cells ina declining proximal to distal distribution, whereas airwaysin the mouse lung are dominated by Clara cells, constituting60–80% of the total epithelial cell population. Clara cells arenon-ciliated secretory cells that (i) display phenotypic adapta-tion and proliferative capacity during injury and inflammation,(ii) are capable of adopting a mucus secretory phenotype [90–92] and (iii) express functional CFTR [93,94]. Clara cells arealso prominent in human bronchioles, which, small in size,but large in number, comprise a major portion of the airwaysurface in the human lung. Like airways in the mouse lung,human bronchioles lack cartilage and submucosal glands.Of note, bronchioles play an important role in the initiationof CF lung disease. For example, Tiddens et al. [95] andStick et al. [96] observed severe distal airway disease in atleast 50% of CF patients before the age of 5 years, includingpatients free of bacterial colonization. Notwithstanding impor-tant functional differences between human and murine airwayepithelia, murine nasal airway epithelia is therefore struc-turally and functionally related to human bronchial airways,whereas murine bronchi are similar to human bronchioles.In alveolar epithelia, CFTR is expressed in type II epithelial

cells [97], where it is involved in cAMP-stimulated fluidreabsorption [98]. Using an ingenious optical method, Lindertet al. [99] demonstrated that basal fluid secretion in thealveolar space was absent in Cftr tm1Unc homozygous mutant


mouse lung and sensitive to the CFTR inhibitor CFTRinh-172in normal mice.

5. Lung pathology in CF mutant mice

CF lung pathology has been studied intensively using CFmutant mice and has been thoroughly reviewed [1–3]. Herewe present a summary of the literature and some relevantrecent data. Two issues are prominent in this field. First, towhat extent can we compare lung pathology between humansand mice? Second, which parameters in a mouse modelprovide relevant information about CF pathology?In view of the major differences in size, cellular archi-

tecture, physiology, host-pathogen interactions, lifestyle andlifespan, it is not surprising that CF mice do not reproduceexactly the lung disease typical of CF patients. However, thereis considerable heterogeneity in the severity and progressionof CF lung disease in humans. Another caveat is that we knowsurprisingly little about the early development of CF lungdisease from direct observations of human airways in situ,especially the distal airways [95]. It is important to take theseconsiderations into account when comparing lung pathologyin mouse models and CF patients.Several groups reported an inflammatory lung phenotype

associated with progressive tissue remodeling in differentunchallenged CF mutant mice [55,100–103]. The severity andpresentation of this phenotype, most prominent in a C57Bl/6backcross of the Cftr tm1Unc mouse [19], appears to be strain-and age-dependent. As discussed above, this phenotype willbe influenced by animal husbandry.Here, we show that the inflammatory score based on quan-

titative histology is moderately, but significantly increased inadult (16–19 weeks) unchallenged Cftr tm1Eur F508del (FVB)mice kept in ventilated cages under pathogen-free conditions(Fig. 8). Inflammation, enhanced levels of the macrophage at-tractant CCL2/MCP-1 and increased numbers of macrophageswere reported in lung lavage fluid of a different colony ofCftr tm1Eur F508del (129/FVB) mice, which were reducedby the macrolide azythromycin [55]. Importantly, this in-flammatory phenotype is not related to detectable pathogencolonization, suggesting that CFTR deficiency by itself issufficient to generate a pro-inflammatory environment in themouse lung. At present it has not been established whetherthis inflammatory lung phenotype is exclusively attributed tothe abnormal behaviour of cells from the myeloid lineage, inparticular macrophages [57,104], or whether abnormal proin-flammatory signaling in CFTR-deficient epithelial cells playsa role [104]. However, it should be noted that these animalsare not kept in a sterile environment. Exposure to fecalbacteria, dust and other allergens are expected to be morechallenging to CF mutant animals than to normal littermates.Therefore, it might be misleading to call the inflammatorylung phenotype “spontaneous”. Moreover, animal husbandry,genetic background and gender likely affect the data, whichexplains why reports from different laboratories differ withrespect to the severity and presentation of the inflammatorylung phenotype.

Fig. 8. Lung inflammatory score is increased in Cftr tm1Eur FVB mutantmice. Paraffin sections were made from paraformaldehyde inflated lungs ofFVB Cftr tm1Eur F508del mice, homozygous normal (+/+, n = 5) and mutant(dd, n = 6), age-matched (16–19 weeks comparable numbers of males andfemales) littermates. The animals were kept in the Erasmus MC animalfacility, pathogen-free (FELASA+), in sterilized ventilated cages, providedad lib with normal chow and sterilized acidified water. Five micron sectionswere stained with hematoxylin/eosin and scored blind by an independentpathologist, for oedema, haemorrhages, recruitment of poly-morphonuclearcells, macrophages, lymphocytes and plasma cells, lesions of alveolitis andbronchitis, area of focal or diffuse consolidation, necrosis and metaplasia ofpneumonocytes. The scores in each category are expressed and averaged ona scale of zero (no abnormalities) through one (mild inflammation) to five(severe, destruction of the lung with lesions of alveolitis, severe bronchitis,with necrosis and large areas of consolidation extended to all lobules). Theresults show mild, but significant inflammation in CF mutant mice comparedto normal, under these conditions.

5.1. A challenged mouse counts for two

Waiting for CF mice to develop lung disease under stan-dard laboratory conditions is a tedious, ineffective method.Therefore, CF mutant mice of various strains have beensubjected to bacterial challenge protocols, resulting in asubstantial, albeit bewildering, body of data.CF mice were infected with Pseudomonas aeruginosa

(PA), because this opportunistic pathogen causes life threaten-ing chronic infections in CF patients. At first sight this seemsa rather naïve approach, since host pathogen interactions arevery complex, differ in mice and humans and are certainlynot dependent on CFTR activity alone. Moreover, PA adaptsrapidly to different environments, from hospital taps andshower heads to patient lungs, forming mucoid variants andbiofilms. Nevertheless, several authors report enhanced mor-tality and reduced clearance of acute PA infection in loss offunction CF mutants [105–108] and G551D mice [109]. Whatcauses mortality in acute PA infection models is not obvious,and should probably be distinguished from the intensity andresolution of inflammation. Interestingly, epithelial, but notmacrophage, specific Cftr gene complementation is necessaryand sufficient to protect G551D CF mice from PA mortality[110].While on the whole data from acute PA infection experi-

ments are satisfactory, several concerns need to be addressed.It proved difficult to establish a true chronic infection withclinical PA isolates. Mice, including CF mice, either rapidlyclear an intranasal or intra-tracheal dose of PA or they die


quickly depending on the dose and method of delivery. Dif-ferent clinical isolates and delivery methods have thereforebeen tested [111,112]. Virulent PA strains embedded in agarbeads elicit a prolonged inflammatory reaction, simulatingchronic infection. Interestingly, responses to this challengeare more severe in CF mutant mice [113], including the gut-corrected version of the Cftr tm1Unc knockout mouse [114].In the latter model, azithromycin reduced inflammation andimproved bacterial clearance after a challenge with alginateembedded PA [115]. Though this is an elegant way to createa prolonged inflammatory challenge, it does not necessarilyreflect accurately the response of human CF epithelia to PAinfection or how biofilms are formed and maintained in CFlungs.In a different approach, Pier and coauthors showed that

delivery of PA in drinking water results in low level chroniccolonization of CF mouse airways, but not those of controls[116,117]. In this model, the inability to clear infectionwas linked to abnormal IL-1β signaling in epithelial cells[118]. However, this model lacked the intense inflammatoryresponses of mice infected with high doses of PA.Intra-tracheal lipopolysaccharide (LPS) isolated from bac-

teria can also be used to induce transient lung inflammation.Similar to bacterial infection, this response is characterizedby activation of alveolar macrophages, cytokine production,a massive neutrophil influx, but little or no mortality. Theresponses seen in these models are primarily triggered by Tolllike receptors (TLR) of the innate immune system. In additionto TLR2 and TLR3 responses to LPS, bacterial flagellin isrecognized by TLR5 and can be used to provoke an inflam-matory response [119,120]. In CF mutant mice responses toLPS challenge are stronger than in normal mice [56,57].

5.2. Mucus production in CF mutant mouse lung

Mucus plugging of the airways is an important aspect of CFlung disease. This has been attributed to abnormal hydrationof CF mucus by the surface epithelium hyperabsorbing Na+

[78] and alternatively to a lack of bicarbonate secretingcells, which prevents normal mucus hydration and secretion[33,34,121]. Furthermore, goblet cell hyperplasia and mucushyper-secretion induced by inflammation are likely to play arole.In the CF mouse nasal cavity, goblet cell hyperplasia

does not correlate with infection [75]. In distal airways ofthe normal mouse lung, we observe no goblet cells usingPAS/Alcian Blue, Muc5AC, and Clca3/Gob5 stains. However,in proximal airways of the lung, second and third bifurcationbronchi, we do observe areas containing PAS/Alcian Blue andClca3/Gob-5 positive cells.Kent et al. [101] observed increased PAS staining together

with other lung alterations in unchallenged congenic C57Bl/6Cftrm1Unc mice. In an independent study, Durie et al. [19]confirmed progressive lung disease and airway accumula-tion of mucus in the same mutant strain. Cftr tm1Hgu micedemonstrate mucus accumulation compared to controls afterchallenge with bacteria [107]. By contrast, Cressman et al.

[122] reported that Cftr tm1Unc mice with different geneticbackgrounds did not exhibit goblet cell hyperplasia com-pared to normal controls, and both groups of mice respondedsimilarly to ovalbumin challenge. More recently, Touqui andcolleagues observed increased numbers of mucus producingcells and MUC5AC antigen in lungs of homozygous mu-tant C57Bl/6 Cftr tm1Unc mice [123]. Of note, the authorsdemonstrated that this response was mediated by abnormalactivation of cytosolic phospholipase A2 and could be reducedby a specific inhibitor of this enzyme [123]. Similar resultswere obtained in Cftr tm1Eur mice (Scholte et al., unpublishedresults).In summary, unchallenged CF mutant mice present a mod-

est, but significant, increase in the number of mucus producingcells in the proximal airways and mucus hypersecretion inlavage. However, overt airway plugging and air trapping suchas that reported in CF patients and recently in CF ferrets andpigs is not observed. Because cells that stain with standardgoblet cell markers are confined to the proximal airways in themouse lung, careful analysis of sections is required. In viewof the known susceptibility of this parameter to environmentaland genetic factors care must be taken to provide properlycontrolled conditions.The molecular biology of this response remains to be

investigated. On the basis of available data it is most likelyan adaptive response of Clara cells to the pro-inflammatoryenvironment created by CFTR deficiency. Whether a similarresponse occurs in human distal airways is not known.Finally, newborn mice overexpressing the β subunit of

ENaC exhibit a considerably stronger version of the mucushypersecretion phenotype (for further information see theaccompanying article [17]).

6. Bioactive lipids and CF lung disease

CFTR mutant mice display distinct abnormalities in themetabolism of several lipids that are known to play arole in intra- and extracellular signaling of inflammationand apoptosis. Freedman et al. [124,125] first reported thatCftr tm1Unc C57Bl/6 knockout mice show an increased ratioof arachidonic acid (AA) to docosahexaenoic acid (DHA),which was reversed by oral DHA. Importantly, this findingcorrelated with a normalization of several phenotypic param-eters in CF mutant mice [126]. More recently, Radzioch andcolleagues confirmed the abnormal AA/DHA ratio in adultCftr tm1Unc C57Bl/6 CF mutant mice [101] and CF patients[127]. Furthermore, these authors reported reduced levels ofmajor ceramide species in plasma and tissue samples in bothspecies, which were reversed by treatment with the vita-min A analogue fenretinide (N-(4-hydroxyphenyl)retinamide;4-HPR) [127]. In CF mutant mice, fenretinide reduced thesensitivity to intranasal challenge with PA [103] and reducedbone abnormalities in CF knockout mice [128]. Whetherfenretinide can reduce the development of progressive CFlung disease in patients remains to be established. Also themolecular mechanism explaining the relationship between thisaspect of the CF phenotype and CFTR deficiency remains


to be determined. It is hypothesized that both oral DHAand fenretinide act by reducing pro-inflammatory signalingthrough the PPAR pathway. In this context, Harmon et al.[129] recently demonstrated that Cftr tm1Unc (B6.129P2) ho-mozygous knockout mice (males and females, four weeksold) have attenuated PPARγ activity due to reduced levels ofPPAR agonists, including 15-keto-PGE2. The authors furthershowed that the PPARγ agonist rosiglitazone, used to treatdiabetes, corrected inflammatory and intestinal pathology inthe mouse model [129]. In accordance with these observa-tions, we have reported previously that expression of the15-keto-PGE2 precursor, PGF2α and Cbr2 (lung carbonylreductase), the enzyme responsible for PGF2α production inairway epithelial cells, are reduced in BALF of homozygousCftr tm1Eur lungs [130]. How these findings relate to defectiveCFTR function is not yet fully understood. Borot et al. [131]presented evidence suggesting that CFTR can form a com-plex with cytosolic phospholipase A2 through the S100A10adaptor protein, which might explain the hyperactivity ofcellular phospholipase A2 in CF mutant mice and its effect onmucin secretion [123]. We hypothesize that CFTR dysfunc-tion affects the enzymatic pathways involved, either directlyor indirectly, through an adaptive process.In a different CF mouse model carrying the Cftr tm1Hgu

partially active knockout allele (Table 1), Gulbins and col-leagues reported an age-dependent accumulation of ceramidespecies in lung tissue, which correlates with progressive lunginflammation and sensitivity to PA challenge [102]. Reductionof acid sphingomyelinase activity (ASM), either by mutation(Smpd−/−) or using the ASM inhibitor amitryptilin normal-ized ceramide levels and reduced lung pathology in these CFmutant mice [102]. Further studies confirmed and extendedthese data in the mouse model [132]; initial experiments inCF patients demonstrated that amitriptylin is safe and mightbe effective in reducing CF disease [133]. At this time,the apparent contradictory changes in ceramide levels in CFmutant mice between the Radzioch and Gulbins groups havenot been resolved using verified experimental protocols. It ispossible that differences in experimental protocols may causea bias towards distinct subsets of ceramide species.

7. Pumping the pipeline, testing experimental therapies inmouse models

A key application of CF mutant mice is to test experimentaltherapeutics. Elsewhere in this issue the development ofpharmacotherapy [134] and gene therapy for CF [135] arereviewed. New antagonists of ENaC have been developed andare being tested in a mouse model of ENaC hyperactivity[17]. Compounds affecting bioactive lipid metabolism anddownstream transcription factor activity were discussed inthe previous section. The cloning of the CaCC TMEM16Aalso presents new opportunities for intervention. As discussedbelow, several experimental therapeutics have been tested inCF mouse models.

7.1. CFTR activators

The mutant strains bearing F508del and G551D were madespecifically to test compounds that aim to correct defec-tive processing and trafficking (CFTR correctors) or defectivechannel gating (CFTR potentiators). A caveat is that these mu-tations are introduced into the mouse CFTR sequence, whichdiffers from the human sequence. Therefore, the properties ofthe mutant protein, including its interaction with therapeuticdrugs, might differ from the human form. Indeed, the murineCFTR Cl− channel exhibits significant differences in its bio-physical properties and regulation compared to human CFTR.These differences include (i) a smaller single-channel conduc-tance, (ii) a markedly different pattern of channel gating and(iii) insensitivity to AMP-PNP and pyrophosphate (PPi), twoagents that potentiate robustly the activity of human CFTR[136–138]. Consistent with these data, a number of CFTRpotentiators (e.g. VRT-532, NPPB-AM and VX-770), whichaugment strongly human CFTR channel gating are withouteffect on murine CFTR [139,140]. Moreover, Scott-Ward etal. [141] demonstrated that transfer of both nucleotide-bindingdomains (NBDs) of murine CFTR to human CFTR endowedthe human CFTR Cl− channel with the gating behaviour andpharmacological properties of the murine CFTR Cl− chan-nel. These data suggest that human-murine CFTR chimerasmight be employed to identify the binding sites of CFTRpotentiators.

7.2. CFTR correctors

Because CFTR correctors target the cellular protein pro-cessing and trafficking machinery, they are less dependenton a direct interaction with the CFTR protein. As a re-sult, a number of CFTR correctors have been successfullytested in the F508del mouse model (De Jonge et al., inpreparation). For example, miglustat, currently under eval-uation as a therapy for Gauchers disease, shows limited,but significant, efficacy in the Cftr tm1Eur mouse model byreducing the amiloride response in the nasal epithelium[142] and increasing F508del CFTR processing efficiency inintestinal epithelium [143]. Moreover, intraperitoneal instil-lation of sildenafil and vardenafil, related phosphodiesterase5 inhibitors, activated nasal Cl− conductance in Cftr tm1Eur

F508del mice, but not Cftr tm1Cam null mice with the samegenetic background [144]. In a recent study, Robert et al.[145] showed that glafenine, an off-patent drug with analgesicproperties, improved the trafficking of human F508del-CFTRin BHK and CFBE41o− cells to 40% of normal values.In isolated intestinal epithelia from Cftr tm1Eur mice, glafe-nine increased the Isc response to forskolin plus genistein,and it partially restored salivary secretion in vivo in mutantmice, without causing adverse effects [145]. These resultsillustrate that mutant CFTR mouse strains can play a signif-icant role in testing experimental drugs that are identified inhigh-throughput screening programs [134].


7.3. Anti-inflammatory drugs

In addition to drugs targeting the bioactive lipid pathways(see above, section 6), anti-inflammatory drugs can be investi-gated successfully in mouse models. The macrolide antibioticazithromycin has anti-inflammatory action and reduced thehyper inflammatory condition in Cftr tm1Eur F508del mutantmice [55], apparently through an action at the level of alve-olar macrophages [146], rather than at the level of epithelialcells [104]. Similar results were reported for the Cftr tm1Unc-TgN(FABPCFTR) gut corrected CFTR null mouse modelchallenged with a clinical PA isolate [115]. The synthetictriterpenoid CDDO reduced the hyper inflammatory pheno-type of a R117H mutant mouse model, presumably throughinhibition of the NF-κB pro-inflammatory pathway [147].

8. Modifier genes and new therapeutic targets

Not only environmental factors but also genetic elementsdetermine CF pathology. First, the many different mutationsin the CFTR gene found in CF patients give rise to variablelevels of residual activity [27,148]. A different category ofgenetic variations, often referred to as “modifier genes”,are polymorphisms in genes that affect CFTR function orpathways involved in CF pathology [149–151]. Identificationof modifier genes not only improves the CF diagnosis, butalso identifies novel therapeutic targets. CF mouse modelsand the toolbox allowing genetic experiments in this speciesoffer unique opportunities to study modifier genes and theirpotential therapeutic effects. First, phenotypic differencesbetween strains in different genetic backgrounds can beanalyzed [152,153] followed by gene mapping [154–156].Current developments in gene and transcriptome sequencingtechnology (“deep sequencing”) are likely to boost this field.Another approach is to create specific mutations in candidategenes that emerge from genetic or physiological studies in theCF patient population. As an example, the identification of theneutrophil marker IFRD1 as a modifier of lung disease in CFpatients was supported by analysis of Ifrd1 mutants in mice[58]. Moreover, using KCNE3 knockout mice, Preston et al.[157] recently demonstrated that the K+ channel β subunitKCNE3 might act as a modifier gene in CF. HeteromultimericKCNQ1/KCNE3 K+ channels provide the driving force forCl− exit across the apical membrane of airway and intestinalepithelia. In KCNE3 knockout mice, CFTR-mediated Cl−secretion is attenuated markedly [157]. The availability of anincreasing number of conditional and inducible mutant mousestrains, allows further validation of putative target genes. TheEUCOMM program (http://www.eucomm.org/), which aimsto generate conditional mutant embryo stem cell lines ofall functional genes in the mouse genome is linked to theEUMODIC program (http://www.eumodic.org/aboutus.html)which coordinates phenotypic analysis of derived strains.Crossbreeding of selected mutations with mutant Cftr allelesis expected to improve our knowledge of CF pathology.

9. Conclusion and perspective

We can conclude that despite their obvious limitations,Cftr mutant mouse models have delivered. First, a betterunderstanding of CF pathology beyond clinical and cellculture studies. Second, novel and promising therapeuticshave been identified, which are now being tested in thesemodels. Large animal models will certainly offer exciting newdimensions in this field. However, the versatility and relativeaffordability of mouse strains in genetic and physiologicalstudies will ensure their continued use.

Acknowledgements

We thank EuroCareCF (LSHM-CT-2005-018932), theDutch CF Foundation NCFS (R.B-O.) and the Dutch MaagLever Darm Stichting MLDS (M.W.), for financial support.We gratefully acknowledge the expert assistance of MichelHuerre (Institut Pasteur, Paris, France) in the quantitativeanalysis of histological data.

Conflict of interest

All authors report no conflict of interest

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