Murine Models of Inflammatory Bowel Disease (IBD): Challenges of Modeling Human Disease

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Murine Models of Inflammatory Bowel Disease (IBD): Challenges of Modeling Human Disease

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Murine Models of Inflammatory Bowel Disease (IBD):Challenges of Modeling Human Disease

JASON DEVOSS1

AND LAURI DIEHL2

1Department of Immunology, Genentech, Inc., San Francisco, California, USA2Department of Pathology, Genentech, Inc., San Francisco, California, USA

ABSTRACT

Animal models of human disease are a critical tool in both basic research and drug development. The results of preclinical efficacy studies often

inform progression of therapeutic candidates through the drug development pipeline; however, the extent to which results in inflammatory bowel

disease (IBD) models predict human drug response is an ongoing concern. This review discusses how murine models are currently being used in

IBD research. We focus on the considerations and caveats for commonly used models in preclinical efficacy studies and discuss the value of models

that utilize specific pathogenic pathways of interest rather than model all aspects of human disease.

Keywords: preclinical research and development; animal models; mouse; gastrointestinal system; inflammation.

INTRODUCTION

Inflammatory bowel disease (IBD) is a complex multigenic

inflammatory disorder affecting approximately 1 to 1.5 million

patients in the United States (Herrinton et al. 2007; Kappelman

et al. 2007; Loftus et al. 2007). IBD incidence has traditionally

been highest in North America and Europe (Gollop et al. 1988);

however, there has been an increase in the global incidence

documented beginning approximately in the middle of the

20th century (Molodecky et al. 2012).

While the introduction of biologic inflammatory mediators

such as tumor necrosis factor (TNF) inhibitors has improved

the IBD therapeutic landscape, there remains considerable

unmet medical need particularly in patients with severe or

complicated clinical manifestations and in pediatric popula-

tions (Yang, Alex, and Catto-Smith 2012; Assaa et al. 2013;

Mehta, Silver, Lindsay 2013). This need continues to drive IBD

research efforts at universities, research foundations, and

biotechnology and pharmaceutical companies spanning the

spectrum from basic science to translational medicine. A com-

mon thread in most of these research efforts is the need for

appropriate animal models. Animal models provide a means

of characterizing physiologic interactions when our under-

standing of such processes is insufficient to allow replacement

with in vitro systems.

UTILIZATION OF ANIMAL MODELS IN IBD RESEARCH

Animal models in IBD research are used in studies that fall

in 5 broad categories: addressing scientific questions, evaluat-

ing preclinical efficacy, evaluating pharmacokinetics (PK),

safety testing, and biomarker studies. There is considerable

variability in how frequently IBD animal models are used in

these categories. For instance, IBD models are rarely used for

safety studies but very frequently used to address scientific

questions and for preclinical efficacy testing. IBD models are

also commonly used for PK studies; however, use of existing

IBD models has raised relatively few concerns in this area

when compared to issues with model use in efficacy, scientific,

and biomarker studies. While this acceptance of current models

for PK studies may need to be revisited at some future date, we

will not discuss PK testing further in this review.

It is challenging to assess the full scope of use of IBD animal

models to address scientific questions. A quick PubMed search

shows greater than 600 IBD publications per year for the past

3 years that were flagged as animal models studies. This

undoubtedly underrepresents the extent of animal model use in

IBD-related studies but does illustrate that this use is both com-

mon and important to scientific research. Animal models are

typically used in these studies as a means of hypothesis testing

but are also utilized in exploratory or hypothesis generating

experiments. For these experiments, scientists may modify

standard models or create new models to enable the analysis.

A typical approach might involve creating a new genetically

modified mouse or using existing lines and subjecting these lines

to a chemical challenge (Ramirez-Carrozzi et al. 2011; Cox

et al. 2012).

The author(s) declared no potential conflicts of interest with respect to the

research, authorship, and/or publication of this article.

The author(s) received no financial support for the research, authorship,

and/or publication of this article.

Address correspondence to: Lauri Diehl, Department of Pathology,

Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA;

e-mail: [email protected].

Abbreviations: AU, adenylate-uridylate; AUC, area under the curve;

CARD15, caspase recruitment domain-containing protein 15; DSS, dextran

sodium sulfate; IL-10, interleukin-10; IACUC, Institutional Animal Care and

Use Committee; IBD, inflammatory bowel disease; KO, knockout; NSAID,

nonsteroidal anti-inflammatory drugs; NOD2, nucleotide oligomerization

domain 2; PK, pharmacokinetics; PD, pharmacodynamics; SCID, severe com-

bined immunodeficiency; TNBS, trinitrobenzene sulfonic acid; TNFa, tumor

necrosis factor alpha;; WT, wild type.

99

Toxicologic Pathology, 42: 99-110, 2014

Copyright # 2013 by The Author(s)

ISSN: 0192-6233 print / 1533-1601 online

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Another major category of animal model use in IBD

research is for preclinical efficacy testing. This is of consider-

able importance to pharmaceutical and biotechnology compa-

nies, and results of preclinical efficacy studies frequently

serve as gating criteria that determine whether a therapeutic

candidate will advance in the drug development pipeline.

Preclinical efficacy studies most commonly utilize well-

established chemically induced or genetic models of intestinal

inflammation (Table 1). It is uncommon for new IBD models to

be developed specifically for preclinical efficacy testing of a

new therapeutic candidate, although models developed in

academic labs are sometimes adopted for testing of specific

therapeutic candidates. The predictive value of results from

preclinical efficacy studies is a major concern with existing

IBD models and will be covered in more depth later in this

review.

An emerging area for animal model use is biomarker devel-

opment. Biomarkers are objective measures of normal or

pathologic processes or, in the case of pharmacodynamics

(PD) markers, of response to therapeutic intervention (Biomar-

kers Definition Working Group 2001; Strimbu and Travel

2010; Goodsaid 2012). Human IBD drug trials, particularly

those in Crohn’s disease, are complicated by both uncertainty

about current disease activity and also by the relapsing and

remitting clinical course. These variables are believed to be

significant contributors to high placebo rates, which can

obscure therapeutic benefits of drug candidates (Su et al.

2004; Sands et al. 2005; Sands 2009; D’Haens et al. 2012). The

need to reduce placebo risk in IBD trials has led to increasing

use of expensive and sometimes invasive imaging modalities to

compensate for the lack of highly reliable indicators of disease

activity (Solem et al. 2008; Loftus et al. 2007; Buisson et al.

2013), so development of reliable and noninvasive disease

activity biomarkers is an ongoing area of interest. In addition

to disease activity biomarkers, predictive biomarkers are an

important area of IBD-related research. Predictive biomarkers

are used to identify individuals within the larger IBD patient

population who are likely to have a positive response to treat-

ment with a specific therapeutic entity (de Castro et al. 2013;

Hanania et al. 2013). A successful predictive biomarker used

to enroll or interpret trial results can make the difference

between a successful or failed trial. In addition, a robust bio-

marker can reduce risk to patients by eliminating those unlikely

to derive clinical benefit from exposure to a drug that is still in

the testing phase (Becker Jr. and Mansfield 2010).

While much IBD biomarker work is done in human patient

populations, the effectiveness of this is limited because it is

often difficult to do hypothesis testing until relatively late in

drug development. If a strong predictive biomarker hypothesis

can be generated from in vitro data, then initial testing can be

done in phase II patients and the results confirmed in phase III.

But, more commonly in IBD trials, biomarker candidates are

not sufficiently robust to be incorporated in phase II study

design and, instead, phase II trial samples are frequently used

to generate a biomarker hypothesis that may get initial testing

in phase III. This longer time line creates challenges for

creation and launch of companion diagnostics (Moore, Babu,

Cotter 2012). These issues underscore the need for animal

models that share pathogenic features and/or utilize molecular

pathways of interest to enable hypothesis testing or even to do

preliminary hypothesis generating studies.

Ideally, a disease model should closely parallel the human

disease in clinical manifestations, pathophysiology, and

response to existing therapeutic reagents. However, this is

rarely possible with complex human diseases such as IBD

where multiple genetic and environmental influences deter-

mine disease onset and clinical course. This creates ongoing

challenges for scientists involved in IBD model development

and analysis. Planning for success in IBD animal model devel-

opment may require that we consider Voltaire’s comment that

‘‘the perfect is the enemy of the good’’ (Voltaire 1772) and

focus our efforts on identifying the specific features we need

to model rather than trying to recapitulate the entirety of human

disease in an experimental animal.

In this review, we discuss some examples of needs that

arise in a drug development environment and the current state

of model development to address those needs. Our intention,

rather than to provide a comprehensive review of every model

and how it may or not meet these needs, is to encourage our

readers to consider the needs specific to their own programs

and pathways of interest with the goal of optimizing IBD

models to address their most important questions. Colitis

models exist in rats, nonhuman primates, and dogs, but, in this

review, we will limit our discussion to only murine IBD mod-

els (Mansfield et al. 2001; Holst et al. 2012; Wadie et al.

2012). The use of mouse models enables relative ease in

incorporating animals with specific genetic modifications,

use of larger cohort sizes, and decreases the quantity of ther-

apeutic reagents required.

CHARACTERISTICS OF HUMAN IBD

IBD encompasses a heterogeneous group of disorders that

have 2 major phenotypic forms, Crohn’s disease and ulcerative

colitis, both with a pattern of chronic relapsing and remitting

intestinal inflammation (Abraham and Cho 2009; Kaser, Zeis-

sig, and Blumberg 2010; Levine et al. 2011). Ulcerative colitis,

the most common of the IBDs (Danese and Fiocchi 2011), is

characterized by continuous mucosal inflammation involving

the rectum and progressing up the colon with increasing dis-

ease severity. Inflammatory infiltrates are confined to the

mucosa and consist primarily of lymphocytes and plasma cells

plus granulocytes in crypt abscesses and present in the mucosa

during disease flares. Ulceration is common during active dis-

ease and colonic epithelial cell changes such as goblet cell

depletion, distorted crypt architecture, and epithelial dysplasia

are associated with chronic disease.

By contrast, Crohn’s disease is characterized by discontinu-

ous inflammation that can affect any part of the gastrointestinal

tract, although ileum and colon are most frequently involved

(Farmer, Hawk, and Turnbull 1975). Inflammation due to

Crohn’s disease can extend throughout the intestinal wall, and

100 DEVOSS AND DIEHL TOXICOLOGIC PATHOLOGY



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this transmural inflammation appears to be responsible for

many of the serious complications associated with Crohn’s

disease, such as fibrostenotic disease, abscesses, and fistula

formation (Keighley et al. 1982; Maeda et al. 1994). Epithe-

lioid granulomas are a characteristic of Crohn’s disease and

provide diagnostic differentiation from ulcerative colitis when

identified histologically. However, these granulomas are found

in only about 20 to 40% of biopsies and 60% of surgical

resection specimens (Scholmerich and Warren 2003).

Current models of IBD pathogenesis hypothesize that

disease arises from interactions between genetically suscepti-

ble hosts and the gut microflora (Xavier and Podolsky 2007).

It is clear that host genetic susceptibility plays an important

role in IBD pathogenesis. The first Crohn’s disease susceptibil-

ity gene, nucleotide oligomerization domain 2 (NOD2), which

is encoded by the caspase recruitment domain-containing

protein 15 (CARD15) gene, was identified in 2001 (Hugot

et al. 2001). Subsequent genome-wide linkage and association

studies have revealed many additional genetic polymorphisms,

and there are currently more than 160 IBD susceptibility genes

or loci recognized (Jostins et al. 2012). A number of these

susceptibility genes are shared between ulcerative colitis and

Crohn’s disease, which suggests the existence of shared

pathways in IBD pathogenesis despite differences in clinical

phenotype (Budarf et al. 2009; Zhernakova, van Diemen, and

Wijmenga 2009). While the functional role of many loci or

single nucleotide polymorphisms is incompletely understood,

many of the susceptibility genes are associated with aspects

of mucosal immunity including immune response to microbial

pathogens (Jostins et al. 2012). These genetic data are in accord

with observations that development of immunologic respon-

siveness to commensal microorganisms is a hallmark of IBD

(Arnott et al. 2004; Lodes et al. 2004; Targan et al. 2005;

Danese and Fiocchi 2011) and that shifts in the bacterial

makeup of intestinal microflora have been documented in IBD

(Frank et al. 2007; Walker et al. 2011; Nagalingam and Lynch

2012).

While the classic phenotypic descriptions of ulcerative

colitis and Crohn’s disease seem to clearly differentiate

these disease conditions, there is a subset of patients in whom

disease differentiation can be problematic. There is a low but

persistent incidence of diagnosis change in patients from

ulcerative colitis to Crohn’s disease or the reverse (Myren

et al. 1988) illustrating that clinical phenotype may change

over time in individual patients. In addition, approximately

10% of patients presenting with symptoms of IBD may have

indeterminate features that preclude an initial diagnosis of

either ulcerative colitis or Crohn’s disease (Guindi and Riddell

2004). This diagnostic uncertainty primarily arises from the

lack of a specific test that can differentiate ulcerative colitis

from Crohn’s disease resulting in heavy reliance on clinical

phenotype which can be heterogeneous. This phenotypic

heterogeneity creates obvious problems for clinical patient

management, but it should also serve as a reminder to those

of us engaged in animal model development of just how com-

plex are the diseases under the broad category of IBD.

GENERAL GUIDELINES FOR IBD MODELS IN PRECLINICAL EFFICACY

STUDIES

Once the decision has been made to initiate studies within a

preclinical model system, a series of thought questions and

tests should be employed to ensure that the model is operating

in a reproducible and consistent manner. Below, we describe a

series of factors that must be thoughtfully considered prior to

experimentation. These factors are arranged in ascending order

according to how easy they are to manage, from easily control-

lable (e.g., study size and powering of groups) to more challen-

ging (e.g., microflora).

Reproducibility is a key component of any scientific experi-

ment, whether it means being able to generate consistent data

repeatedly within the same laboratory, or being able to repeat

published work performed at other sites. Typically, the former

is more easily constrained than the latter. However, recent

publications have highlighted several major pharmaceutical

companies’ struggles in attempting to reproduce published lit-

erature (Arrowsmith 2011; Prinz, Schlange, and Asadullah

2011; Begley and Ellis 2012). While this review cannot address

all of the issues involved in data reproducibility, we will try to

highlight those factors that critically influence preclinical IBD

models such as study size, mouse strain and gender, study

reagents including positive and negative treatment controls,

and study group randomization.

STUDY SIZE

Preclinical efficacy studies should be of sufficient power to

draw a meaningful conclusion. Ideally, this would be deter-

mined through review of the study design and past model

results with a biostatistician. While our typical group size is

approximately 10 mice, it is not possible to make a blanket

recommendation that will be applicable to all institutions even

for a single model. The number of mice needed is largely deter-

mined by the variability typically observed within a group. This

varies widely between institutions and by specific model so

must be determined locally. In general, the fewer animals per

group, the more likely subtle outliers will influence the

interpretation of an experiment and the lower the likelihood

of generating reproducible data. Despite the fact that inbred

strains of mice are typically used in preclinical models of IBD,

variability in the response is frequently observed and must be

taken into account. In our experience, models of chemically

induced colitis have a higher degree of variability than genetic

models. This may be a reflection of the relatively short disease

course in acute chemical models, which can highlight early

kinetic differences in disease onset that might equilibrate over

time. In contrast, chronic or progressive models of disease

often develop, over sufficient periods of time, a rather homoge-

neous disease burden.

GENDER

Gender can also provide a source of variability, and gender-

specific effects have been documented for dextran sodium




sulfate (DSS) colitis (Mahler et al. 1998), 2,4,6-trinitrobenzene

sulfonic acid (TNBS) colitis (Bouma, Kaushiva, and Strober

2002), and inflammatory disease in senescence accelerated

mouse prone 1 (SAMP1)/Yit Fc mice (Pizarro et al. 2011).

Regardless of whether the induction of disease is possible in both

genders, we would recommend using female animals whenever

possible. Male animals are more prone to fighting and the inflam-

mation and stress associated with such behavior often has a neg-

ative impact on studies. In long-term or chronic studies, fight

wounds may also fester and require euthanizing the animals in

accordance with Institutional Animal Care and Use Committee

(IACUC) protocols. While single housing of experimental ani-

mals is an option, it becomes challenging as study sizes increase.

REAGENT VALIDATION

For proper interpretation of a scientific study, the reagents

and materials used in the study should be rigorously validated.

For genetically engineered animals, this should include

confirmation that the genetic targeting is correct and complete

(e.g., gene and protein expression for knockout animals). In

chemically induced models of disease, such as DSS and TNBS

colitis, each new lot of chemical inducer should be titrated prior

to the study to understand the dose–response relationship. If the

amount of DSS is too little, the overall histological burden will

be insufficient to provide a window to see differences between

treatments. If the amount of DSS is too high, the dose may be

fatal or may result in damage too extreme to reasonably expect

a detectable protective effect, regardless of treatment reagents.

MOUSE STRAIN

Rodents, in particular mice, have been the model of choice

for immunologists, and inbred mice are available in a wide

range of strains. However, each of these strains has a unique

genome that makes it susceptible or resistant to intestinal

inflammation. DSS colitis has been attempted using many

different mouse strains with varying degrees of success. The

current hypothesis of how colitis-inducing injury occurs is that

DSS is directly toxic to the gut epithelium (Laroui et al. 2012).

The loss of epithelial integrity and subsequent migration of

bacteria into the crypt results in inflammation and triggers a

pathologic loss of structure. Despite this rather broad postu-

lated mechanism of action, BALB/c mice and C57BL/6 mice

require significantly different doses of DSS to induce disease.

At present, the reasons behind this difference are unknown, but

it must be taken into account when choosing an animal model.

In contrast, induction of colitis with TNBS results in a similar

degree of acute toxicity to the epithelium and subsequent

inflammation; however, SJL and BALB/c strains are suscepti-

ble to disease induction while C57BL6 are resistant (Scheiffele

and Fuss 2002).

Another aspect to consider in choosing a mouse strain and

preclinical model is the availability of genetically engineered

animals for the gene of interest. The majority of genetically

engineered animals are constructed on the C57BL6 back-

ground. Thus, if an investigator wishes to determine the effects

of their particular genetically engineered animal on a particular

model system, they must either choose a model compatible

with this genetic background or spend significant time and

resources backcrossing their animals to a different strain.

TREATMENT CONTROLS

In order to draw meaningful conclusions from any scientific

experiment, positive and negative controls must be incorpo-

rated. In preclinical models of IBD, this can be challenging

as the number of clinically validated therapies is relatively low

and the number of models that respond to these therapies is also

low. Although they are not typically used in the clinic due to

safety issues, high-dose immunosuppressants can be used in

most inflammation-mediated models as a positive control

(Hoshino et al. 1992; Melgar et al. 2008; Hirano and Kudo

2009; McNamee et al. 2010, 2013). In Table 1, we summarize

the standard controls used in various animal models and their

respective effects on disease.

RANDOMIZATION

Once the model has been chosen and the number of animals

is decided, animals should be randomized into experimental

groups based on factors identified as important to the model

in question. This process, termed initial balancing or randomiz-

ing, ensures that subtle differences across the cohort will be

less likely to influence the experimental outcome. For IBD

models, this typically consists of genotype, date of birth or age,

body weight, and if animals are to be treated after the onset of

disease, some accounting for disease burden prior to start of

treatment. Understanding the degree of pathological change

without histology is technically challenging, however. In our

experience, the pathological changes that occur in colitis mod-

els can be positively correlated with body weight (Figure 1). In

contrast, disease activity scores such as diarrhea and presence

of occult blood are typically not quantitative enough to be

meaningfully correlated with pathology (i.e., these are binary

readouts) and are not used in balancing cohorts. When

available, more sophisticated techniques such as endoscopy

can be employed to assess colon damage and group animals

(Laroui et al. 2012). In contrast to colitis models, ileitis models

do not positively correlate with body weight loss, and visuali-

zation of the mouse ileum is not possible using endoscopy

methods. For this instance, we are investigating the use of

disease biomarkers as a means of balancing cohorts. However,

the number of predictive biomarkers in preclinical models of

IBD is limiting, although recent studies have been published

looking at fecal biomarkers such as calprotectin and lipocalin

(Chassaing et al. 2012; Cury et al. 2013).

ANIMAL SOURCE

Despite efforts to properly control studies, variability may

derive from a myriad of other external sources. The gut micro-

flora, a collective term for the various species of bacteria inha-

biting the intestinal tract, has been demonstrated in multiple

Vol. 42, No. 1, 2014 EVALUATING MURINE MODELS OF IBD 103



model systems to be important in disease. Antibiotics decrease

the severity of DSS- and TNBS-induced colitis (Aranda et al.

1997; Hans et al. 2000; Fiorucci et al. 2002), and the immune

response in CD45RBhi transfer colitis is believed to be largely

targeting endogenous bacteria (Stepankova et al. 2007). Simi-

larly, the emergence of intestinal disease in the SAMP1/Yit

mouse corresponded with the transfer of the colony from a

native flora facility to a specific pathogen free facility (Matsu-

moto et al. 1998), and gnotobiotic animals are often resistant to

disease induction. Since different vendors, and even different

facilities of the same vendor, are known to harbor slightly

different microflora (Ivanov et al. 2009), the same source of

animals should be used across experiments. However, other

recent studies have suggested that the microflora of genetically

deficient animals may be unique but also transferrable to wild-

type littermates, suggesting such effects may mask phenotypes

observed within a colony (Elinav et al. 2011). Further compli-

cating this matter, there is also an emerging role for nonmicro-

bial pathogens, such as norovirus (Cadwell et al. 2010), and the

fact that the microflora diversity and frequency changes in

response to the induction of colitis (Okayasu et al. 1990).

Despite these challenges, we still believe that wild-type litter-

mate controls should be used in experiments whenever possi-

ble, since differences in disease induction can be observed

within ‘‘wild-type’’ animals of different colonies but the same

strain (Figure 2).

HISTOLOGIC PROCESSING AND ANALYSIS

Finally, because histologic analysis is still the gold standard

for evaluating the extent of intestinal lesions and protective

effect of therapeutic candidates, maintaining quality and con-

sistency during preparation of histologic specimens is impor-

tant. In our opinion, there is not a single ‘‘perfect’’ system

for preparing tissue samples for histologic analysis. Instead,

we find that maintaining internal consistency in how intestinal

tissues are collected and prepared allows for best results. At our

facility, we fix either colon or small intestine held in a Swiss

roll configuration by a standard plastic histology cassette using

10% buffered formalin. Our technique is a variation of that

described in the Moolenbeek which we have modified by not

making the longitudinal opening made in the intestinal wall and

instead removing fecal material using a saline flush (Moolen-

beek and Ruitenberg 1981). This allows for rapid evaluation

of the entire colon or small intestine by the pathologist or using

an automated algorithm (Kozlowski et al. 2013).

Reported histologic scoring systems vary considerably

between institutions, pathologists, and the various disease mod-

els. While it might be useful to develop some ‘‘best practices’’

in regard to scoring common preclinical efficacy models, thatFIGURE 1.—Correlations between body weight and histology in various

IBD models are shown for various preclinical models of IBD. Body

weight loss is reflected as percentage body weight (relative to base-

line) area under the curve (AUC) for the portion of the study indicated.

(A) DSS; results are a meta-analysis of 5 studies; diamond ¼ cyclos-

porine A (CSA), circle ¼ no DSS control, square ¼ isotype control

antibody. (B) TNBS; results are a meta-analysis of 2 studies; diamond

¼ CSA, circle ¼ FIGURE 1.—(CONTINUED) ethanol only, square ¼TNBS. (C) CD45RBhi transfer colitis; results are a meta-analysis of

3 studies; diamond ¼ aP40 Ab, circle ¼ unsorted control cells, square

¼ isotype control antibody. IBD ¼ inflammatory bowel disease; DSS

¼ dextran sodium sulfate; TNBS ¼ trinitrobenzene sulfonic acid.




task is beyond the scope of this review. At our institution, we

use a robust and rapid histologic scoring system that we apply

to all colitis studies with some model-specific adjustments. The

scoring system is designed to capture both extent and severity

of lesions by scoring 4 anatomic regions of colon (proximal,

middle, distal colon, and rectum) on a severity scale from 0

to 5. Criteria for scoring severity are model-specific. For

instance, DSS colitis is primarily scored based on loss of colon

crypts, while transfer colitis is scored based on inflammatory

infiltrate and change in intercryptal distance. But the scoring

output is consistent and, as such, is readily interpretable by a

variety of collaborating investigators. Examples of scoring out-

put can be seen in Figures 1 and 2.

CONSIDERATIONS FOR CHOOSING A PRECLINICAL MODEL

The above guidelines apply to all preclinical IBD models. In

this section, we will discuss strategies for determining the

model of greatest relevance to the particular scientific question

to be addressed. It would be outside of the scope of this article

to discuss every model published to date. To simplify, we will

focus on a set of standard preclinical models frequently used in

pharmaceutical companies to assess therapeutic efficacy.

These models span a diverse landscape of biology and are

summarized in Table 1.

As described previously, Crohn’s disease and ulcerative colitis

can occur in different anatomic regions of the gastrointestinal

tract. For many years, the majority of animal models published

focused on the large intestine, with DSS, TNBS, CD45RBhi cell

transfer, and interleukin-10 (IL-10) gene deficiency induced

inflammation all largely affecting the colon (Neurath, Fuss, and

Strober 2000; Read and Powrie 2001; Perse and Cerar 2012). The

colon and the small intestine, however, differ in anatomical and

cellular structure as well as bacterial diversity and burden (Eck-

burg et al. 2005). Thus, it is reasonable to postulate that the

mechanisms governing disease in these distinct locations may

be similarly unique. Fortunately, the emergence of a number of

models that develop inflammation in the small intestine, includ-

ing the Tumor necrosis factor alpha delta AU rich element

(TNFDARE) and the SAMP1/Yit (Fc) has provided an additional

series of platforms to test therapeutic candidates (see Table 1).

IBD models tend to segregate by the nature of the response

that is evoked. To better determine whether a model would suit

a particular scientific question, it is helpful to ask what is the

dominant pathological response that is observed. For example,

does the postulated therapeutic mechanism of action target pre-

vention of damage to the epithelium or intestinal architecture,

inflammation caused by innate immune cells, or inflammation

mediated by an adaptive immune response?

Chemically induced models of colitis, such as DSS and

TNBS, induce acute damage to the epithelium. These models

are useful to explore pathways or therapeutics that are thought

to protect intestinal epithelial cells from damage or stress

response as well as pathways that maintain gut integrity in the

presence of a biological insult. Animals that have genetic

deficiencies in permeability through a variety of mechanisms

(Laukoetter et al. 2007; Kong et al. 2008; Chogle et al. 2011;

Williams et al. 2012) have been shown to be more susceptible

to DSS colitis. Consistent with this, therapeutics that maintain

or increase gut permeability are protective in DSS colitis

(Ukena, Singh, and Westendorf 2007; Chogle et al. 2011).

However, damage to the epithelium is only the opening salvo

of DSS colitis. Upon loss of barrier integrity, bacteria rapidly

penetrate the epithelium and mediate both direct and

inflammation-associated toxicity (Johansson et al. 2010;

Johansson et al. 2013). Thus, therapeutic agents that moderate

the gut flora or modify subsequent inflammatory responses to

gut flora also have the potential to demonstrate efficacy in the

DSS colitis model. Illustrating this point, treatment of specific

pathogen-free colonized animals with antibiotics improves

colitis (Hans et al. 2000; Rath, Schultz, and Sartor 2001). Sur-

prisingly, however, the administration of DSS to gnotobiotic

animals results in severe disease—suggesting that normal flora

is required to establish those mechanisms that protect the

epithelium from insult (Kitajima et al. 2001; Hudcovic et al.

2007). Consistent with this hypothesis, germ-free animals have

significantly decreased mucus layer thickness in the lumen of

the colon, and animals with a genetic deficiency in mucus

production have exacerbated responses to DSS (Petersson

et al. 2011). From this standpoint, DSS colitis and other acute

biological insult models represent a meaningful means of

assessing gut integrity, host responses to microflora mediated

by intestinal epithelial cells, and innate immune cells.

Innate immune cell response dominates in acute inflamma-

tion models when there is insufficient time to develop an

FIGURE 2.—Disparity in disease induction in wild-type (WT) and

knockout (KO) animals. In experiment 1, wild-type animals from

another colony were used. In experiment 2, littermate controls were

used. Symbols indicate individual animals within the experiment. Bars

are mean histology score þ/� standard deviation.




adaptive immune response. This is also true of intestinal

inflammation models that have been performed in commonly

used immunodeficient mouse strains. These models have been

used to ascertain the contribution of innate immune cells to the

maintenance of immune tolerance and responses to inflamma-

tion in the gut. While several studies have suggested a role for

T cells in the DSS response, disease induction is similar in mice

homozygous for the severe combined immunodeficiency

(SCID) mutation or the Rag1 mutation which is devoid of B

and T cells, clearly demonstrating that these cells are neither

necessary nor sufficient for disease development (Axelsson

et al. 1996; Kim et al. 2006). Within the damaged epithelium,

neutrophils and macrophages are the most common immune

cells present. Therapies that target the downstream mediators

of these cells, such as reactive oxygen species, are therefore

protective (Krieglstein et al. 2001; Amrouche-Mekkioui and

Djerdjouri 2012; Vong et al. 2012; Yasukawa et al. 2012).

While the acute chemical-induced colitis models typically

only incorporate features of innate biology (both epithelial and

immune), the chronic and progressive models typically illus-

trate a complex interplay between innate biology and adaptive

immune responses. The TNFDARE mouse model, in which

loss of translational repressors result in chronic TNFa overex-

pression (Kontoyiannis et al. 1999, 2002), provides an example

of this complexity and the resulting opportunities for testing

therapeutic entities that target various aspects of immune

function. In normal animals, the regulation of TNFa expression

is tightly controlled, including regulation through mRNA

destabilization mediated by a 30 adenylate-uridylate (AU)-rich

region (Anderson 2000). When the AU-rich region is removed

from the gene, TNFa overexpression is observed in all cell

types normally capable of expressing TNFa and these

TNFDARE mice develop spontaneous arthritis and ileitis

(Kontoyiannis et al. 1999). A series of genetic crosses suggest

that ileitis observed in this model is dependent on CD8þ T

cells and the effector cytokines IL-12/23 p40 and interferon-

g (Kontoyiannis et al. 2002). However, while disease was

greatly diminished in animals that lack the b2-microglobulin

gene, a necessary component of major histocompatibility class

1, and subsequently display greatly diminished CD8þ T cells,

lymphocyte-specific overexpression of TNFa resulted in only

mild ileitis. Thus, while CD8 T cells are effector cells neces-

sary for disease, they are not sufficient. In contrast, driving

TNFa overexpression in the myeloid compartment using

LysM-cre was sufficient to cause ileitis comparable to a

TNFDARE animal indicating that myeloid immune cells are

an important mediator of disease (Kontoyiannis et al. 2002).

Finally, an additional study suggested that TNF receptor

expression solely via the collagen VI promoter, restricting the

ability to respond to TNF to the epithelial layer of the gut and

other stromal tissues, was sufficient to induce disease (Armaka

et al. 2008). This model highlights the complex interplay

between the various facets of an immune response—myeloid

production, epithelial sensing, and an effector response

mediated by CD8 T cells and the production of interferon-g all

contribute to induce a disease process controlled by TNFa.

The TNFDARE model example illustrates the complexity of

an intact immune response, highlighting the difficulty in mod-

eling such a system in an in vitro fashion. It also demonstrates

how detailed knowledge of the mechanistic basis of disease can

help to determine what therapeutics may be active in a disease

context. Neutralization of TNFa in the TNFDARE model is

very efficacious (McNamee et al. 2013); however, targeting

of downstream effector functions allows the interrogation of

the events set in motion by this single cytokine.

INTERPRETING PRECLINICAL DATA FROM IBD MODELS

The previous sections have discussed decision making

during IBD model development and the use of knowledge

about the underlying biology to determine when to employ

such a model. In this section, we will discuss the interpretation

of data generated from these models and recommend how to

weigh the predictive value of study data. In this context, it is

useful to consider whether a model is intended to serve as a

translatable model or a pathway model.

A truly translatable preclinical model of human disease

would incorporate several features including similar pathologi-

cal hallmarks of disease, shared underlying mechanisms and

biology, a response to standard of care therapeutics that mirrors

what is observed in human patients, and accurate prediction of

response to experimental therapeutics in clinical trials. Unfor-

tunately, there are no models that currently exist which fulfill

these criteria. We choose to view preclinical models of disease

as pathway models that will inform whether or not modulation

of a specific pathway in the disease state will have an effect on

the overall process. To the extent that the mechanism of action

for a given therapeutic is known, the downstream effect of

modulation can be queried. This information can then be linked

to developing the knowledge of human disease biology and

genetics from which therapeutic candidates arise. When a spe-

cific pathway is determined to be activated or repressed in

human disease, we then determine whether or not it is similarly

modulated in an animal model using many of the principles

articulated earlier in this review.

If a given pathway is modulated in both human and mouse

disease and therapeutic targeting of that pathway in a relevant

preclinical model results in disease amelioration, how much

does this inform our understanding? Does this need to be

demonstrated in a single model or multiple models? To what

extent should a preclinical model be used for deciding whether

or not to advance to a clinical trial? The answers to these ques-

tions are complex and will likely vary depending on goals of

the individual scientist posing the question as well as the

current goals of the sponsoring organization. Given that we

have already addressed the limitations of preclinical animal

models and do not consider them as truly translatable, we view

preclinical efficacy as a criterion for advancing candidate

therapeutic agents to the next stage of development. However,

preclinical efficacy cannot serve as the only criteria.

If an experimental therapeutic is advanced into clinical

trials, does preclinical data accurately predict success? In a




recent review of 108 phase II clinical trials, over half of the trials

failed due to efficacy (Arrowsmith 2011). Similarly, a review of

83 phase III clinical trials noted that 66% of the trials failed due

to efficacy (Arrowsmith 2011). It is reasonable to assume that

some form of preclinical data was generated to support these

studies, suggesting that either the model was not translatable

to human disease or the effect to which target was modulated

in mouse and human was not comparable. The latter hypothesis,

that a given drug fails due to insufficient target coverage, can be

addressed in both rodents and humans provided a PD marker is

identified. PD markers provide a context in which to understand

relationships between the dose of drug administered and the

biological effect that the dose produces. Ideally, the same PD

marker is informative in both the preclinical model and the

human model, although distinct PD markers can be used if they

both faithfully report on the downstream biology in each species.

In IBD, however, the most relevant organ in which to collect

samples for PD interrogation is the gut and frequent biopsies

may not be desirable to all IBD patients. A blood-based PD

marker, therefore, is desirable but extensive validation studies

must be performed to ensure that the PD readout in the blood

is reflective of target modulation in the end organ.

Other factors that some investigators choose to assess within

a preclinical model include immunogenicity and safety.

However, both of these are difficult to interrogate within the

context of an IBD model. The predictive value of preclinical

models for immunogenicity is a controversial subject and many

animal models show limited predictive values (Norlander, Got-

thard, and Strom 1990; Brinks et al. 2013; Thway et al. 2013),

owing to differences in treatment regimens, dosing routes, and

host immune response. In addition, human subjects may be on

immunosuppressant regimens that will further complicate the

anticipation of antidrug responses. Safety assessment within

an animal model is similarly contentious, especially if baseline

values typically assessed in a safety study are significantly

affected by the disease process. For this reason, we choose to

assess immunogenicity and safety in separate studies per-

formed in healthy animals.

We view the current landscape of IBD model development

for scientific as well as preclinical efficacy testing as challen-

ging but one in which careful decision making can enhance the

opportunities to derive meaningful data from these studies.

Both the increasing understanding of factors contributing to

human disease and the expanding repertoire of mouse models

of intestinal inflammation provide opportunity for investigators

to dissect mechanistic pathways and response of those path-

ways to experimental intervention. While no single mouse

model recapitulates all of the features of human IBD, it is worth

remembering that there is considerable genotypic and phenoty-

pic variation even within humans with a similar diagnosis of

either Crohn’s disease or ulcerative colitis. Therefore, it is

unlikely and possibly not even desirable to be able to develop

a single prototypic model of disease. Careful utilization of

pathway models to query specific scientific or efficacy ques-

tions is likely to be the more successful approach to existing

needs.

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