Alternatives for Lung Research: Stuck Between a Rat and...

Introduction

At the beginning of the new millennium, respi-ratory disorders are the leading cause of deathsworldwide, with further increases in mortalityexpected in the future (1–3). Chronic respiratorydiseases (CRDs) of the airways and other struc-tures of the lung include asthma, chronicobstructive pulmonary disease (COPD), respira-tory allergies, occupational lung diseases,obstructive sleep apnoea, tuberculosis, bronchi-tis and pulmonary hypertension (Figure 1).CRDs are under-recognised, under-diagnosedand under-treated, and insufficiently prevented(4). In addition, they cause a substantial socio-economic burden to both individuals and soci-eties. In the past 25 years, no significantadvances have been made in diagnostic tools ordrug treatments for lung disease (5), and as

such, lung cancer, asthma, pneumonia andCOPD are now killing more people in WesternEurope and the UK than heart disease — in fact,1 in every 4 people. The World HealthOrganisation (WHO) expects CRDs to increaseby 30% in the next 10 years (3, 6).

Although the health of the world is generallyimproving, and fewer people are dying from infec-tious diseases, more people are living longenough to develop chronic diseases (7). These dis-eases erode the health and well-being of thepatients and have a negative impact on familiesand societies, and are usually associated withtobacco smoking or prolonged exposure to othernoxious indoor/outdoor particulate matter andgaseous air pollutants (8–12). Women and chil-dren are particularly vulnerable, especially thosein low and middle income countries, where theyare exposed on a daily basis to poor indoor air

Alternatives for Lung Research: Stuck Between a Rat and aHard Place

The Twelfth FRAME Annual Lecture (the Sixth Bill Annett Lecture) presented atthe Kennel Club, London, on 4 November 2010

Kelly A. BéruBé

School of Biosciences, Cardiff University, Wales, UK

Summary — The respiratory system acts as a portal into the human body for airborne materials, whichmay gain access via the administration of medicines or inadvertently during inhalation of ambient air (e.g.air pollution). The burden of lung disease has been continuously increasing, to the point where it now rep-resents a major cause of human morbidity and mortality worldwide. In the UK, more people die from res-piratory disease than from coronary heart disease or non-respiratory cancer. For this reason alone, gainingan understanding of mechanisms of human lung biology, especially in injury and repair events, is now aprincipal focus within the field of respiratory medicine. Animal models are routinely used to investigatesuch events in the lung, but they do not truly reproduce the responses that occur in humans. Scientistscommitted to the more robust Three Rs principles of animal experimentation (Reduction, Refinement andReplacement) have been developing viable alternatives, derived from human medical waste tissues frompatient donors, to generate in vitro models that resemble the in vivo human lung environment. In the spe-cific case of inhalation toxicology, human-oriented models are especially warranted, given the new REACHregulations for the handling of chemicals, the rising air pollution problems and the availability of pharma-ceutically valuable drugs. Advances in tissue-engineering have made it feasible and cost-effective to con-struct human tissue equivalents of the respiratory epithelia. The conducting airways of the lowerrespiratory system are a critical zone to recapitulate for use in inhalation toxicology. Three-dimensional (3-D) tissue designs which make use of primary cells, provide more in vivo-like responses, based on the tar-geted interactions of multiple cell types supported on artificial scaffolds. These scaffolds emulate the nativeextracellular matrix, in which cells differentiate into a functional pulmonary tissue. When 3-D cell culturesare employed for testing aerosolised chemicals, drugs and xenobiotics, responses are captured that mirrorthe events in the in situ human lung and provide human endpoint data.

Key words: air pollution, animal alternatives, human, inhalation toxicology, lung disease, tissue-engineering.

Address for correspondence: Kelly BéruBé, Director of the Lung and Particles Research Group, School ofBiosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, Wales, UK.E-mail: [email protected]

ATLA 39, 121–130, 2011 121

122 K.A. BéruBé

quality from the use of solid biomass fuels forcooking and heating (Figure 2; 4). In high incomecountries, tobacco is the most important risk fac-tor for CRD, and in some of these countries,tobacco use among women and young people isstill increasing (3). Moreover, outdoor air pollu-tion has increased globally, due to the boomingeconomies of China and India, and more peoplenow live in areas of poor air quality than everbefore (4).

Lung disease exacts a tremendous financialburden on the patient, the care-giver and society.The annual financial burden of lung disease inEurope amounts to nearly €102 billion, and $15billion in the United States and the UnitedKingdom combined (4). In 2009, the US NationalHeart, Lung and Blood Institute estimated thatthe annual cost of providing healthcare related toall respiratory conditions, excluding lung cancer,was $113 billion (3). Lung disease leads to reduc-tions in income, owing to loss of productivitycaused by illness or death, and can lead to lost

opportunities for young household members wholeave school to act as care-givers or who are illthemselves (1, 2).

Despite tremendous advances in intensive carein general, and respiratory care in particular,chronic lung disease still remains a major causeof morbidity and mortality, in both the prema-ture infant and the adult. This is primarily due toa lack of understanding of the molecular mecha-nisms involved in normal and abnormal lungdevelopment. This, in turn, is the result of thefailure of animal models to act as representativesurrogates for the cellular/molecular mechanismsinvolved in physiological lung development andpathophysiology of related diseases. Added tothis predicament is the paucity of human tissue-based lung models for basic respiratory research.As a means to conquer this problem, and bringsafer drugs to CRD patients at a much quickerrate, human tissue-based screening platforms forcandidate drugs are urgently required.

Figure 1: The definition of lung disease

Lung disease is any disease or disorder that occurs in the lungs, or that causes the lungs not to work properly. Thereare three main types of lung disease. 1) Airway diseases, which affect the tubes (airways) that carry oxygen and othergases into and out of the lungs, and cause a narrowing or blockage of the airways. They include asthma, emphysema,and chronic bronchitis. People with airway diseases sometimes describe the feeling as ‘trying to breathe out through astraw’. 2) Lung tissue diseases, which affect the structure of the lung tissue. Scarring or inflammation of the tissuemakes the lungs unable to expand fully (‘restrictive lung disease’), and less capable of taking up oxygen (oxygenation)and releasing carbon dioxide. Pulmonary fibrosis and sarcoidosis are examples of lung tissue diseases. Peoplesometimes describe the feeling as ‘wearing a too-tight sweater or vest’ that will not allow them to take a deep breath.3) Pulmonary circulation diseases, which affect the blood vessels in the lungs. They are caused by clotting, scarring,or inflammation of the blood vessels, and affect the ability of the lungs to take up oxygen and to release carbondioxide. These diseases may also affect heart function. Most lung diseases actually involve a combination of thesetypes, and the most common lung diseases include asthma, chronic bronchitis, emphysema, COPD, pulmonaryfibrosis, and sarcoidosis. RT = respiratory tract.

Airway disease:narrowing of tubes (asthma, emphysema,

chronic bronchitis)Lower RT:

bronchi

Lower RT:bronchioles

Distal RT:alveoli

Tissue disease:inability to expand lungs (fibrosis)

Circulation disease:blood vessel clotting, scarring, ↓O2 uptake (↓heart function)

R RRR

Challenges and Limitations of In VivoModels for Inhalation Toxicity Testing

In vivo studies have traditionally been consideredto be the most useful basis for the toxicologicalanalyses of inhalation hazards, since they provide aphysiologically-relevant response that can be usedfor predicting the impact of inhaled materials onhuman health. The current differences between invivo and in vitro testing of respiratory hazards can

be grossly simplified as differences in the deliveryof materials and in the biological responses. Theprimary advantage of in vitro studies is that unin-tended consequences can be assessed, and analysiscan be performed with a higher degree of experi-mental control than can be achieved with in vivotests. In addition to the reasons for implementingthe Three Rs, the practical scientific reasons formoving away from animal studies in the field ofinhalation toxicology include their extreme costand time requirements, which result in a very low

The 12th FRAME annual lecture 123

Figure 2: Field emission scanning electron microscope (FESEM) images of particles present inambient air

Common combustion-derived particles that dominate ambient air, i.e. diesel exhaust, carbon black and coal fly ash,were collected onto a polycarbonate filter (pore size 0.65μm diameter). Diesel and carbon black singlet particles aretypically ∼7–10nm in diameter and aggregates are ≤ 2.5µm in diameter, rendering them ‘highly-respirable’, i.e. ableto penetrate into the distal lung regions (i.e. alveoli). Individual coal fly ash particles are usually 1–2μm in diameterbut are still respirable. Other ambient geogenic (i.e. mineral and calcium sulphate [gypsum]) and biogenic (i.e.organic [plant matter]) particles are shown as a reference for size.

a) diesel exhaust b) carbon black c) fly ash

d) mineral e) gypsum f) organic

Acc.V Spot Magn Det WD —————————25.0kV 4.0 65,000× SE 7.5 1μm






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sample throughput. The inherent difficulties inextrapolating across species must also be consid-ered. There are also significant problems in extrap-olating high-dose animal studies to low-dosechronic exposures (inhalation and instillation),since the capacity of the airway epithelium to deac-tivate compounds can be saturated at the high con-centrations and short times often used inlaboratory animal testing (13).

The additive and synergistic effects of heteroge-neous materials are infrequently assessed, but arelikely to play a role in the observable effects. Forexample, humans are exposed to a variety of chem-icals in their everyday lives, through their interac-tion with the environment. There are a multitudeof chemicals in the environment, such as ambientindoor and outdoor air pollution mixtures, whichhave not been thoroughly tested due to the cum-bersome, expensive and uncertain nature of theanimal tests on which current toxicity testing isfounded. Moreover, the evaluation of a wide rangeof chemical mixes, representing more realisticexposure scenarios, is impossible when using cur-rent animal-based methods, because of the widevariety of interactive combinations and quantitiesof chemicals which could potentially be assessed.Overall, the major in vivo challenge lies in assess-ing the full array of information provided by theexposed respiratory tissue, e.g. the release of sig-nalling compounds, the role of complement activa-tion, the permissive transport of particulate/gasphase components (crossing a cell barrier), and thephysical changes which take place (e.g. loss of lungcompliance and cell tight junctions, inactivation ordeath) — all of which can be addressed at the invitro level (Figure 3).

Human Tissue-based Alternatives forLung Research

Bringing a new medicine to patients requires, onaverage, 10 to 15 years of testing (14). Along theway, many drugs fail to prove efficacious and mayhave serious side-effects, regardless of a patient’sgenetic circumstances. Much of the screening forefficacy and toxicity occurs outside the species ofinterest, in laboratory animals or their tissues.Obviously, extrapolating data between species ismuch more hazardous than within a species.Indeed, according to the United States Food andDrug Administration (FDA), a staggering 92% ofall drugs found safe and therapeutically effectivein animal tests fail during human clinical trialsdue to their toxicity and/or inefficacy, and aretherefore not approved (15). Furthermore, overhalf of the mere 8% of drugs which do gain FDAapproval, later have to be withdrawn or re-labelleddue to severe and unexpected side effects (16).

Historically, lung research is first conducted on

animals (e.g. rodents, rabbits and dogs) or humancell lines derived from bronchial (e.g. BEAS andCalu-3) or alveolar (e.g. A549) cells, but both ofthese methods have limitations (17). There is nosuitable animal model to mimic the human lung,and the production of permanent or immortalisedcell lines for long-term use requires altering theirbehaviour, leading to cancer-like cells. Altern -atively, researchers can tissue-engineer ‘normal’cells isolated from human donors to grow outsidethe body, in order to develop safety screeningstrategies. The required alternative must advancescience to the point where pre-clinical tests arebased on human biology, which would better pre-dict what would happen to real human volunteersor patients in the clinical trials. In vitro techniquesare now seen as an important adjunct to in vivostudies. They allow specific biological pathways tobe tested under controlled conditions, as well asthe isolation of pathways which are not feasible invivo. For example, it is difficult to discriminate invivo whether complement activation plays a role inany pro-inflammatory effects of particles (18).

Today, cutting-edge technologies in human tis-sue-engineering permit the better prediction ofcomplex human reactions. They remove many ofthe in vivo caveats and produce standardised plat-forms for basic research and toxicity testing.Specific biological and mechanistic pathways canbe isolated and tested under controlled conditions,in ways that are not feasible with in vivo tests. Asa specific case study, air pollution particles (rang-ing from combustion-derived products to transitionmetals [18]) have been investigated extensivelythrough in vivo exposures, by using various animalmodels, as well as cell culture experiments.Collectively, the animal studies revealed increasedpulmonary inflammation, oxidative stress and dis-tal organ involvement upon respiratory exposureto inhaled or implanted ultrafine particulate mat-ter (18). Numerous human cell-based analyseshave supported these physiological responses, andyielded data which point to an increased incidenceof oxidative stress, inflammatory cytokine produc-tion, and apoptosis, as well as increases in theexpression of certain genes and the activation ofcertain cell signalling pathways in response toexposure to ultrafine particles (18).

Another case in point is the requirement in thepharmaceutical, cosmetic and biomedical indus-tries for a lung model to screen aerosolised formu-lations for safety. An in vitro testing platform, inwhich primary, human-derived cells are used tocreate three-dimensional (3-D) organotypic mod-els, could improve drug development by providinghuman endpoint data, and could also improve therate of bench-to-bedside progression through thedesign of high-throughput platforms (19, 20). Anexemplar platform would involve the use of amixed-cell phenotype culture grown in a physiolog-

ically-relevant manner, i.e. at the air–liquid inter-face (ALI), to account for the interactions of apotential toxin in the respiratory environment.Cells grown in ALI bioreactors experience a simi-lar dynamic exposure to that which occurs withinwhole animals, but with the added value of thepotential for capturing unsuspected effects (e.g.disruptions to the epithelial barrier, apico-lateralor baso-lateral paracellular transport, or specifictight junctional complexes) and user-defined geneand signalling pathway interactions (21–23). Invitro cell culture analogues would direct more-rational chemical/drug development processes,improve treatment procedures, and reduce theneed for animal experiments.

3-D for the Three Rs

Toxicity testing is approaching a scientific pivotpoint, and is poised to take advantage of the cur-rent revolutions in biology and biotechnology.Advances in toxicogenomics, bioinformatics, sys-tems biology, epigenetics, and computational toxi-cology could transform toxicity testing from asystem which is based on whole animal testing, toone founded primarily on in vitro methods thatevaluate changes in biological processes by usingcells, cell lines, or cellular components, preferablyof human origin (24). As a stopgap in the firstinstance, human tissue equivalents (HTEs) can beemployed to bridge the void between two-dimen-sional (2-D), i.e. monolayer, cell culture methodsand cells and tissues in vivo. Cells grown in 2-Dcultures lack many of the in vivo-like propertiesobserved in 3-D cultures, due to their inability toachieve a fully-differentiated phenotype. Forinstance, with regard to the proximal or lower lungregion where bronchial epithelial cells persist,fully-differentiated 3-D models of this regionexhibit a muco-ciliary phenotype characterised bymultiple cell layers, denoted by ciliated cells withbeating cilia, mucus-secreting goblet cells, toxi-cant-responsive Clara cells, and functional basalprogenitor cells (i.e. cells which replace injuredepithelial cells). This creates a ‘human biology sub-environment’ that mimics the critical zones andcomponents of the proximal lung (Figure 4; 23).This model not only represents an innovativeopportunity to replace, refine and reduce animalsin basic research and safety testing, but also offersa reductionist approach to remove confoundingphysiological factors (e.g. systemic inflammation,biotransformation and hormonal activities), per-mitting resolution of the finer details of a giventoxic response. The use of a standardised platformfor the production of 3-D lung constructs based onfilter-well (micro-porous membrane) technologycan also result in a harmonisation of dosing regi-mens and testing protocols. The interpretation of

in vitro studies can be difficult, as researchersfrom different laboratories use their own uniquemethods for cell culture, dosing, and the analysisof cell responses. The culture of 3-D lung con-structs on micro-porous membranes could pro-vide the means for more concordance since it is ahighly-accessible technology, given that the con-sumables involved for their production can bepurchased as ‘off-the-shelf’ plasticware, and thatno specialist knowledge or training is required toreproduce high-fidelity cell cultures.

Replacement Strategies

Requirements under the new European Unionrules relating to the REACH (Registration,Evaluation and Authorisation and Restriction ofChemicals) system, demand extensive safety test-ing of existing and new chemicals (25). Obtaininginhalation toxicity testing data for new andalready-in-service chemicals that fall under thisnew EU rule, has unique challenges when com-pared to most bio-testing regimes, due to the com-plexity, time and expense involved in conductingstandardised whole animal inhalation assess-ments. There are numerous in vitro approachesthat could be drawn on to obtain respiratory sys-tem-related information (17), but there is currentlyno universally accepted model or system, partlydue to the numerous considerations (e.g. selectionof cell types, dosage and fundamental study proce-dures) that must be satisfied before validating andadopting any in vitro bioassay(s) or batteries ofsuch assays, to obtain data which can substitutefor whole animal inhalation studies.

The lungs represent a potential target for anyairborne detritus (e.g. particles and gases). Ongaining access via this portal of entry, the debrisdeposits in the conducting airways or alveolarepithelium and encounter mucus or epitheliallining fluid. Airborne detritus (Figure 2) maythen interact with macrophages, which mightresult in its clearance, or it could enter the inter-stitium, making contact with fibroblasts andendothelial cells or certain cells of the immunesystem. Therefore, lung epithelial cells, macro -phages, immune cells and fibroblasts representkey target cells for observing toxicant effectswith specific regard to inflammation, immuno-pathology, fibrosis, genotoxicity, microbialdefence and clearance. The creation of co-culturemodels with different combinations of these celltypes, according to the potential targets of agiven inhaled hazard, will offer more-realisticsystems (e.g. 26–31) for studying the possiblecellular/molecular interactions of inhaled xeno-biotics and their toxic potentials in the humanlung — all of which bodes well for the future oflung research.


Conclusions

Human lung diseases cause morbidity, mortality,and socio-economic burdens. Susceptibility to thepathogenesis of most lung diseases results from acomplex interaction between a relevant challengefrom the environment, an individual’s geneticbackground, and the nature of the resultant hostresponses. Cutting-edge research into lung diseasenow centres on understanding the lung as a genet-ically-determined biological organ that functions tomediate gas exchange and defend against a hostileenvironment. A major challenge is to determine

the hierarchy of gene expression that integratesthe functions of multiple cell types in this complexscenario. Emerging therapies that will play amajor role in the future treatment of lung diseasewill have greater success, if they are developed viathe route of human tissue-based model systems.

References

1. ERS & ELF (2003). Lung Health in Europe, Facts &Figures, 60pp. Sheffield, UK: European RespiratorySociety & European Lung Foundation. Available at:

Figure 4: Pseudo-coloured FESEM images of the apico-lateral surface of a normal humanbronchial epithelium model

a) A pseudo-coloured FESEM image of the apico-lateral surface of a fully-differentiated normal human bronchialepithelium (NHBE) model grown at the ALI in 3-D on a micro-porous membrane (Millicell). Some of the key defencecells of the muco-ciliary phenotype are visible, i.e. ciliated cells (CC; yellow and green) and goblet cells (GC;polygonal, flat cells covered in white microvilli). The raised borders of microvilli denote junctions between cells. b) Ahigher magnification image of the cilia from ciliated cells. The cilia are ∼10μm in length and there are ∼250 cilia percell. They play a crucial role in the muco-ciliary escalator to remove debris trapped in the mucus. c) A close inspectionof the mucus (seen as red spheres) released from its resident goblet cell. Note that goblet and ciliated cells arecomponents of the muco-ciliary system and are always immediate neighbours.

a) b)

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128 K.A. BéruBé

GC

CC

http://www.european-lung-foundation.org/uploads/Document/WEB_CHEMIN_13411_1222853571.pdf(Accessed 07.04.11).

2. Loddenkemper, R., Gibson, G.J. & Sibille, Y. (2004).The burden of lung disease in Europe: Data fromthe first European White Book. Breathe 1, 7–9.

3. WHO (2011). Air Quality: Facts and Figures. Cop -en hagen, Denmark: World Health Organisation.Available at: http://www.euro.who.int/en/what-we-do/health-topics/environmental-health/air-quality/facts-and-figures (Accessed 07.04.11).

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