Animal Models of Fibrotic Lung Disease
Bethany B. Moore1, William E. Lawson4,5, Tim D. Oury6, Thomas H. Sisson1, Krishnan Raghavendran2,
and Cory M. Hogaboam3
1Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,2Department of Surgery, and3Department of Pathology,
University of Michigan, Ann Arbor, Michigan;4Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt
University School of Medicine, Nashville, Tennessee;5Department of Veterans Affairs Medical Center, Nashville, Tennessee; and6Department of
Pathology, University of Pittsburgh Medical School, Pittsburgh, Pennsylvania
Interstitial lung fibrosis can develop as a consequence of occupa-
tional or medical exposure, as a result of genetic defects, and after
trauma or acute lung injury leading to fibroproliferative acute
respiratory distress syndrome, or it can develop in an idiopathic
manner. The pathogenesis of each form of lung fibrosis remains
poorly understood. They each result in a progressive loss of lung
function with increasing dyspnea, and most forms ultimately result
in mortality. To better understand the pathogenesis of lung fibrotic
summarizes the common and emerging models of lung fibrosis to
highlight their usefulness in understanding the cell–cell and soluble
have allowed for the development of models to study targeted inju-
ries of Type II alveolar epithelial cells, fibroblastic autonomous ef-
has more closely mimicked the pathology of human fibrotic lung dis-
ease. We also have a much better understanding of the fact that the
aged lung has increased susceptibility to fibrosis. Each of the models
of fibrotic lung disease.
Keywords: fibrosis; collagen; fibroblast; aging; cytokines
Interstitial lung disease is often associated with the development
of chronic fibrosis. These diseases are characterized clinically by
progressive dyspnea, cough, restrictive physiology, and impaired
gas exchange. Humans manifest many types of fibrotic lung
disease (1). Among the diffuse parenchymal lung disorders
(DPLDs) are diseases of known cause (e.g., drug-related, envi-
ronmental exposures, or those associated with collagen vascular
disease), the idiopathic interstitial pneumonias (IIPs), the gran-
ulomatous DPLDs (e.g., sarcoidosis), and rare noncategorized
diseases, such as lymphangioleiomyomatosis. Idiopathic pulmo-
nary fibrosis (IPF) is the most common disease within the cat-
egory of IIPs, and is histopathologically identified as usual
interstitial pneumonia (UIP). Additional diseases within the
IIP category include desquamative interstitial pneumonia,
respiratory bronchiolitis interstitial lung disease, acute intersti-
tial pneumonia, cryptogenic organizing pneumonia, lymphocytic
interstitial pneumonia, and nonspecific interstitial pneumonia
(NSIP). IPF carries a poor prognosis, with a mean survival time
of less than 5 years after diagnosis (2–5). Biopsies from a single
patient can show heterogenous patterns consistent with both UIP
and NSIP (4, 6, 7), suggesting that NSIP shares common patho-
genic mechanisms with UIP.
Diagnoses of patients with IPF who do not exhibit classic
high-resolution computed tomography scan changes are con-
firmed by histopathologic evaluations of surgical lung biopsies,
which demonstrate the pattern of UIP. Hallmark features of
UIP include epithelial cell hyperplasia, basement membrane de-
that is spatially and temporally heterogeneous (8). Attempts to
understand the pathogenesis of IPF and other fibrotic lung dis-
orders have relied on animal models. Unfortunately, no animal
model exhibits progressive disease, and most animal models do
not fully recapitulate the histologic pattern of UIP or other
interstitial lung diseases. This, however, does not mean animal
models are dispensable for research in pulmonary fibrosis. Tra-
ditional animal models of lung fibrosis have generated impor-
tant insights into the pathobiology of lung injury, inflammation,
and fibroproliferation (9). In addition, animal models have re-
cently been refined to reflect better the known pathogenic
mechanisms in IPF. Although it is appreciated that the sponta-
neous development of fibrosis in other species (e.g., cats, horses,
and dogs) (10–12) can be instructive for comparative medicine,
the most tractable models for studies of pathogenesis involve
rodents. Many traditional and newly developed models of ex-
perimental lung fibrosis offer opportunities to study cell–cell
interactions and the soluble mediators driving pathologic fi-
brotic remodeling. A better understanding of these pathways
in experimental models provides a critical approach to identi-
fying novel targets to assess and validate in human studies. We
will review several established and newer models of lung fibro-
sis, and highlight particularly useful aspects of each. We include
environmental and genetic models because these offer insights
into the development of fibrosis by known causes, and also shed
(Received in original form February 28, 2013 and in final form March 12, 2013)
This research was supported by National Institutes of Health grants HL087846
and HL091745 (B.B.M.), National Institutes of Health grant HL105479 (W.E.L.),
the Department of Veterans Affairs (W.E.L.), National Institutes of Health grant
R21HL09549 (T.D.O.), National Institutes of Health grant RO1 HL-102013 (K.R.), and
National Institutes of Health grant RC2 HL101740 (C.M.H.)
Correspondence and requests for reprints should be addressed to Bethany
Moore, Ph.D., Division of Pulmonary and Critical Care Medicine, Department
of Internal Medicine, University of Michigan, 109 Zina Pitcher Place, 4053 BSRB,
Ann Arbor, MI 48109-2200. E-mail: email@example.com
Am J Respir Cell Mol Biol
Copyright ª 2013 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2013-0094TR on March 22, 2013
Internet address: www.atsjournals.org
Vol 49, Iss. 2, pp 167–179, Aug 2013
This review summarizes common and recently developed
animal models of lung fibrosis. It highlights the pathologic
features of these models, and will aid basic science and
translational science investigators in choosing the most
appropriate model system to be used for their own fibrosis
light on the pathogenesis of intractable fibrotic diseases such as
IPF. In addition, we include discussions on animal models of
interstitial fibrosis secondary to specific risk factors for acute
lung injury (ALI) and acute respiratory distress syndrome
ANIMAL MODELS REPLICATING IPF
Asbestosis continues to be an important fibrotic lung disease in
exposed humans. Human asbestosis and IPF are similar in their
gross distribution, and share a common UIP histopathology (13,
14). In terms of their main difference, asbestosis involves fewer
myofibroblastic foci than does IPF, and this may explain the
slower progression of asbestosis compared with IPF (14). As-
bestosis is distinguished from IPF by histologic findings of as-
bestos bodies in the lungs, indicating the cause of disease. These
features are largely recapitulated in inhalation models in ani-
mals, and these models are useful for understanding the patho-
genesis of both asbestosis and UIP. An intratracheal instillation
of asbestos fibers promotes the rapid development of fibrosis
with a single administration. One caveat about intratracheal
models involves the distribution between lung lobes, which
can be uneven, and the pattern of fibrosis tends to be central
rather than subpleural, as is more prominent in the inhalation
model. However, the ability to visualize asbestos fibers histo-
logically can confirm an appropriate deposition, and can be used
to locate areas where fibrosis is likely to develop. As indicated,
inhalation models develop a more peripheral pattern of disease,
but they require special inhalation chambers and the time for
disease development can be long, especially if using nonamphibole
(chrysotile) fibers. All asbestos should be baked before in vivo
instillation to destroy common contamination by lipopolysac-
charide. The intratracheal models with amphibole fibers show
fibrosis by Day 7, which is mature by Day 14 (Figure 1). Inha-
lation models may take over a month to develop fibrosis. The
fibrosis that does develop is persistent and may be progressive
with large enough dosing, especially when using amphibole
fibers. The deposition of asbestos fibers specifically induces
oxidative stress and alveolar epithelial cell injury (15).
Macrophages, lymphocytes, eosinophils, and neutrophils have
also been implicated in promoting injury in these models. Mu-
rine asbestosis comprises one of the few animal models to de-
velop fibrotic foci, and therefore is particularly useful for
understanding the development of this pathologic lesion. Table
1 summarizes the pathogenic features that are especially well
studied, using the asbestos model and other models of lung
The instillation of silica into murine lungs results in the develop-
ment of fibrotic nodules that resemble simple silicotic nodular
fibrosis, which develops in humans after some occupational
exposures (16), but are generally more cellular than the fibrotic
nodules seen in humans. Silica can be delivered to rodents via
aerosolization (17), intratracheal administration (18), or oro-
pharyngeal aspiration (19). The fibrotic response to silica instil-
lation is strain-dependent. C3H/HeN, MRL/MpJ, and NZB
mice are all susceptible in the aerosolized model, whereas Balb/c
mice show little response (17). Similarly, C57Bl/6 mice are more
susceptible than CBA/J mice after intratracheal silica (20). Silica is
retained in the lung, and the response is characterized by a persis-
tent, toxic, and inflammatory response. Fibrotic nodules develop
around silica deposits, and silica particles are easily identified both
histologically and by polarization microscopy. The intratracheal
models are easier and more cost-efficient, whereas inhalation mod-
els more closely mimic human exposure but take longer to develop
(40–120 d) (18) than the intratracheal models (14–28 d) (19). Care
must be exercised when choosing formulations of silica particles,
because different formulations vary widely in their potency of
stimulating fibrotic responses. Silica should be baked before in vivo
instillations, to destroy contamination by lipopolysaccharide. One
important feature of silicosis involves the strong influence of mac-
rophage NACHT, LRR, and PYD domains-containing protein 3
(NALP3) inflammasome activation (21), making it an important
model for studying the innate immune regulation of lung fibrotic
responses. Figure 2 demonstrates the histopathologic findings in
the silica model.
Among currently applied models of experimentally induced pul-
monary fibrosis, the administration of bleomycin is used most
frequently (9, 22). The recognition that bleomycin could result
in pulmonary fibrosis in humans led to its use in experimental
models, and for four decades it has been the most commonly
applied model of experimental lung fibrosis. It has been used in
multiple animals, including mice, rats, guinea pigs, hamsters, rab-
bits, dogs, and primates, but mice are most common (9, 23). Fur-
thermore, it has been delivered by multiple methods, including
intratracheal, intraperitoneal, subcutaneous, intravenous, and inha-
lational (9, 23). Although each method of delivery has its own
strengths and weaknesses, the intratracheal delivery of bleomycin
has emerged as the most frequent route (22). Whatever the route
of administration, the delivery of bleomycin results in direct cell
Figure 1. Asbestos induced pulmonary fibrosis. (A) Control lung shows
normal terminal bronchi and alveolar parenchyma. (B) Intratracheal
instillation of crocidolite asbestos induces robust peribronchial fibrosis
with extension into the adjacent alveolar parenchyma (14 d after
exposure). Scale bars ¼ 50 mm.
168AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 49 2013
injury through the induction of DNA strand breaks, the generation
of free radicals, and the induction of oxidative stress (24). Cell
necrosis and/or apoptosis follow, with subsequent inflammation
and the development of fibrosis (9). When delivered systemically
(intravenously, intraperitoneally, or subcutaneously), the
initial site of injury is the pulmonary vascular endothelium,
which is thought to reflect similar processes at play in humans
affected by bleomycin-induced pneumonitis (25). With this initial
endothelial cell damage, the drug can then gain access to the alve-
olar epithelium to induce damage. In contrast, with lung-specific
delivery, primary alveolar epithelial cell (AEC) injury from bleomy-
cin is the inciting event (9, 22, 26). The delivery of bleomycin di-
rectly to the airways can be accomplished by direct intratracheal
injections after surgical neck cutdown (27, 28), by injections of dry
powder (29), or by endotracheal intubation, which allows for re-
peated dosing. One repeated dosing regimen administered 0.04 of
a unit of bleomycin every other week for eight doses (30). This route
of delivery offers the advantage of mimicking a repetitive or chronic
injury with more robust fibrosis noted than with single doses. Neu-
trophilic inflammation is attenuated later in the repetitive dosage
model, compared with single dose. AEC hyperplasia is promi-
nent in areas of fibrosis, and fibrosis remains prominent (as does
AEC hyperplasia) 20 to 30 weeks after the final bleomycin dose
in the eight-dose, every-other-week model (22, 30).
For the systemic delivery of bleomycin, a common intrave-
nous dosing regimen administers 20 mg/kg twice weekly for 4
to 8 weeks, resulting in initial endothelial cell injury followed
by epithelial cell injury, inflammation, and fibrosis. Fibrosis is
first noted by Week 4, with progression through Week 12 for
mice treated for 8 weeks (31). For intraperitoneal injections,
one dosing regimen administers bleomycin at 0.035 U/g twice
weekly, and lungs are subsequently harvested 33 days after the
first dose of bleomycin (32), a time point at which fibrosis is
prominent. Another subcutaneous dosing regimen administers
0.05 mg bleomycin three times a week for 4 weeks (33). Bleomycin
can also be delivered via miniosmotic pumps, providing another
means of subcutaneous delivery (34).
Bleomycin can induce fibrosis in a relatively short time period
(2–4 wk in an intratracheal model, and 4–12 wk in a systemic
delivery model). The histopathology is not fully consistent with
UIP, and by most accounts, the single-dose model is thought to
resolve over time, although reports are conflicting (26, 35–39).
Because the bleomycin model resolves in some cases, this offers
an opportunity to study the natural resolution of fibrosis. The
response to bleomycin is strain-dependent, with C57Bl/6 mice
demonstrating more susceptibility than Balb/c mice. The inflam-
matory component is considerable within the first week. Interven-
tions that interrupt the inflammatory phase are often protective
(24). It is recommended that when using the bleomycin model to
study the effects of an antifibrotic therapeutic agent, it is best if the
therapy is delivered after the inflammatory phase (at least 7 d after
bleomycin) for assessment (24). Multiple cell types have been
shown to be involved in the development of bleomycin-induced
fibrosis, including Type I and Type II AECs, fibroblasts,
myofibroblasts, fibrocytes, macrophages, lymphocytes, neutro-
phils, endothelial cells, pericytes, airway epithelial cells, and
TABLE 1. PATHOGENIC MECHANISMS IN ANIMAL MODELS OF LUNG FIBROSIS
AsbestosEpithelial cell injury
Macrophage oxidative stress
Ability to visualize fiber deposition
Ability to visualize particle deposition
Macrophage NALP3 inflammasome activation regulates disease development
Direct cell injury via DNA damage
Initial site of injury can be determined via method of delivery (intravenous or intraperitoneal vascular endothelium,
intratracheal alveolar epithelial cells)
AEC hyperplasia in repetitive dosing model
Resolution in single-dose model
Ability to visualize injured areas of the lung
Epithelial stress in response to injury
Fibroblasts in aged lungs are poised to respond well to TGF-b signaling
Fibroblast loss of Thy-1, but gain of EDA-fibronectin
Natural infections with herpesvirus cause fibrosis in aged, but not young, mice
AEC and airway epithelial cell injury
Fibrosis develops in absence of significant inflammation
Opportunities to study cross-regulation of the cytokine with TGF-b activation and responsiveness
AEC ER stress responses
Opportunities to study “two-hit” models of pathogenesis
Directed injury of Type II AECs
Allows studies of how other cell types respond to directed Type II AEC injury
Pattern of fibrosis involves interstitial rather than alveolar consolidation
Allows studies of hypoxemia, permeability injuries, and effects of hyperoxia
Models fibroproliferative changes seen with ALI and ARDS
Vascular remodeling (reminiscent of pulmonary arterial hypertension)
Mesenchymal stem cells regulate repair responses
Opportunity to study phenotype of human IPF fibroblasts in vivo
Studies of how fibroblast-autonomous alterations affect other lung cell types are possible
Currently does not allow studies of immune cell regulation of disease development
Familial IPF models
Targeted Type II AEC Injury
Direct forms of lung injury (acid/hyperoxia)
Humanized mouse models
Definition of abbreviations: AEC, alveolar epithelial cell; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; EDA, extra domain A containing fibronectin;
FITC, fluorescein isothiocyanate; IPF, idiopathic pulmonary fibrosis; NALP3, NACHT, LRR, and PYD domains-containing protein 3; TGF-b, transforming growth factor–b;
Thy-1, thymocyte differentiation antigen 1.
Translational Review 169
stem/progenitor cells. Among the major advantages of repet-
itive dosing models, the site of initial injury can be controlled
via the delivery route, and the histopathologic changes are
similar to those in human IPF (22). Figure 3 demonstrates
the typical histologic patterns seen after various routes of
Fluorescein isothiocyanate (FITC) has also been used as a model
of lung injury leading to fibrosis (40). The intratracheal delivery
of FITC to the lungs results in alveolar and vascular permeabil-
ity, culminating in lung fibrosis within 14 to 21 days (9, 41). In
what is perhaps the most useful aspect of the model, FITC
conjugates to parenchymal proteins and remains persistently
localized to the areas of initial injury. This allows investigators
to use immunofluorescence to localize areas of injured lungs (9).
Fibrosis has been shown to correlate closely with areas of FITC
deposition (9), as shown in Figure 4. In terms of pathogenesis,
FITC is associated with acute lung injury and the development
of edema and inflammation (including neutrophils), followed by
the development of fibrosis. Doses ranging from 0.007 mg per
gram body weight dissolved in PBS to an intratracheal delivery
of 50 ml of a 1.4-mg/ml solution have been used in mice (28, 40,
42). Advantages of the model include a robust effect in both
Balb/c and C57Bl/6 mice and durable fibrotic response, lasting
for months (9). The FITC model has been shown to be depen-
dent on Th2 cytokines (IL-13) (43), and is also regulated by the
Figure 2. Intratracheal silica injury leads to multinodular
fibrosis (A), predominantly in the alveolar parenchyma
and terminal bronchioles (C). Activated macrophages
are predominant in these lesions (D, arrows). (B) Control
Figure 3. Bleomycin can be administered in different
manners to induce lung fibrosis in mice. (A) Trichome
blue–stained lung sections from normal wild-type
C57BL/6 mice. (B) Lung section at 3 weeks after 0.08
of a unit of intratracheal bleomycin demonstrates the
development of an area of fibrosis typically seen with
this model. (C) Lung section from a mouse at 2 weeks
after the eighth biweekly repetitive intratracheal 0.04-
unit bleomycin dose. This repetitive intratracheal
model not only induces prominent lung fibrosis,
but also results in regions with prominent alveolar ep-
ithelial cell (AEC) hyperplasia lining areas of fibrosis.
Arrow points to hyperplastic AECs. (D) Lung section
from a mouse harvested on Day 33 in a twice-weekly
intraperitoneal 0.035-U/g bleomycin study. With sys-
temic delivery modalities such as intraperitoneal injec-
tions, fibrosis develops prominently in the subpleural
regions. Arrowhead points to pleural edge. All sections,
170AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 492013
chemokine (C-C) motif ligand (CCL12)-mediated recruitment
of fibrocytes in response to the lung injury (44). This model also
has its disadvantages. They include the stipulation that the so-
lution must be made fresh each time, and its efficacy can vary,
depending on the lot of FITC used and the size of the particle
created via sonication. Longer periods of sonication, resulting in
smaller particle sizes, can increase the acute toxicity leading to
early death from lung injury (9).
Age-Related Models of Lung Fibrosis
Progress has been made in identifying models that demonstrate
an age-dependent increase in fibrosis, more closely mimicking
shown thatolder mice are moresusceptible than youngermice to
bleomycin-induced injury (45), and that aged male mice may be
more sensitive than female mice in the same model (46). This is
especially intriguing because IPF also has a male predominance
(8). Moreover, senescence-prone mice develop more severe fi-
brosis in response to bleomycin than do senescence-resistant
mice (47). The transgenic deletion of genes for the receptor of
the advanced glycation end products (RAGE2/2mice) (48) or
relaxin (relaxin2/2mice) (49) results in the spontaneous age-
related development of lung fibrosis. In relaxin2/2mice, males
developed worse age-dependent fibrosis than did female mice
(49). An age-dependent development of fibrosis has also
been noted in murine models of Hermansky-Pudlak disease
(50). Finally, the infection of aged, but not young, mice with
g-herpesvirus–68 results in the development of lung fibrosis, which
is associated with epithelial cell endoplasmic reticulum stress and
increased transforming growth factor–b (TGF-b) signaling in fibro-
blasts (51, 52). This is particularly intriguing because it suggests
that a common viral infection may be able to promote the devel-
opment of fibrosis in aged individuals. This could conceivably in-
volve a fibrotic response to a new infection or to a reactivation of
a previous herpesvirus infection. Interestingly, g-herpesvirus (both
as a latent and as a lytic infection) has been shown to augment the
fibrosis caused by FITC or bleomycin (53, 54). Patients with IPF
tend to harbor herpesviral genomes within their lung tissue,
whereas this is uncommon in normal individuals (55–58). This
may indicate that certain individuals are genetically inclined to
harbor virus in their lung epithelium, which may lead to reac-
tivation and fibrosis as they age. In addition, fibroblasts from
aged mice display decreased thymocyte differentiation antigen
1 (Thy-1), a hallmark of human myofibroblasts (59), and lungs
from aged mice display an increased expression of matrix
metalloproteinase–9 and extra domain A (EDA)-containing fi-
bronectin (45), a molecule associated with TGF-b activation
and responsiveness in myofibroblasts (60). Many cellular pro-
cesses involved in fibrosis likely operate differently in aged ver-
sus young lungs. As such, there is enthusiasm for promoting the
use of aged mice in all of these models of lung fibrosis. Of course,
in terms of the downside of this strategy, aged animals are not
readily available to all investigators, and the costs associated with
housing an animal until the appropriate age could be prohibitive.
Another difficulty with the model involves choosing the appropri-
ate age. With the bleomycin model, studies have been published
on 24-month-old (45), 15- to 18-month-old (51), and 12-month-old
mice (46) to show the differential effects of age. Additional stud-
ies are needed.
CYTOKINE OVEREXPRESSION MODELS
The overexpression of cytokines, including TGF-b, TGF-a,
IL-13, TNF-a, and IL-1b, using both gene-transfer and trans-
genic approaches, results in lung fibrosis. We will briefly detail
these important models of cytokine overexpression.
TGF-b is a potent profibrotic cytokine that is elevated in most
forms of lung fibrosis (61). As such, models that rely on the
overexpression of TGF-b are especially relevant for dissecting
the downstream signaling pathways involved in multiple cell
types. The overexpression of TGF-b can be achieved by adeno-
viral delivery (62) or by doxycycline-regulated transgenic ex-
pression in epithelial cells (63). In the original rat models of
adenoviral overexpression, significant elevations of active
TGF-b were seen in the lung by Day 1, and reached a peak
concentration of 13 ng/ml by Day 7. Notably, the concentration
of latent TGF-b also peaked in these experiments around Day 7
at a concentration of 72.3 ng/ml, suggesting that the expression
of the constitutively active transgene induced endogenous pro-
duction. The expression of TGF-b in the lung was accompanied
by mononuclear cell infiltration (Days 3–7), followed by the
development of alveolar consolidation. Lung collagen concen-
trations increased 2-fold by Day 14 (62). Subsequent to these
original reports, adenoviral vectors have been used to demon-
strate a dose-dependent increase in fibrosis among mice. Inter-
estingly, this model was also strain-dependent, in that C57Bl/6
mice demonstrated increased responsiveness to TGF-b overex-
pression, compared with Balb/c mice (64). Other features of this
model include epithelial cell apoptosis and changes in soluble
mediators that mimic human disease (63). Adenoviral-mediated
TGF-b overexpression is also unique in that it leads to persis-
tent scarring, which may more closely mimic the fibrotic changes
Figure 4. Fibrosis develops in areas of fluorescein isothiocyanate (FITC)
deposition. Serial lung sections were prepared on Day 21 after FITC
intratracheal instillation in C57Bl/6 mice. Hematoxylin-and-eosin stain-
ing shows patchy areas of fibrosis and consolidation (A), which line up
well with areas of FITC deposition, as seen in the immunofluorescent
image captured of a serial lung section (B).
that occur late in IPF. In the doxycycline-inducible, club cell
(Clara cell) 10 (CC10) promoter–driven model of TGF-b over-
expression, the treatment of mice with doxycycline resulted in
a rapid up-regulation of TGF-b (z 1 ng/ml) within the lungs at
12 hours. Induction over the course of 2 months resulted in an
approximately 2-fold increase in lung collagen concentrations
(63). Although not progressive per se, the degree of fibrosis con-
tinued to worsen over the duration of the doxycycline exposure.
In these models, the overexpression of TGF-b has been associ-
ated with airway and alveolar cell apoptosis, myofibroblast accu-
mulation, and the induction of the epithelial-to-mesenchymal
transition. Studies of the overexpression model have also eluci-
dated the contributions of alternatively activated macrophages to
disease development (65).
TGF-a, similar to TGF-b, is also overexpressed in the lungs of
patients with IPF (66). The overexpression of TGF-a in the lung
epithelium results in lung fibrosis in rodent models (67, 68). In
addition, TGF-a overexpression results in pulmonary hyperten-
sion, and therefore is instructive in elucidating the pathogenic
changes of the vascular architecture. This model illustrates the
importance of regulatory crosstalk between lung epithelial cells
and mesenchymal cells. For example, the overexpression of
TGF-a in the lung epithelium results in a persistent up-regulation
of the mitogen-activated protein kinase kinase (MEK)/extracellular
regulated kinase signaling pathway in lung mesenchyme (69). When
the inducible expression of TGF-a is discontinued, lung remodeling
is partly reversed, allowing for studies of the mediators involved in
this reversal process (67).
The lung-specific overproduction of IL-13 has been accom-
plished through a transgenic approach in which the IL-13 gene
is constitutively expressed by the CC10 promoter. These trans-
genic mice develop both airway and parenchymal fibrosis (as
measured by trichrome staining and Sircol assay) (70). Al-
though the time course of collagen accumulation in this model
is not well characterized, the development of airway and pa-
renchymal scarring is mediated by an increase in TGF-b ac-
tivity. The requirement for TGF-b activity in modulating the
profibrotic effects of IL-13 was established by demonstrating
a decrease in collagen accumulation after treatment with
a soluble TGF-b receptor. Furthermore, the administration of
aprotinin, a serine protease inhibitor, to IL-13–overexpressing
mice decreased TGF-b activity and limited scarring (70).
A single administration of an adenoviral vector containing the
IL-1b gene was also shown to induce lung fibrosis (71). Com-
pared with rats treated with a control vector, the instillation of
5 3 108plaque-forming units (pfu) of adenoviral (Ad). IL-1b
resulted in a progressive increase in lung collagen, as measured
by hydroxyproline from Day 21 to Day 60. The administration
of the IL-1b vector also resulted in an early neutrophilic inflam-
matory response in bronchoalveolar lavage fluid and an increase
in lung macrophages, and this influx in inflammatory cells was
evident on lung histology. By Day 14, lung histology also
revealed an increase in a–smooth muscle actin (a-SMA)–positive
cells within fibroblast foci. Of note, IL-1b overexpression also
increased TGF-b concentrations, TGF-b activity, and platelet-
derived growth factor (PDGF) concentrations, suggesting they
are potential downstream mediators for the profibrotic effects
of IL-1b (71).
The adenoviral-mediated gene transfer of TNF-a has also been
found to induce lung fibrosis (72). Similar to IL-1b, the over-
expression of TNF-a resulted in an early inflammatory response,
with an influx of neutrophils, macrophages, and lymphocytes.
Although the extent of collagen accumulation was not quanti-
fied in this initial study, lung sections demonstrated an accrual
of a-SMA–positive cells that were present as early as Day 7, and
that had increased in number on Day 14. The gene transfer of
TNF-a was also associated with an increase in TGF-b. Although
this study implicated TNF-a as a profibrotic molecule, a follow-
up study of transgenic mice that overexpressed this cytokine off
the surfactant protein–C promoter suggested that it may exert
antifibrotic properties in the setting of a second insult with
bleomycin or TGF-b (73). Interestingly, in the study showing
an inhibitory effect of TNF-a overexpression, concentrations of
the antifibrotic mediator prostaglandin E2in the bleomycin-
injured lungs were increased, suggesting a mechanism of pro-
tection against fibrosis. These studies point out the complex
Figure 5. Targeted Type II AEC injury induced fibrosis.
Trichrome-stained sections of lungs from wild-type
(WT) or surfactant protein–C–diphtheria toxin receptor
(SpC DTR) mice harvested on Day 28 after diphtheria
toxin (DT; 100 µg/kg) treatment on Days 0–14. Mag-
172AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 492013
role that TNF-a may play in both promoting and inhibiting
FAMILIAL MODELS OF IPF
Over the past decade, reports have linked cases of familial inter-
stitial pneumonia (FIP) to mutations in four genes: surfactant
protein–C (SFTPC) (74–76), surfactant protein–A2 (SFTPA2)
(77), telomerase reverse transcriptase (TERT) (78, 79), and
telomerase RNA component (TERC) (78, 79). Depending on
the particular reports, FIP may account for anywhere from 2–
20% of cases of IPF (32, 80–82). Furthermore, these four genes
probably account for 15–20% of cases of FIP (83). Thus, the
primary underlying genetic defects in most cases of FIP, which
is most often encountered in an autosomal dominant inheri-
tance pattern (84), have yet to be determined. In addition to
searches for rare alleles underlying cases of FIP, common alleles
may also be important, serving as risk factors for disease devel-
opment. In 2011, a common allele in the promoter of mucin 5B
(Muc5B) was found to be significantly more frequently encoun-
tered in cases of both FIP and sporadic IPF (85). In another
genetic correlate of interest, some forms of Hermansky-Pudlak
syndrome (HPS), an autosomal recessive disorder, lead to pul-
monary fibrosis, which is often the cause of death in these sub-
types (86, 87).
ics is based not only on the fact that individual genes may be im-
portant to specific families, but also that key pathways may be
identified that could be important to the pathogenesis of IPF
in general (83). As such, the identification of SFTPC mutations
(as well as SFTPA2 mutations) suggests that endoplasmic retic-
ulum (ER) stress responses may be important in the alveolar
epithelium, and indeed ER stress markers have been shown to
be prominent in AECs during IPF in general, even in the absence
of known surfactant protein mutations (88, 89). In a similar fash-
ion, telomere shortening is prominent in IPF in general, even
among populations without defined TERT or TERC mutations
(90, 91). Thus, delineating the mechanisms at play in FIP may
help considerably in improving our ability to understand the
pathogenesis of IPF. As such, models based on known FIP muta-
tions may serve as paradigms for modeling key aspects of IPF.
SFTPC mutations have been linked to both pediatric and adult
interstitial lung disease, including individuals with UIP according
to pathologic criteria, and IPF according to clinical definitions (74).
Surfactant protein–C (SPC)–deficient mice in a 129/Sv strain do
develop mild interstitial lung disease spontaneously, but also ex-
press an emphysematous phenotype. Thus, the findings do not
recapitulate the pathology noted in human forms of the disease
(92). SPC2/2mice in an outbred Black Swiss background have a
normal lung appearance, but with enhanced fibrosis after bleomycin
(26). In 2011, a model was reported in which a mutant form of
SFTPC identified in a large FIP family (L188Q SFTPC) was
expressed in AECs, resulting in ER stress in Type II AECs (88).
ER stress alone did not cause disease, but did predispose the mice
to bleomycin-induced pulmonary fibrosis, raising suspicion that ER
stress leads to a vulnerable AEC population, and that a “second
hit” then unmasks the fibrotic tendency (88).
Mutations in one of the surfactant protein–A genes (SFTPA2)
have been linked to cases of FIP (77), and in vitro modeling
suggests that such mutations may cause ER stress, as is seen
with SFTPC mutations. Specific SFTPA2 mutations have not
been modeled in vivo, but studies have revealed greater AEC
injury/death and greater lung inflammation in SP-A–deficient
mice, compared with control mice (93).
TERT and TERC
TERT and TERC mutations have been linked to cases of adult
FIP and IPF (78, 79). However, no studies have recapitulated
a pulmonary phenotype of increased susceptibility to lung
fibrosis in these mice that might be expected, given the genetic
observations (94). In the bleomycin model, whether single-
dose or repetitive, a recent study revealed that despite signif-
icant telomere shortening, bleomycin-induced lung fibrosis
was similar between telomerase-deficient mice (whether
TERT2/2or TERC2/2) and control mice (94). In contrast, a
separate study presented data that telomerase deficiency was
actually protective against bleomycin-induced fibrosis (95). It
is hard to know exactly why these mutations are not generating
a consistent phenotype in murine models, but one may specu-
late that these mutations will only be relevant with particular
environmental cues that are not adequately modeled by bleo-
Some types of HPS develop pulmonary fibrosis, and this man-
ifestation often leads to pulmonary insufficiency and death (86).
HPS is characterized by trafficking defects in different cell
populations, and in the lung, the Type II AEC appears to be
the culprit cell. For each human form of HPS, a mouse corre-
late exists, and accordingly, those that are fibrosis-prone in
humans also experience greater bleomycin-induced lung fibro-
sis (87, 96). Similar to SFTPC and SFTPA2, HPS mutations
point to the role of the alveolar epithelium in disease patho-
genesis, and provide suggestive evidence for a “two-hit” model
of fibrotic pathogenesis. As new genetic links to disease are
identified, either as rare variants in familial cases or as com-
mon variants in population studies, mouse modeling will
hold promise in dissecting the pathways pertinent to disease
TARGETED TYPE II CELL INJURY
Because IPF is known to involve AEC injury and hyperplasia,
a model was recently developed to target injury specifically to
alveolar Type II cells. This model involves the expression of
the diphtheria toxin receptor (DTR) under the control of a Type
II AEC promoter (surfactant protein–C) (97). In this manner,
the repetitive delivery of diphtheria toxin (DT, administered
daily for 14 d intraperitoneally) injures AECs specifically, in-
duces a hyperplastic proliferative response, and results in a model
of interstitial thickening that bears similarity to the changes
noted in IPF (Figure 5). The development of fibrosis is evident
by Day 21 and persists through Day 28. At a dose of 8.0 mg/kg,
DT-treated DTR-expressing mice demonstrate an approxi-
mately 2-fold increase in lung collagen content (compared
with untreated mice and wild-type mice treated with DT)
and a mortality rate of 25% by Day 21. Higher doses of DT
increase mortality (a dose of 10 mg/kg causes 50% mortality by
Day 21), without substantially exacerbating the accumulation
of lung collagen. The development of a lung-scarring response
is associated with weight loss and ruffled fur. This model is also
interesting because a selective inflammatory response, com-
posed mostly of lymphocyte antigen (Ly)6Chighmonocytes
and exudative macrophages that exhibit a phenotype of alter-
native activation, is associated with the development of fibro-
sis (98). This model is particularly useful for studying the
downstream pathways that translate epithelial cell–specific
defects into fibrosis. In this regard, targeted epithelial injury
results in the significant up-regulation of plasminogen activation
inhibitor–1, a known profibrotic mediator (98).
MODELS REPLICATING INTERSTITIAL FIBROSIS
AS A RESULT OF DIRECT LUNG INJURY
Interstitial fibrosis as a result of severe acute lung injury (ALI)/
ARDS in human subjects is a devastating complication, resulting
in significant secondary pulmonary hypertension and high mor-
tality. The development of associated multifactorial pulmonary
vasculopathy in ARDS is an independent risk factor for mortal-
ity (99). The risk factors for ALI/ARDS include a multitude of
direct pulmonary injuries (such as aspiration or lung contusion)
and indirect factors (e.g., sepsis and pancreatitis) (100). Most of
the animal models to study this phenomenon are related to
direct injuries, and a few of these models will be discussed.
Acid Instillation–Associated Fibroproliferative Lung Injury
Acid instillation recapitulates many clinical features of ALI, and
with certain modifications of delivery and/or recovery, rodents
can survive to develop interstitial thickening. This type of model
tem to be used to study fibrosis, modifications (e.g., a fluid bolus,
supplemental oxygen, and careful monitoring to be assured of
surviving the procedure) are imperative because without them,
the animals die of lung injury before the development of lung
scarring. This model recapitulates the hypoxemia that is clini-
cally relevant in ARDS. This model can be performed in ham-
sters as well as rats and mice (101, 102). Epithelial cell responses
(secretory cell metaplasia) have been characterized in hamsters
in response to three different types of acid instillation, and
these cellular changes are long-lasting (up to 17 wk) (103).
Although some investigators have used rodent ventilators to
provide supplemental oxygen, a recent study provides a modi-
fication that uses a box with humidified supplemental oxygen
(with an inspiratory oxygen fraction reduced gradually from
1.0 to 0.21) to improve survival and allow for studies of the
fibroproliferative phase after ALI. This modification should be
widely accessible to most laboratories (104) wanting to study
fibroproliferation as a result of ALI/ARDS. Similar models in
both rats and mice have used a combination of acid and par-
ticulate matter to mimic clinically relevant aspiration events in
human subjects (101, 105). A combination insult of acid with
particulate matter induces synergistic and progressive lung in-
jury, and therefore is likely to result in severe interstitial pul-
monary fibrosis (101).
Radiation-induced fibrosis is a clinically relevant injury to the
respiratory tract that results in the development of fibrosis. Sus-
ceptibility to this injury is genetically based in mice, and most
notably C3H/HeJ and CBA/J mice are resistant in comparison
with the more susceptible C57Bl/6 strain (106). A single dose
of 12 to 15 Gy of total body irradiation can result in lung fibrosis
by 20 weeks (107). However, if other organs are not shielded,
fibrotic responses will be systemic. Regimens to deliver thorax-
limited radiation are commonly applied to avoid this complica-
tion, and these models result in fibrosis by 24 weeks (108, 109).
The inflammatory responses appear to dictate the severity of
lung remodeling, but overall, this form of lung fibrosis is asso-
ciated with low mortality (106). Regarding a major feature of
this model, the pulmonary vascular remodeling that occurs is
reminiscent of pulmonary arterial hypertension (110). The
pathobiology of irradiation-induced fibrosis is believed to in-
volve free radical–mediated DNA damage and the induction
of TGF-b. This was one of the first models used to demon-
strate a reparative role for bone marrow mesenchymal stem
Lung Contusion–Induced Fibrosis
An understudied area of fibrotic lung disease involves the devel-
opmentof fibrosisaftertraumatic injury.Duringthe pastdecade,
multiple small-animal models of lung contusion–induced fibrotic
injury have been established to fill this gap (102, 105, 112, 113).
Using rats, a bilateral lung contusion without associated cardiac
injury is induced in anesthetized animals by dropping a 0.3-kg
hollow cylindrical weight onto a mobile plate with a precordial
shield (102). The height from which the cylinder is dropped can
be changed, to generate an impact energy of 2.0–2.45 Joules.
The model results in the development of lung contusion char-
acterized by hemorrhagic injury, alveolar disruption, and a subse-
quent neutrophilic inflammatory response, peaking by 48 hours
after injury (112). This injury is neutrophil-dependent and is
characterized by severe hypoxia that resolves over a period of
48 hours. A similar murine model of unilateral closed-chest
injury, using a cortical-contusion impactor, was first developed
in 2007, and is currently being used with various transgenic
animals (113). These rodents go on to develop a form of fibro-
sis seen with bronchiolitis obliterans organizing pneumonia by
7 d after the initial insult (112). Figure 6 depicts representative
hematoxylin-and-eosin staining of a rat lung on Day 7 after
contusion injury. This model may be especially useful for un-
derstanding how acute inflammatory and injury responses can
lead to fibrotic outcomes.
HUMANIZED MODELS OF LUNG FIBROSIS
Because none of the animal models of experimental fibrosis fully
recapitulate IPF pathology, recent attempts have been made to
“humanize” these models. The best-characterized humanized
model involves the intravenous instillation of human IPF fibro-
blasts into immunodeficient nonobese diabetic/severe combined
immunodeficiency (NOD/SCID/beige) mice (114, 115). These
mice lack many features of innate and adaptive immune re-
sponse, thus allowing for the growth of human cells in the lung.
However, the lungs of these mice show no evidence of fibrotic
pathology before the instillation of IPF fibroblasts. Thus, this
model has provided important insights into the pathogenic
Figure 6. Lung contusion induced fibrosis. Hematoxylin and eosin–
stained lung sections from a rat on Day 7 after contusion injury show
the development of fibrotic lesions, especially around bronchioles.
174 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 49 2013
potential of IPF fibroblasts. The instillation of IPF fibroblasts,
but not of fibroblasts from normal lungs, results in focal fibrotic
alveolar remodeling (114, 115), such as that seen in Figure 7A,
but no fibrotic changes in other organs. Interestingly, although
human fibroblasts directly contribute to the pathologic remod-
eling in the mouse lung, these cells also activate murine epithe-
lial cells and fibroblasts to undergo pathologic remodeling and
proliferation. This is perhaps the best evidence to date for
a pathologic phenotype that is fibroblast-autonomous. This
model offers advantages in that the transferred fibroblasts can
be easily labeled with cell-permeable dyes to follow trafficking,
and the development of the fibrosis is relatively rapid (occurring
within 30–35 d after injection) and persists for months. This
model also offers an opportunity to study cells derived from
multiple patients, thus providing insights into IPF heterogeneity
in fibroblast phenotypes. Finally, this model has created the
opportunity to explore antifibrotic agents with human specific-
ity. As shown in Figures 7B–7G, the pathologic remodeling in
this model can be markedly attenuated after the administra-
tion of a monoclonal antibody (mAb) directed against human
platelet-derived growth factor receptor (PDGFR)–a (Figure
7C), but not PDGFR-b (Figure 7D), compared with an IgG
control (Figure 7B). The antifibrotic effect of the anti-
PDGFRa mAb in this model was confirmed by hydroxyproline
analysis (Figure 7E). Although the PDGFRa mAb treatment
significantly reduced human TGF-b protein expression, it
exerted no effect on human PDGF-BB expression in whole-
lung samples compared with the other treatment groups. This
model also involves the disadvantage of modeling the development
of fibrosis in the absence of immune cells, which is not what
occurs in humans. This model involves the further disadvantage
that these immunodeficient mice are quite expensive and require
specialized housing. Expanded analyses of such humanized
mouse models, including mice with humanized immune systems
(e.g., bone marrow, liver, thymus [BLT] mice) (116), may offer
unique insights in future studies.
EVALUATION OF EXPERIMENTAL MODELS
Each experimental model of lung fibrosis includes its own time-
line for best evaluations of fibrotic responses. Nonetheless, they
fibrosis. Importantly, when analyzing outcomes in all of these
models, care must be taken to ensure the changes in collagen
Figure 7. Humanized model of lung fibrosis. Pulmo-
nary fibroblasts were grown from idiopathic pulmonary
fibrosis (IPF) surgical lung biopsies and labeled with
PKH26. After the PKH26 labeling, 1 ml of the fibroblast
suspension and PKH26 dye solution containing 1 3
106fibroblasts were then injected intravenously into
each mouse, using a 26-gauge needle and tuberculin
syringe. The histological appearance of the lungs of
these mice on Day 63 after injection is shown in A
(trichrome staining; original magnification, 340).
The effects of a human IgG1-l isotype control (anti–
chi-lysozyme–MOR03207), anti-PDGFRa monoclonal
antibody (mAb) (IMC-3G3; ImClone, Summerville,
NJ), or anti-PDGFRb antibody (IMC-2C5; ImClone)
were examined using histological (trichrome staining)
(B) IgG1, (C) anti-PDGFRa mAb, and (D) anti-PDGFRb
mAb (original magnification, 340), biochemical anal-
ysis (hydroxyproline; E), and transforming growth
factor–b and PDGF-BB (ELISA; F and G, respectively)
analyses for the presence of pulmonary fibrosis, per-
formed at various times after the intravenous injection
of human fibroblasts. All antibodies and control IgG
were administered at 1 mg per mouse every other
day, beginning on Day 35. Groups of mice were killed
on either Day 49 or Day 63. All data shown in E–G
represent means 6 SEMs for groups of n ¼ 5 mice.
*P < 0.05. **P < 0.01. PDGFR, platelet-derived growth
factor receptor; hPDGFR, human platelet-derived growth
expression are truly caused by fibrosis. Appropriate analyses of
of matrix deposition (e.g., hydroxyproline assays, Sircol assays,
or Western blots) with histologic evaluations of the pathology
associated with those changes. For example, collagen accumula-
tion has been noted in emphysema models (117), but the pa-
thology is clearly different from that of IPF. Thus, to provide
both biochemical and histologic analyses is important for a full
understanding of how modulations of cell–cell interactions and
soluble mediators may be affecting fibrosis. Additional modal-
ities to complement histologic and biochemical evaluations
include evaluations of lung mechanics and lung imaging. Unfor-
tunately, these techniques alone are not sufficient for delinea-
tions between lung inflammation and lung fibrosis. However, we
hope that small-animal imaging techniques will continue to
evolve and allow live-animal imaging that permits detailed eval-
uations of interstitial lung disease in these models.
In conclusion, recent advances have led to more persistent mod-
els of experimental fibrosis and have created systems to allow for
studies of targeted epithelial injury, fibroblast-specific alter-
ations, inflammatory cell modulations of fibrosis, and epithelial–
mesenchymal crosstalk. Although these models still do not
recapitulate all features of IPF pathogenesis, they allow for
specific analyses of signaling pathways and interactions among
various cell types. More persistent models contain the further
advantage of enhancing our ability to study mechanisms oper-
ative during the fibrogenic stage, and this approach increases
the likelihood of translating findings to human disease as well
as allowing for the efficacy testing of therapeutics on fibrotic
remodeling. This may permit more accurate predictions of
which compounds have the capacity to improve outcomes after
fibrosis has been established. Taken together, animal modeling
provides an important tool as the pulmonary fibrosis research
community seeks important clues to the pathogenesis of IPF
and other fibrotic lung diseases, while also seeking therapies
for these devastating diseases.
Author disclosures are available with the text of this article at www.atsjournals.org.
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