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C H A P T E R

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Animal Models of Inflammatory BowelDisease for Drug Discovery

Atsushi Mizoguchi*, Akira Andohy*Department of Pathology, Harvard Medical School, Boston, MA, USA

yDepartment of Mucosal Immunology, Shiga University School of Medicine, Shiga, Japan

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O U T L I N E

Introduction
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Classification of Mouse IBD Models
501
Etiology and Mouse IBD Models
501
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The Hygiene Hypothesis 5
01 Appendectomy 5 02 Cigarette Smoking 5 03
Classical Therapy and IBD Models
503 Corticosteroids 5 03 Cyclosporine/Tacrolimus 5 04 Nonsteroidal Anti-Inflammatory Drugs 5 04 5-Aminosalicylic Acid 5 05
Biological Therapy and IBD Models
505 Anti-TNF-a Therapy 5 05 Anti-Integrin-a4 Therapy 5 06 Anti-Integrin-b7 Therapy 5 07 Anti-Interleukin-12p40 and Anti-Interleukin-23
Therapy 5
07
499l Models for the Study of Human Disease

dx.doi.org/10.1016/B978-0-12-415894-8.00022-1

Anti-Interleukin-17A Therapy 5
08 Anti-Interleukin-6R Therapy 5 09 Anti-CCR9 Therapy 5 09 Recombinant Interleukin-10 Therapy 5 09 Stem Cell Therapy 5 10 Anti-CTLA4 Therapy 5 11 Anti-CD3 Therapy 5 11 Anti-CD25 Therapy 5 13 Anti-Interferon-Gamma Therapy 5 13 Recombinant GM-CSF Therapy 5 13 Recombinant Interleukin 11 Therapy 5 14 Recombinant Keratinocyte Growth Factor Therapy 5 14 Anti-CD20 Therapy 5 14
Conclusion
518
Acknowledgments
518
References
518
INTRODUCTION

Inflammatory bowel disease (IBD) is a chronicrelapsing inflammatory condition that affects over1.5 million Americans.1 The incidence and prevalenceof IBD are increasing with time and in different regionsaround the world, indicating its emergence as a globaldisease.2 IBD is classified into two major forms, Crohn’s

disease (CD) and ulcerative colitis (UC), which aremediated by both common and different mechanismsand have distinct clinical features.3–6 For example, CDis characterized by transmural inflammation affectingthe entire depth of the intestine throughout the digestivetract (frommouth to anus), while UC is characterized bymore focal inflammation restricted to the mucosa of thecolon. A T-helper cell 1 (Th1)-dominant cytokine profile

Copyright � 2013 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/B978-0-12-415894-8.00022-1

22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE500

is often seen in CD, while UC tends to have contribu-tions from the Th2 response.5

The mucosal immune system is distinct from itssystemic counterpart by virtue of its enormous anti-genic exposure (commensal flora, food antigens, path-ogens), making IBD pathogenesis more complicated.7

Indeed, many kinds of knockout (KO) and transgenic(Tg) mouse strains have been shown to develop colitisand/or ileitis spontaneously. So far, at least 69 kindsof animal model are available to study IBD.8 Thesemodels can be classified into four groups dependingon the location of inflammation (Fig. 22.1). The “colitismodel” develops inflammation only in the colon, the“ileitis model” only in the small intestine, the “pan-enterocolitis model” in both colon and small intestine,and the “systemic model” in multiple organsincluding the intestine. Importantly, new therapeuticideas for IBD have been stimulated by preclinicalstudies in the wide variety of animal models ofIBD.9,10

SAMP/YitEpithelial-specific N-cadherin KI

TNFR1-deficient, epithelial-specific TGFβ activation kinase

Src-homology-2-containing inositol 5 phosphatase (SHIP)1 KO

TNF(ARE) KI

CD2-specific TNFS

Epithelial-sp

Epithelial-specific

Runt-related transcription factor RUNX3 KO

Inducible enteric glia KO

Epithelial-specific X-box-binding protein (XBP) 1

Glutathione peroxidase (GPX) 1

Ileitis

OxazolonTNBSCotton-top tamarin

C3H/HeJBir CD3eT

Janus family tyrosine kinase ( T-cell-specific suppressor of cytokine signaling (SOC

Epithelial-specific core 1β1,3-galactosyltransfera

IL-7 Tg

RAG KO treated with anti-CD40

CD45RB IL-10 KO

CD1

TCRα K

Wiskott-Aldrich syndrome protein (WASP) K

Phosphatidyl inositol 3 kinase (PI3K) p110δ KI

CD4-specific phosphoinositide-dependent kinase (PD

T cell-specific B-lymphocyte-induced maturation protein 1

G protein Gαi2 KO

Liver-specific soluble C

CD11c-specific integin β8 KOInducible STAT4 Tg

Myeloid-specific STAT3 KO

Inducible STAT3 KO

24αβ TCR Epithelium polarized sorting factor AP1B KO

IL-6 receptor gp130 KI

Epithelial-specific IκB kinase NEMO KO

T

K

Multiple drug resistanc

RAG KO with deficiency of nuclear factor of activated T ce

RAG KO with deficiency of T-box transcription facto

Colitis

FIGURE 22.1 The different types of IBD models. These include chemspontaneous models (black), congenital models (brown), and genetically einto conventional transgenic models (light blue), conventional knockoutknockout models (pink), knock-in models (orange), and innate models (pu(colitis, red square), small intestine (ileitis, blue square), or multiple orga

VI. URINARY TRACT, K

Over 71 susceptibility loci/genes in CD and over 47 inUC have already been identified in humans.3,11,12 There-fore, one of the current topics in the field of IBD modelsis to identify the biological function of these IBD suscep-tibility genes, and these studies have brought manynovel concepts to further understand IBDpathogenesis.3,11–13 Another topic is to identify thecommensal bacterial composition associated with IBD.The importance of this topic has been highlighted bythe fact that development of colitis was abolished inthe majority of murine IBD models when they weremaintained in a germ-free condition.14 The relevanceof this field to human IBD is supported by recent meta-genome analyses of the human intestinal tract, whichhave identified that CD, UC, and healthy individualsall had distinct bacterial composition patterns.15

Interestingly, Neurath and colleagues have proposed“10” mysteries with IBD, some of which are associatedwith the etiological factors.16 Among them, the hygienehypothesis (probiotics), appendectomy, and cigarette

1-deficient DKO

F15 (TL1A) Tg

ecific IL-15 Tg

Caspase-8 KO

KO

IL-2 KO CD25 KOCD122 KO

TGFβ KO

Dominant negative TGFβR II Tg

Casitas B-lineage lymphoma protein cb1-b KO

Ubiquitin-editing enzyme A20 KO

CD2-specific TNFSF14 (LIGHT) Tg

B-cell-specific CD40L TgEndothelial-specific integrin αV KO

CD4-specific TGFβ KO

Autophagy-related gene (Atg) 5- deficient thymus-implanted nu/nu mice

/2 DKO

DSS

g model

JAK) 3 KOS) 1 Tg

se KO

Mucin 2 KO

32 KO

O

O

K) 1 KO

(Blimp1) KO

D86 Tg

Tg x CD1d Tg

LR5 KO

eratin 8 KO

e (Mdr) 1a KO

lls (NFAT)c2

r T-bet

Systemic Inflammation

ically induced models (dark green), cell-transfer models (light green),ngineered models. Genetically engineered models are further classifiedmodels (red), conditional transgenic models (dark blue), conditionalrple). These models spontaneously develop inflammation in the colonns including the colon and small intestine (systemic, green square).

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ETIOLOGY AND MOUSE IBD MODELS 501

smoking may have potential for IBD therapy. In addi-tion, several conventional and biological drugs havebeen successfully, or unsuccessfully, applied for IBD,or are currently in clinical development.1,17 Therefore,this chapter focuses on these fields (etiological factorsand drugs) that may have immediate therapeutic poten-tial. The authors hope that the datadsummarized in thischapterdwill direct clinicians who are unfamiliar withanimal models of IBD to be familiarized on interpretingdata from IBD models, for the consideration of futureclinical trials of candidate therapies. In addition, thesedata may be helpful for basic researchers to realizesome clinically relevant fields for bridging the gapbetween the bench and the bedside.

CLASSIFICATION OF MOUSE IBDMODELS

In addition to the location of inflammation (seeabove), IBD models can be classified into five majorgroups depending on the method of disease induction(Fig. 22.1)d“chemically induced models” such as thedextran sodium sulfate (DSS) model, in which acutecolitis can be induced by administration of DSS indrinking water; “cell-transfer models,” such as theCD45RB model, in which colitis can be induced inimmunodeficient hosts by adoptive transfer ofCD4þCD45RBhigh naıve T cells from wild-type (WT)donors; “spontaneous models,” such as the cotton-toptamarin (a marmoset native to a small geographic regionof Colombia) that develops colitis presumably throughenvironmental stress when maintained at US; “congen-ital models,” such as SAMP/Yit mice that develop ileitisin a senescence-prone strain derived from 24 genera-tions of siblings from a litter of AKR/J mice; and “genet-ically engineered models” that develop colitis and/orileitis by genetically engineered deletion or overexpres-sion of certain molecules.8,18,19 In addition, geneticallyengineered models are further classified into sixsubgroups.8 “Conventional transgenic” or “conven-tional knockout” mice are genetically engineered tocontinuously overexpress or lack the gene of interest inall cell types, respectively. “Conditional transgenicmice” are genetically engineered using cell-specificpromoters to overexpress the gene of interest in a specificcell type. “Conditional knockout mice” are geneticallyengineered to lack a gene of interest in a specific celltype or to make the gene deletion inducible at anytime. “Knock-in (KI) mice” are genetically engineeredto carry a mutation in the gene of interest. “Innatemodels” are immune-deficient mouse-based systems,such as recombinant activation gene (RAG) KO micethat lack both T and B cells.


At least 66 kinds of IBD model had been establishedby early 2011,8 and 3 new genetically engineered IBDmodels have since been established (Fig. 22.1). Amongthe new models, epithelial-cell-specific deletion ofcaspase-8 (under control of the villin promoter) resultedin the spontaneous development of ileitis but not colitisin more than 80% of mice by inducing “necroptosis”.20

Polarity of epithelial cells is, in part, established by anepithelium-specific polarized sorting factor adaptorprotein, AP1B. AP1B KOmice spontaneously developedantibiotic-treatable colitis at 8 weeks of age.21 24ab trans-genic (Tg) mice carry a T-cell receptor (TCR) froma CD1d-reactive natural killer T (NKT) cell line withexpression of TCRVa3.2 and Vb9. When 24ab Tg micewere crossed with CD1d Tg mice, 70% of them devel-oped colitis at 6 months of age.22 The spontaneousdevelopment of colitis was still observed when theywere further crossed with RAG1 KO mice (24ab Tg �CD1d Tg � RAG KO) that had only a mono-associatedT-cell population, TCRVa3.2þ Vb9þ NKT cells.

ETIOLOGY AND MOUSE IBD MODELS

The Hygiene Hypothesis

Environmental factors are essential components ofthe pathogenesis of IBD.23 The hygiene hypothesis hasbeen proposed because of the fact that better livingconditions during childhood have been associatedwith an increased risk of IBD.1,23 North–south gradientsexist within individual countriesdCD is less common inthe southern US and in the south of France.24 Givena more favorable environment (warm temperaturewith high humidity) for microbes in the southern parts,these north–south gradients may support the hygienehypothesis.

Analysis of the recent evolutionary history of humaninterleukin (IL) genes through integrating informationon environmental variables suggested that helminthinfections represent a major selective force of thehygiene hypothesis in IBD.25 In contrast, a large,population-based cohort study suggested that helminthinfections do not reduce the risk for IBD.26 Although theinvolvement of prior helminth infections in the hygienehypothesis of IBD is still controversial, preventive andtherapeutic effects of helminth infections on colitishave been well explored using mouse IBD models.27

Pre-exposure to Schistosoma mansoni eggs, Trichinella spi-ralis, or Hymenolepis diminuta made mice more resistantto Th1-mediated acute colitis induced by trinitroben-zene sulfonic acid (TNBS) or dinitrobenzene sulfonicacid (DNBS).28,29 A therapeutic effect of S. mansoni andAncylostoma caninum extracts has also been reported inthe TNBS colitis model.30 The beneficial effect of

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helminths on colitis may be mediated by suppression ofinterferon gamma (IFN-g) and IL-17 production,enhancement of IL-4, IL-10, and transforming growthfactor beta (TGF-b) production, induction of regulatoryCD8þ T cells, and/or inhibition of macrophageactivation.30–33 TGF-b-signaling in T cells may berequired for the suppression of the Th1 response byhelminthic parasites.34 Different effects of helminths onTh2-mediated colitis in humans versus mice have beenreported. A randomized double-blind placebo-controlled human trial has demonstrated some benefi-cial effect of Trichuris suis ova on UC that has significantcontributions from Th2 responses.35 On the other hand,pre-exposure toH. diminutamademice more susceptibleto Th2-mediated acute colitis induced by oxazolonethrough enhancement of IL-5 production.36

A helminth-independent hygiene hypothesis may besupported by a Th2-mediated chronic colitis model ofTCRa KO mice. Gaskins et al. reported in 1997 thatTCRa KO mice spontaneously developed colitis whenmaintained under specific pathogen-free conditions(SPF), but failed to do so when maintained in a conven-tional facility (CF).37 This observation, although unex-pected, has been confirmed by a subsequent study.38

Enhanced exposure to microorganisms in CF preventedthe development of colitis in TCRa KO mice by stimu-lating the expansion of peritoneal B1 B cells producinglarge amounts of natural IgM that can recognize a broadspectrum of microorganisms. However, no significantdifference in the bacterial colonization pattern betweenSPF and CF TCRa KO mice was found using terminal-fragment-length polymorphism (T-RFLP) analysis,a powerful approach for assessment of diversity ofbacterial flora. In addition, helminth was undetectablein both SPF and CF TCRa KO mice. These findings raisethe possibility that the prevention of colitis in CF ismediated by enhanced exposure to viruses and/orfungi. Indeed, involvement of viruses in the hygienehypothesis has been proposed.39 In addition, adminis-tration of chitin, which is a major component of fungi,has recently been shown to prevent the developmentof colitis in TCRa KO mice by inducing immunetolerance.40

Appendectomy

One of the mysteries with IBD is the negative associ-ation of development of UC, but not CD, with a priorhistory of appendectomy.16 This negative associationwas first demonstrated in 198741 and has since beenconfirmed by many, but not all, case-control studiesand meta-analyses.42–44 Speculations have been raisedto explain the mysterious but consistent finding.A possibility is that the appendix is actively involved


in the development of UC. A second possibility is thatpatients who are more susceptible to appendicitis carrygenetic loci protective against UC. A third possibility isthat appendicitis rather than appendectomy serves asthe preventive factor, presumably by generatingimmune tolerance against UC development.45

Accumulating data from mouse models of IBD mayfavor the first possibility. As mice lack an appendixanatomically, resection of cecal patches (analog ofhuman appendix lymphoid follicles) was performed inTCRa KO mice (a model of human UC) as a mimic ofhuman appendectomy. Interestingly, development ofcolitis of TCRa KO mice was suppressed when theappendectomy was done before 3 weeks of age, whereasappendectomy after 3 weeks of age had only minimaleffect on this colitis.46 A similar finding has since beenconfirmed in humans; a stronger negative associationof development of UC with appendectomy wasobserved in patients who underwent appendectomybefore 20 years of age.42,47 A preventive effect of appen-dectomy on DSS-induced acute colitis has also beenreported.48 Cecal patches of TCRa KO mice provideda place for the priming of restricted CD4þ Tcell popula-tions carrying a negatively charged amino acid residueat a specific region within an antigen-recognitiondomain (complementary determinant region 3) ofsome TCRb chains, such as the Vb8.2 chain.49 Interest-ingly, the CD4þ T cells were shown to possess colito-genic ability.49 Indeed, colitogenic CD4þ T cells weredemonstrated in the CD45RB colitis model to firstmigrate into the cecal patch rather than the colon.50

Since the appendix and cecum serve as a major reservoirof commensal microbes that actively participate in thepathogenesis of IBD,14 it is possible that lymphoid folli-cles, such as those of the appendix, and cecal patches inbacteria-rich locations provide a place for the initialpriming of colitogenic CD4þ T cells. The primedCD4þ Tcells may then migrate into the colon to expandthe inflammation. Indeed, an area in the cecum withfocal inflammatory activity surrounding the appendicalorifice, referred to as the “peri-appendical red patch,”has been recognized in 7.9% of patients with distal ulcer-ative colitis, so-called “left-side UC.”51 Similar findingswith much higher incidence have been reported sepa-rately by different group.52–54

In addition to the preventive effect, a potential thera-peutic effect of appendectomy on UC was proposed in2000 by a case report showing the drug-free remissionof a patient with ulcerative proctitis (inflammationrestricted to the rectum) after appendectomy.55 A similartherapeutic effect of appendectomy has since beendescribed in scattered case reports.56,57 In contrast, a clin-ical trial in six patients with refractory UC failed toconfirm the therapeutic effect of appendectomy.58 Inter-estingly, another study showed that appendectomy

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CLASSICAL THERAPY AND IBD MODELS 503

transiently improved endoscopically recognized inflam-mation in five patients with left-side UC, whereasinflammation remained unchanged or was exacerbatedin four patients with pancolitis after appendectomy.59

The effect of appendectomy on left-side UCmay be sup-ported by a recent prospective case series study of30 patients with ulcerative proctitis; 40% patients expe-rienced a complete remission (as judged by a “simpleclinical colitis activity index” score of 0) after appendec-tomy.60 The remission was also paralleled by endoscopicscores. In mouse IBD models, a preventive, but not ther-apeutic, effect of appendectomy has been explored.

Cigarette Smoking

Another mystery with IBD is the different effects ofcigarette smoking on UC versus CD.1,16 Smoking isa risk factor for CD, whereas it serves as a suppressivefactor against UC. Several animal studies have been con-ducted to dissect the mysterious mechanism. One studyproposed an ability of nicotine to induce the differentia-tion of CD4þ T cells into regulatory T cells (Treg)through inhibition of their differentiation into Th17T cells.61 IL-4 induced the expression of a7 nicotinicacetylcholine receptors (a7nAChR) on colonic CD4þ Tcells, and thus pretreatment with nicotine (total dosesof 7.5 mg/k g through subcutaneous (s.c.). injections)enhanced Treg development and simultaneouslyinhibited Th17 development, making mice resistant toTh2-mediated acute colitis induced by oxazolone. Incontrast, IL-12 (an inducer of the Th1 response) sup-pressed the expression of a7nAChR on colonic CD4þT cells, allowing nicotine to exacerbate a Th1-mediatedcolitis induced by TNBS through inhibition of Tregdevelopment.61 Similarly, pre-exposure to cigarettesmoking made rats more susceptible to Th1-mediatedacute colitis induced by TNBS. The increased suscepti-bility was associated with a decreased colonic super-oxide dismutase activity.62 Colonic dorsal rootganglion (DRG) neurons form peripheral afferent path-ways that carry sensory information from the colon tospinal cord. The ability of nicotine to suppress thehyperexcitability of colonic DRG was recently shownin a DSS-induced acute colitis model.63

However, the mechanism of smoking may be morecomplicated. In IL-10 KO mice (another model of CD),administration of nicotine (12.5 mg/ml) in drinkingwater suppressed chronic colitis.64 This suppressionwas associated with an increased production of intes-tinal trefoil factor, which is required for epithelialrepair.65 One component of cigarette smoke is thegaseous molecule carbon monoxide (CO). Exposure ofIL-10 KO mice to 250 ppm CO for 4 weeks improvedchronic colitis, and this improvement was mediated by


heme oxygenase (HO)-1-dependent inhibition of IFN-g-induced IL-12p40 expression in macrophages throughIFN regulatory factor (IRF) 8.66 In addition, exposure toCO improved Th2-mediated chronic colitis of TCRa KOmice (a model of UC).67 Treatment with the pharmaco-logical HO-1 inducer cobalt protoporphyrin was alsobeneficial for this colitis. The improvement of the colitiswas associated with enhanced production of IL-10 byCD11bþ cells and of IL-22 by CD11b� cells.

CLASSICAL THERAPY AND IBD MODELS

Corticosteroids

Corticosteroids have been widely but cautiously usedto treat the acute exacerbation of UC and CD.1 As seen inIBD patients, the therapeutic effect of dexamethasone(Dex) has been demonstrated in experimental modelsof both UC and CD. Intraperitoneal administration ofDex (100 mg) for 3 days attenuated the ileitis ofSAMP-1/Yit mice (a model of CD).68 A beneficial effectof Dex has also been shown in Th1-mediated acutecolitis induced by TNBS.69 The glucocorticoid-inducedleucine zipper (GILZ), which can be induced by Dex,was shown to improve chronic colitis of IL-10 KOmice.70 Dex therapy also improved Th2-mediatedchronic colitis of TCRa KO mice71 and Th2-mediatedacute colitis induced by administration of oxazolone.72

In DSS colitis models, the effect of Dex seems todiffer depending upon the disease stage. Dex therapyexacerbated DSS-induced acute colitis, whereas itimproved DSS-induced chronic colitis that was inducedby repeated administrations of DSS.73,74

Although Dex has a strong immune suppressiveeffect, the mechanism has been largely unknown. Anattractive mechanism in this regard has been proposedby IBD models. Combined stimulus with Dex and anactive form of vitamin D3 induced the differentiationof human and mouse naıve T cells to an IL-10-producing CD4þ Tcell subset termed “Tr1.”75 Adop-tive transfer of the in vitro-generated Tr1 was able tosuppress the colitis of a CD45RB model, which wasinduced by effector CD4þ T cells.76 Dendritic cells(DCs) generated in vitro by the same regimen (Dexand vitamin D3) were also reported to possess suppres-sive ability against this colitis.77

Glucocorticoid-induced tumor necrosis factorreceptor (TNFR)-family-related protein (GITR) was firstidentified in 1997 by comparing the gene expressionprofile between Dex-treated versus untreated mouseT-cell hybridoma (3DO) through a differential displayanalysis.78 GITR has since been found to be expressedat high levels by a CD4þCD25þ T cell population thatcontains both recently activated CD25þ effector T cells

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and Foxp3þ regulatory Tcells (Treg).79 Interestingly, theauthors found that the ligation of GITR abolished theimmune suppressive ability of Treg. Consistent withthis observation, GITR deficiency protected mice fromTNBS-induced acute colitis,80 and ligation of GITR byan agonistic antibody led to the exacerbation of thiscolitis.81 Alternatively, a GITRþ, but not GITR�, CD4þT-cell population was shown to suppress the colitis ofa CD45RB model,82 raising the possibility that a GITRþCD4þ T-cell subset serves as a regulatory cell popula-tion when GITR is not activated. An alternative possi-bility has been raised by a recent reportdGITR, whichis expressed by antigen-presenting cells but not by Tcells, was required for the suppression of colitis ina CD45RB model.83 The authors suggested that thesuppressive function of GITR in this colitis may bemedi-ated by the induction of tolerogenic CD103þ PDCA(plasmacytoid marker)þ plasmacytoid DCs that arerequired for the development of Treg.

A therapeutic effect of Dex on colitis/ileitis hasbeen shown in seven kinds of IBD models, includingTh1-mediated chronic and acute colitis, Th2-mediatedchronic colitis, and chronic ileitis. Corticosteroids repre-sent a conventional drug that has been widely used fortreatment of UC and CD.1

Cyclosporine/Tacrolimus

Cyclosporinedwhich serves as an inhibitor ofcalcineurindis effective in hospitalized patientswith severely active UC, but not CD.1 Tacrolimus(FK506)dwhich exhibits similar pharmacological ac-tion to cyclosporinedalso induced rapid clinicalresponse and mucosal healing in hospitalized patientswith steroid-refractory UC.84 The current data, al-though needing further evidence from high-qualityrandomized controlled trials and careful attention tonephrotoxicity, also support the use of tacrolimus inCD that is refractory to conventional therapies.85,86

Calcineurin is required for the activation of nuclearfactor of activated T cells (NFAT) by allowing the trans-location of NFATc into nucleus.87 Deficiency of NFATc2has been shown to suppress Th2-mediated acutecolitis induced by oxazolone.88 Acute colitis in TNBSmodels is mediated by different cytokine responsesdepending upon the mouse strain used. TNBS colitisin the majority of mouse strains, such as SJL, was depen-dent on the Th1 response, whereas TNBS colitis in theBALB/c strain was mediated by an IL-4-dependentTh2 response.89 Treatment with cyclosporine (7.5 mg/kg) suppressed the Th2-mediated TNBS colitis inBALB/c mice.69 In contrast, no therapeutic effect ofcyclosporine on colitis has been proposed in IL-10 KOmice90 or CD45RBmodels.91 Since NFATs have an ability


to recruit the transcription factor GATA3 to Th2-typegene promoters such as IL-5,92 this may be one ofreasons why cyclosporine is beneficial to UC and Th2--mediated experimental colitis but not to CD or Th1/Th17-mediated experimental colitis.

Avery rapid therapeutic effect of cyclosporine enemaon DSS-induced acute colitis was shown in 1993,93 anda beneficial effect of intraperitoneal cyclosporine injec-tions on this acute colitis was also demonstrated.94 Theauthors showed the ability of cyclosporine to enhancethe expression of TGF-b, which in turns suppressesepithelial cell apoptosis. Leucine-rich-repeat kinase 2(LRRK2)dwhich is encoded by a major susceptibilitygene for CD3,11,12dwas shown recently to possess anability to serve as a negative regulator of NFAT-driveninnate immune responses in myeloid cells.95 Deficiencyof LRRK2 enhanced nuclear localization of NFAT1 andmade mice more susceptible to DSS-induced acutecolitis.

A beneficial effect of tacrolimus on experimentalcolitis was first proposed in 1995 by Asakura andcolleagues, who utilized a rat DSS colitis.96 Tacrolimusdwhich was entrapped into nanoparticlesdhas sincebeen shown to have a more beneficial effect on rat DSScolitis when it was rectally, rather than orally, adminis-tered.97 In a mouse TNBS colitis model, pretreatmentwith tacrolimus suppressed local inflammation (asjudged by histology of colitis) but not systemic inflam-matory response (as judged by body weight loss).98

Intrarectal administration of tacrolimus had a beneficialeffect on acute colitis in a mouse DSS model and chroniccolitis in IL-10 KO mice, presumably through inductionof apoptosis in activated macrophages.99 In contrast, noeffect of tacrolimuswas shown in chronic colitis that wasinduced in immune-deficient mice by adoptive transferof IL-10-deficient CD4þ T cells.90

The therapeutic potential of cyclosporine and tacroli-mus on colitis has been shown using three kinds of IBDmodels in mice and rats, with some exceptions. Cyclo-sporine and tacrolimus have been used for treatmentof hospitalized patients with severely active UC butnot CD.1

Nonsteroidal Anti-Inflammatory Drugs

Nonsteroidal anti-inflammatory drugs (NSAIDs)dwhich inhibit the production of prostaglandinsand thromboxanes through cyclooxygenase (Cox)-1and 2dhave been reported to aggravate or reactivateIBD.1,100 Enteric commensal bacteria play a major rolein chronic colitis of IL-10 KO mice.14 Development ofcolitis in IL-10 KO mice was abolished or reduced whenthey were maintained under germ-free conditions or inthe absence of Helicobacter species, respectively.14,101

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BIOLOGICAL THERAPY AND IBD MODELS 505

Since the incidence of spontaneous colitis developmentin IL-10 KO mice was low in the absence of Helicobacterspecies, NSAIDs have been used to rapidly and repro-ducibly induce the development of this colitis. Contin-uous administration of piroxicam (200 ppm) in mousechow for 2 weeks induced the rapid development ofcolitis in IL-10 KO mice within 4–6 weeks after termina-tion of piroxicam treatment.100 Administration of eithera selective Cox-1 inhibitor (SC-560) or a selective Cox-2inhibitor (SC-58236 orNS-398) failed to induce the devel-opment of colitis in these mice, but the combination ofthese inhibitors was able to do it.100 Eicosanoidsdwhichare derived from arachidonic aciddinclude both Cox-derived products and 5-lipoxygenase (5-LO)-derivedproducts, also known as leukotrienes. However, 5-LO-derived leukotrienes were not required for thepiroxicam-induced colitis of IL-10 KOmice.102 Enhancedmucosal exposure to enteric bacteria due to the increasedapoptosis of colonic epithelial cells may be responsiblefor the piroxicam-induced colitis.103 An IL-13 decoyreceptor IL-13Ra2dwhich serves as an inhibitor of IL-13 activitydhas been proposed to mediate thepiroxicam-induced colitis by enhancing Th17responses.104 In addition, the rapid and reproducibleinduction of colitis in the piroxicam system has allowedinvestigators to develop a methodology for the assess-ment of colitis. For example, the applicability of2-deoxy-2-[18F]fluoro-D-glucose (FDG) uptake by posi-tron emission tomography (PET) for evaluation of colitiswas demonstrated using a piroxicam-induced colitissystem.105 Activity-based probesdwhich covalentlymodify activated cathepsinsdhave recently been sug-gested as a biomarker of colitis using this system.106

Exacerbation of inflammation by NSAIDs has alsobeen demonstrated in other IBD models. Administra-tion of piroxicam induced the rapid development ofTh2-mediated colitis of TCRa KO mice within 14 daysafter termination of treatment.71 Development of ileitisin SAMP1/Yit mice was elicited by piroxicam treatmentfor 3 weeks.107 In DSS-induced acute colitis, oral admin-istration of a non-selective NSAID (indomethacin) ora combination of Cox-1 (SC-560) and Cox-2 (celecoxib)inhibitors exacerbated the disease.108

NSAIDs have been shown to elicit chronic colitis andileitis in three kinds of chronic IBD models and an acutecolitis model. NSAIDs have been a high-alert drug inIBD patients.

5-Aminosalicylic Acid

5-aminosalicylic acid (5-ASA) preparations are thefirst drugs to be used for UC. This drug, although itsprecise mechanism of action is still unclear, has beensuccessfully applied to treat mild to moderately activeUC and to prevent UC relapse.1


Therapy with 500 mg/kg 5-ASA (mesalamine) inmouse chow suppressed epithelial dysplasia withoutaffecting colonic inflammation in piroxicam-treated IL-10 KO mice, while therapy with 1650 mg/kg 5-ASA inmouse chow reduced the dysplasia in conjunction withimproving colonic inflammation.109 Of note, theauthors discovered the ability of 5-ASA to reduce Aktand b-catenin signaling in epithelial cells, particularlywithin middle-to-upper crypts. A nitric-oxide-releasingderivative of mesalamine (NCX-456), but not conven-tional mesalamine, was reported to have a beneficialeffect on TNBS-induced acute colitis.110 The authorsproposed that NCX-456 inhibited the proliferation ofand simultaneously induced the apoptosis of activatedTh1 T cells. A beneficial effect of 5-ASA has also beenreported in DSS-induced and oxazolone-induced acutecolitis models.72,111

A beneficial effect of 5-ASA on colitis has been sup-ported by four kinds of IBD models, and 5-ASA hasbeen widely used for the treatment of UC.

BIOLOGICAL THERAPY ANDIBD MODELS

Anti-TNF-a Therapy

The most successful biological therapy for IBD is thehumanized anti-TNF-a (tumor necrosis factor alpha)antibody infliximab, which has been approved since1998 by the US Food and Drug Administration (FDA)for treatment of IBD.17 Infliximab is effective at inducingremission in moderate to severely active CD, and is usedas a maintenance therapy. This therapy is also effectiveat inducing remission in moderate to severely activeUC outpatients.1

TNF-a is one of the most studied cytokines in IBDmodels. The pathogenic role of TNF-a is well high-lighted by the spontaneous development of ileitis inTNF(ARE)mice, in which TNF-a expression is stabilizedby the absence of functional adenosine-uracil multimers(AU-rich elements, ARE).112 In addition, the pathogenicabilities of TNF-a to initiate and promote intestinalinflammation have been demonstrated using both loss-of-function and gain-of-function approaches in manyIBD models, including Th17-mediated chronic colitisof IL-10 KO mice, Th1-mediated chronic colitis ofCD45RB and CD3ε transgenic models, Th1-mediatedacute colitis of TNBS models, and ileitis of SAMP/Yitmice.8 In contrast, there was no significant beneficialeffect of anti-TNF monoclonal antibody (mAb) on theearly stage of Th2-mediated chronic colitis of TCR-a KO mice,113 but absence of the TNF receptor TNFR2improved this colitis.114 On the other hand, bothTNF-a and TNFR1 were dispensable in the development

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of colitis in A20 KO mice.115 Interestingly, administra-tion of TNF-a ameliorated Th2-mediated acute colitisinduced by oxazolone through induction of extraadrenal glucocorticoid synthesis,116 suggesting a protec-tive role of TNF-a in this acute colitis model. Similarly,TNF-a KO mice were more susceptible to DSS-inducedacute colitis.117,118

Clinical trial data have suggested that 30–40%patients fail to have an initial response if anti-TNFtherapy is introduced after the failure of nonbiologicaltherapies.119 There are also some safety issues withanti-TNF-a therapy, such as an increased risk of infec-tions.119 A two-fold increased risk of hospitalizationwith bacterial infections was seen among patients whoreceived anti-TNF-a therapy as compared to thosewho received methotrexate therapy.120 The ineffective-ness in a subset of patients and increased risk of infec-tions may be causeddin partdby the ability of TNF-ato stimulate both TNFR1 and TNFR2, which may playdistinct biological functions. In DSS-induced acutecolitis, the disease was exacerbated by absence ofTNFR1 but not TNFR2.121 In addition, after DSS chal-lenge, mortality, presumably due to bacterial invasion,was significantly increased in TNFR1-deficient RAG-1(a recombination activating gene) double KO mice lack-ing both T and B cells as compared to TNFR2-deficientRAG-1 double KOmice and RAG-1 KOmice, suggestingthat TNFR1 expressed by innate cells such as epithelialcells plays a protective role against enteric microorgan-isms under an acute intestinal inflammatory condi-tions.122 Indeed, TNFR1 has been shown to promoteepithelial cell survival by protecting TNF-a-inducedapoptosis.123 In addition, a requirement for TNFR2,but not TNFR1, for TNF-a-induced epithelial barrierdysfunction has been demonstrated.124

The roles of TNFR1 versus TNFR2 in colitis have alsobeen explored using TNBS models. TNFR1 KO micedeveloped a more severe form of colitis with increasedmortality after TNBS challenge, while the colitis wasimproved in TNFR2 KO mice.125 Another groupreported that both TNFR1 KO and TNFR2 KO micewere resistant to TNBS-induced acute colitis, buta systemic inflammatory response was seen in TNFR1KO, but not TNFR2 KO, mice after TNBS challenge.126

An interesting observation was made from epithelial-cell-specific TGF-b-activated kinase 1 (TAK1) KOmice. These mice developed very severe epithelialdamage in the small intestine by 3 days of age.127 Theepithelial damage was rescued by absence of TNFR1,but development of ileitis and colitis was in turn eli-cited in TNFR1-deficient epithelial-specific TAK1double KO mice. On the other hand, deficiency ofboth TNFR1 and TNFR2, but not each individually,inhibited the development of ileitis of TNF(ARE)mice.128 In addition, lack of TNFR2 on CD4þ T cells


may exacerbate colitis of CD45RB models.129 Takentogether, these findings highlight much a more compli-cated role played by TNFR1 and TNFR2 in IBD thanpreviously predicted.

The pathogenic role of TNF-a in colitis has been sup-ported by many IBD models, with some exceptions. Onthe other hand, some acute colitis models have proposeda protective role of TNFR1 against microbial infections,which may provide a clue to understand how anti-TNF-a therapy increases the risk of infections. Anti-TNF-a therapy has been approved by the FDA fortreatment of IBD.

Anti-Integrin-a4 Therapy

There are at least 18 a-subunits and 8 b-subunits ofintegrins, which link together as heterodimers to formmore than 24 different functional integrin receptors thatallow leukocytes to travel into different tissues.130

Among them, a4 integrin forms heterodimers with b7and b1 integrins. The heterodimer of a4b7 is a pivotalmediator of leukocyte homing into the gastrointestinal(GI) tract through its interaction with mucosal addressincell-adhesion molecule (MAdCAM)-1. On the otherhand, the heterodimer of a4b1 binds to the vascularcell-adhesion molecule (VCAM)-1, which mediatesleukocyte homing into the central nervous system(CNS). The feasibility ofmodulation of leukocyte homingfor IBD therapywas first demonstrated in 1993 by Podol-sky et al.131 They showed that treatment with anti-a4--integrin mAbs attenuated spontaneously developingcolitis in the cotton-top tamarin, a marmoset native toa small geographic region of Colombia.131 This observa-tion has prompted several human trials, and anti-a4--integrin therapy (natalizumab) has been approvedsince 2008 by the FDA for inducing andmaintaining clin-ical response and remission in patients with moderatelyto severely active CDwho had an inadequate response toconventional and anti-TNF therapies.17,132

After the discovery of the beneficial effect of anti-a4-integrin therapy on colitis of cotton-top tamarins, role ofa4 integrin in colitis has been explored in other IBDmodels. A small molecule antagonist against a4 integrinwas shown to have therapeutic potential inDSS-induced acute colitis.133 Interestingly, treatmentwith anti-a4-integrinmAbsalonehadno significant bene-ficial effect on the ileitis of SAMP/Yitmice, but a combina-tion therapy of anti- a4-integrin and anti-ICAM1(intercellular adhesion molecule) mAbs improved theileitis in 70%of thesemice.68 In contrast, a long-term treat-mentwith anti-a4-integrinmAbswasproposed to exacer-bate chronic colitis and increase the risk of cancerdevelopment in Gai2 KO mice (a model of UC).134 Theexacerbationof colitismaybe causedby impairedhomingof Ab-producing B cells into the inflamed colon.134

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In addition to anti-a4-integrin therapy, therapeuticmeasures targeting its ligand MAdCAM have alsobeen explored. Anti-MAdCAM therapy improvedTh1-mediated chronic colitis of a CD45RB model,135

and an antisense oligonucleotide capable of inhibitingMAdCAM activity improved Th1-mediated acute colitisinduced by TNBS.136 Preventive and therapeutic effectsof anti-MAdCAM mAbs were also shown inDSS-induced acute colitis.137,138

The therapeutic benefit of targeting the a4 integrin/MAdCAM interaction on colitis/ileitis has been sup-ported by five kinds of animal models, in particular inmodels of CD. Anti-a4-integrin therapy has beenapproved by the FDA for the treatment of moderatelyto severely active CD.1

Anti-Integrin-b7 Therapy

A concern with anti-a4 integrin therapy is the risk ofan unexpected complicationdprogressive multifocalencephalopathy (PML)da serious, often fatal, infectionof the brain caused by reactivation of JC polyomavirus.17,132 This adverse effect is caused by inhibitionof leukocyte recruitment into the CNS through blockageof the a4b1 pathway by anti-a4-integrin therapy.17,132 Inorder to minimize this serious problem, anti-b7-integrintherapy, which blocks the intestine-specific a4b7pathway but not the brain-specific a4b1 pathway, hasbeen under clinical development. A multicenterdouble-blind placebo-controlled trial on 181 patientswith active UC showed that treatment with a mAb(MLN0002) targeting a4b7 heterodimers was effectivefor the induction of clinical and endoscopic remission.139

In addition, a randomized double-blind controlled trialon 185 patients with active CD reported a dose-dependent beneficial effect of MLN0002 (intravenousinfusion of 2 mg/kg Ab at days 1 and 29) on the clinicalremission.140 Of note, worsening of CD was noted as themost common serious adverse effect.140

Administration of an Act-1 mAbdwhich was initiallyreported to recognize CD25 but has subsequently beenidentified to recognize b7 integrin but not CD25dimproved colitis in cotton-top tamarins.141 Adoptivetransfer of b7-integrin-deficient naıve CD4þ T cellswas unable to induce colitis in a severe combinedimmune deficient (SCID) host.142 Similarly, treatmentwith an anti-b7-integrin mAb suppressed the colitisof a CD45RB model.135 Alternatively, this therapy mayhave a preventive, but not therapeutic, effect on thiscolitis.91 Development of ileitis in TNF(ARE) mice wascritically dependent on the integrin b7 pathway.143 Incontrast, the role of b7 integrin in the colitis of IL-2 KOmice did not reach a consensus. One group reportedthat pretreatment with anti-b7-integrin mAb as well as


anti-aE-integrin mAb suppressed an antigen (OVA)-induced colitis in IL-2 KO mice.144 In contrast, anothergroup showed that b7-integrin-deficient IL-2 doubleKO mice still developed colitis comparable withthat seen in IL-2 KO mice.142

A novel cell-specific therapy was also tested by tar-geting b7 integrin. An anti-b7-integrin-specific nanopar-ticle approachdwhich was engineered to suppress theproliferation of b7 integrinþ cells through blockage ofcyclin D1dwas shown to improve acute colitis inducedby DSS.145 In addition, the effect of anti-b7-integrintherapy on leukocyte homing to the intestine has beenexplored by several cutting-edge approaches in mouseIBD models. MicroPET imaging of mice with injectionof 64Cu-labeled anti-b7 integrin showed the specificaccumulation of b7 integrinþ cells in the inflamedcolon after challenge with DSS.146 Treatment with anti-b7-integrin mAb improved the colitis of a CD45RBmodel through inhibition of leukocyte homing into thecolon, but this therapy had no effect on experimentalautoimmune encephalomyelitis owing to the lack ofability of this mAb to inhibit leukocyte homing intothe brain.

A potential concern with anti-b7-integrin therapy israised by the observation that some leukocytes were stillable to home into the inflamed colon in a b7-integrin-independent manner.147 Therefore, the therapeutic effectof anti-b7 integrin may be limited as compared to anti-a4-integrin therapy. On the other hand, an attractivepiece of news is that b7-integrin-deficient Treg was stillable to prevent colitis in a CD45RB model, suggestingthat anti-b7-integrin therapy may not affect Treg func-tion, which is well known to contribute to the suppres-sion of colitis.148

The therapeutic potential of anti-b7-integrin therapyin IBD has been supported by four kinds of IBD models,although there is inconsistency in data from IL-2 KOmice. Anti-b7-integrin therapy has been in clinicaldevelopment.17

Anti-Interleukin-12p40 and Anti-Interleukin-23 Therapy

IL-12p40 (p40) is a subunit of the IL-12 family of cyto-kines, which associates with the p35 subunit to form IL-12p70 (IL-12) and with the p19 subunit to form IL-23.IL-12 is a well-known inducer of the Th1 response,and polymorphisms of the IL-23 receptor are negativelyassociated with the development of IBD.3,11,12 A multi-center randomized placebo-controlled double-blindphase II clinical trial in 79 active CD patients reportedthat anti-IL-12p40 therapy (ABT-874/J695, briakinumab)induced a clinical response and remission.149 However,a recent trial with CD patients had a disappointing

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outcome.17,150 Ustekinumab (human IgG1 against p40)has been approved by the FDA for the treatment ofmoderate to severe psoriasis. A double-blind cross-over trial of ustekinumab in 104 patients with moderateto severe CD did not achieve the primary end point.Post-trial analysis, however, found that this therapyinduced a clinical response in patients who had previ-ously received anti-TNF therapy.151

A beneficial effect of anti-p40 mAbs on colitis wasinitially proposed in a Th1-mediated acute colitis ofa TNBS model by Neurath et al.152 and it has sincebeen confirmed in chronic colitis of IL-10 KO mice.153

Anti-p40 therapy was also demonstrated to have bothpreventive and therapeutic effects on colitis ofa CD45RB model.91 Genetically engineered deficiencyof p40 improved the ileitis of TNF(ARE) mice128 andthe colitis of CD3ε transgenic model.154 In contrast, over-expression of p40 in epithelial cells (under the control ofthe T3b promoter) prevented the development of colitisin IL-10 KO mice,155 suggesting a protective role ofectopically expressed p40 in this colitis model.

The ability of p40 to form both IL-12 (p40/p35) and IL-23 (p40/p19) tends to make its function more compli-cated. Indeed, IL-12 and IL-23 may play distinct roles incolitis, depending on the mechanism involved. InTh1-mediated acute colitis of a TNBS model, p35 KOmice lacking IL-12 developed mild inflammation,whereas p40KOmice lacking both IL-12 and IL-23 devel-oped a more exacerbated form of inflammation.156 Simi-larly, TNBS colitis was exacerbated in IL-23 (p19) KOmice.157 These findings suggest a pathogenic role of IL-12p70 and a protective role of IL-23 in TNBS-inducedcolitis. In contrast, a pathogenic role of IL-23 and aprotec-tive role of IL-12 has been shown in the chronic colitis ofIL-10 KO mice.158 IL-12 (p35)-deficient IL-10 double KOmice developed colitis at an earlier age as compared toIL-10 KO mice, while IL-23 (p19)-deficient IL-10 doubleKO mice were still colitis-free at 12 months of age. Inaddition, a pathogenic role of IL-23 and its receptor hasbeen well documented in Th1-mediated chronic colitisof a CD45RB model,159,160 and in innate-immune-mediated acute colitis that can be induced in RAG KOmice by anti-CD40 mAb administration.161 Similarly,both preventive and therapeutic effects of anti-IL-23(p19) mAb were demonstrated in the colitis that wasinduced by a colitogenic bacterial-antigen-specificCD4þ T-cell subset.162 In addition, deficiency of p19dramatically improved the colitis of CD4þ T-cell-specifictransgenic mice of dominant negative TGFbRII.163 Acontribution of IL-23 in the development of intestinalgranulomas (a specific feature of CD) has also beenproposed.164 In Th2-mediated chronic colitis of TCRaKOmice, IL-12 (p35) has been proposed to play a protec-tive role.165 On the other hand, IL-12 may play no role inTh2-mediated acute colitis induced by oxazolone.166


Mouse IBD models have suggested differing roles(inflammatory versus protective) of IL-12 (p40/p35)versus IL-23 (p40/p19) in colitis, depending on thefundamental mechanism involved. Anti-IL-12p40 andanti-IL-23 therapies for IBD are currently under clinicaldevelopment.

Anti-Interleukin-17A Therapy

IL-17 is a cytokine that stimulates the recruitment ofneutrophils and monocytes into inflamed areas. In theinflamed colon, IL-17A is produced primarily by a T-cellsubset termed Th17, and by innate lymphoid cells (ILC)that are characterized by the expression of Thy1, stemcell antigen (SCA)1, retinoic-acid-related orphanreceptor (ROR)g, and IL-23R.167,168 Although Th17 Tcells were initially believed to produce IL-17 but notIFN-g, and to differentiate from the same precursor ofTreg, more complicated biology with Th17 has recentlybeen unveiled. CD4þ T cells expressing both IL-17Aand IFN-g were detectable in the inflamed colon of CDpatients,169 and CD4þ T cells expressing both IL-17Aand Foxp3 (a marker of Treg) also appeared in theinflamed colon of IBD patients.170,171 In addition, recentstudies suggest that Th17 T cells are an immediateprecursor of IFN-g-producing Th1 cells.172 These find-ings suggest more complicated functions of IL-17 inIBD. A double-blind placebo-controlled proof-of-concept clinical study in 59 patients with moderate tosevere CD showed anti-IL-17A therapy (secukinumab)exacerbated the disease in a subset of patients.173

Anti-IL-17 mAb together with anti-IL-6 mAb signifi-cantly improved the chronic colitis of IL-10 KOmice.158 IL-17 receptor KO mice were protected fromTNBS-induced acute colitis.174 In response to a colito-genic bacterial antigen, a more potent ability of IL-17-producing effector CD4þ T cells was demonstratedwhen compared to IFN-g-producing effector CD4þ Tcells.175 On the other hand, the role of IL-17 inDSS-induced acute colitis is still controversial. A protec-tive role of IL-17 in DSS colitis was initially suggested bythe exacerbation of colitis after administration of anti-IL-17 mAb.176 In contrast, a beneficial effect of anti-IL-17mAb was shown in the DSS colitis that was induced inCX3CR1 KO mice,177 and resistance of IL-17A KO miceto DSS colitis was also proposed.178 In addition, IL-17may play a more complicated role in the Th1-mediatedchronic colitis of a CD45RB model. Since IL-23dwhichis required for the maintenance of Th17 cellsdcausedthe development of colitis in a CD45RB model, a patho-genic role of IL-17 and Th17 in this colitis was initiallyassumed.179 However, the IL-23-dependent colitis ofthe CD45RB model did not require IL-17 secretion byCD4þ T cells.180 Indeed, adoptive transfer of CD4þ Tcells from IL-17F KO mice or from IL-17A-deficient

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and IL-17F-deficient double KO mice still inducedsevere colitis in recipient RAG KO mice.181,182 In addi-tion, a protective role of CD4þ T-cell-derived IL-17Ain this colitis has been demonstrated.183 Deficiency ofIL-17A did not affect the severity of colitis in transgenicmice in which TGF-b signaling was impaired specificallyin CD4þ T cells.163 Similarly, administration of anti-IL-17A mAb did not improve Th2-mediated chronic colitisof TCRa KO mice.165

Therapeutic potential of anti-IL-17 therapy in colitishas been supported by some IBD models, but a criticalconcern has also been raised by other IBD models.Anti-IL-17 therapy is currently under clinical develop-ment but an initial clinical trial has had an unfavorableoutcome.

Anti-Interleukin-6R Therapy

IL-6 activates various cell types carrying themembrane-bound IL-6R/gp130 heterodimer receptors(classical IL-6 signaling) as well as IL-6R� gp130þ cellsvia the soluble IL-6R (IL-6 trans signaling). A pilotrandomized clinical trial on 36 patients with active CDshowed that bi-weekly treatments with a humanizedmAb to the IL-6 receptor normalized the acute phaseresponse and induced clinical, but not endoscopic orhistological, remission in 20% of patients.184

A proinflammatory role of IL-6 in colitis has been welldocumented in several IBD models. Blockage of IL-6trans signaling had a therapeutic effect on theTh1-mediated colitis of a CD45RB model.185 Like theCD45RB model, a single transfer of CD44low CD62Lþnaıve CD8þ T cells from WT mice induced colitis inrecipient RAG2 KO mice. This colitis development wasabolished by the absence of IL-6.186 Anti-IL-6 therapysignificantly improved the chronic colitis of IL-10 KOmice,158 and IL-6 KO mice were resistant to TNBS-induced acute colitis.187 In addition to Th1-mediatedcolitis models, IL-6 was essential for Th2-mediated acutecolitis of an oxazolone model,88 and deficiency of IL-6improved Th2-mediated chronic colitis of TCRa KOmice.114 In contrast, inconsistent findings with thesusceptibility of IL-6 KO mice to DSS-induced acutecolitis have been reported;8 some groups have proposedsusceptibility of IL-6 KOmice to DSS colitis, while othersbelieve IL-6 KO mice are resistant to such treatment.

A concern with anti-IL-6 therapy is raised by theability of IL-6 to serve as a strong activator of signal trans-ducer and activator of transcription 3 (STAT3). Absenceof STAT3 in CD4þ T cells abolished the development ofTh1-mediated colitis in a CD45RB model.188 In contrast,absence of STAT3 in innate immune cells such as macro-phages induced the spontaneous development ofcolitis.189 Similarly, absence of STAT3 in epithelial cellsdelayed the recovery from DSS-induced acute colitis.190


Consistent with these findings, gp130 knock-in (KI)mice, which carry a mutated gp130 lacking all STAT3binding sites, spontaneously developed ulceration inthe anorectal region.191 These findings may suggestopposite roles of STAT3 in colitis, depending on the celltypes (e.g., adaptive versus innate) targeted.

A pathogenic role of IL-6 in colitis has been supportedby six kinds of IBD models, but these models have alsosuggested a “double-edged sword” function of STAT3(a downstream event in IL-6 signaling) in colitis. Anti-IL-6 therapy is currently under clinical development.17

Anti-CCR9 Therapy

Interactions of chemokines and their receptors are crit-ical for leukocyte accumulation, including that of Tcells,B cells, eosinophils, and macrophages in the intestinesduring inflammation.192 Among 47 chemokines and 19chemokine receptors, CCL25 (also called Teck) is selec-tively chemotactic for the small, but not the large, intes-tine.193 Ligation of CCR9 by CCL25 results inconformational changes in another intestine-specifichoming receptor pair (integrin a4b7), leading to firmadhesion of a4b7 to its ligand MAdCAM1.193 TheProspective Randomized Oral Therapy Evaluation inCD trial demonstrated the effectiveness of a small mole-cule CCR9 inhibitor (CCX282-B) in maintaining theremission of CD patients over 36 weeks.194

CCL25-deficient TNF(ARE) mice and CCR9-deficientTNF(ARE) mice both developed ileitis, suggesting thatCCL25/CCR9 is dispensable in the ileitis of TNF(ARE)mice.143 On the other hand, pretreatment with a CCR9antagonist (CCX282-B) was reported to suppress thedevelopment of ileitis in TNF(ARE) mice.195 In anotherileitis model of SAMP1/Yit mice, preventive administra-tion (before onset of disease) of neutralizing anti-CCR9or anti-CCL25 mAbs inhibited the development ofileitis, while therapeutic administration of these mAbs(after onset of disease) failed to improve the ileitis.196

Interestingly, a recent report suggested that expressionof CCL25 and CCR9, which is generally undetectablein normal colon, is elicited in the inflamed colon in thecontext of DSS challenge.197 The authors showed thatCCR9 KO mice were more susceptible to DSS-inducedacute colitis than WT control mice, suggesting a protec-tive role of ectopically expressed CCR9 in colitis.

A concern with anti-CCR9 therapy in CD has beenraised by two kinds of ileitis models and an acute colitismodel. CCR9-antagonist therapy in human CD iscurrently under clinical development.

Recombinant Interleukin-10 Therapy

IL-10 is an immune regulatory cytokine that repre-sents a major IBD susceptibility gene.3,11,12

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Polymorphisms within IL-10 receptor genes (IL-10R1and IL-10R2) are also associated positively with thedevelopment of both CD and UC.198 In addition, loss-of-function mutations within the genes of IL-10, IL-10R1, or IL-10R2 are closely associated with early-onset IBD,199 in particular with a very-early-onsetUC.200 Multicenter prospective randomized double-blind placebo-controlled and sequential-escalating-dose trials in 95 patients with Crohn’s disease activityindex (CDAI) scores between 200 and 250 and in 329therapy-refractory patients with CD reported thatsubcutaneous daily administrations of recombinanthuman IL-10 for 28 days did not achieve the primaryend point.201,202

In contrast to the situation in humans, a protective roleof IL-10 in colitis has been reproducibly demonstrated byseparate groups usingmanydifferent kinds of IBDmodels.Genetically engineered deficiency of IL-10 induced thespontaneous development of colitis in mice.203 IL-10served as a protective factor in Th1-mediated acute colitisof a TNBS model.204 Similarly, a protective role of IL-10has been well documented in Th1-mediated chronic colitisof a CD45RB model.205,206 Interestingly, deficiency of IL-10R conferred the ability on Foxp3þ Treg to cause thecolitis of a CD45RBmodel.207 Impaired regulatory functionof Foxp3þ Treg by absence of Wiskott–Aldrich syndromeprotein (WASP) also represented a causative factor ofTh2-mediated chronic colitis in WASP KO mice.208 Th2-mediated chronic colitis of TCRaKOmicewas also exacer-bated by genetic deletion of IL-10.209 In addition, IL-10 KOmice exhibited an increased susceptibility to DSS-inducedacute colitis.210 Mice that are genetically engineered tocarry a point mutation in the P110d gene locus spontane-ously developed focal inflammation restricted to therectum and cecum,211 and this colitis was exacerbated byabsence of IL-10.212 Similarly, deficiency of IL-10 exacer-bated the colitis developing in toll-like receptor (TLR5)KO mice213 and in mucin (Muc2) KO mice.214

Although many IBD models have reacheda consensus regarding the protective role of IL-10 incolitis, clinical trials of subcutaneous recombinant IL-10 injections in CD had a disappointing outcome. Thereare several possibilities to explain these distinctoutcomes between mice and humans.17 The first possi-bility is that the biological function of IL-10 differsbetween mice and humans. However, this possibilitymay be unlikely because in vitro systems using humanand mouse materials generally tend to exhibit similarpatterns of immune responses. A second possibility isan impaired response of IBD patients to IL-10 due topolymorphisms within IL-10 receptors. Since a recentstudy showed that the majority of IBD patients can nor-mally respond to IL-10,215 this possibility may berestricted to a subset of IBD patients. On the otherhand, very attractive observations with a hope for future


IBD therapy have recently been reported. Allogeneictransplantation of hematopoietic stem cells with intactIL-10 and IL-10R genes improved CD in a patientcarrying a mutation within the IL-10R gene.198 In addi-tion, among 66 patients with early-onset IBD (< 5 yearsof age), 16 patients carried loss-of-function mutationswithin the IL-10 or IL-10R genes; 3 patients had an IL-10 mutation, 5 patients an IL-10R1 mutation, and8 patients an IL-10R2 mutation.199 Among them,5 patients were subjected to allogeneic hematopoieticstem cell transplantation carrying intact IL-10 signaling.Of note, sustained clinical remission (so far, for 2 years)was seen in all of these 5 patients.199 A third possibility isthat the therapeutic efficacy of recombinant IL-10ddelivered by subcutaneous injectiondis limited byits poor bioavailability to intestinal tissues.17 This possi-bility is supported by the short half-life and instability ofrecombinant cytokines as compared to antibodies thathave been successfully used for IBD therapy. Forexample, recombinant IL-4dwhich was subcutaneouslyinjected or continuously delivered to the subcutaneousspace through an osmotic pumpddid not alter theimmune response in the colon. The problem was over-come when IL-4 was continuously delivered into theperitoneal cavity through an osmotic pump.216 In orderto clarify this possibility, delivery of IL-10 into the intes-tinal lumen through Lactococcus lactis genetically engi-neered to produce human recombinant IL-10 wasconfirmed in a phase I trial to be safe, but the efficacyhas not been reported.217

Stem Cell Therapy

Stem cell therapy is an emerging and promising area ofresearch that has potential to open a new avenue for IBDtherapy. Mesenchymal stromal/stem cells (MSCs) arenon-hematopoietic stromal cells exhibiting multi-lineagedifferentiation capacity and ability to mediate immuno-suppressive and anti-inflammatory effects.218 A phase Iclinical trial of MSC therapy has been conducted in ninepatients with refractory CD. After infusion of ex vivoexpanded autologous bone marrow (BM)-derived MSCs,three patients exhibited an improved clinical response(CDAI decrease > 70) without reaching remission, andanother three patients required surgery owing to diseaseworsening.219

The therapeutic potential of systemic infusion withnot only syngeneic but also allogeneic adipose-derivedmesenchymal stem cells (ASCs) was shown in Th2--mediated acute colitis that was induced in BALB/cmice by TNBS.220 The therapeutic effect may be medi-ated by several mechanisms, including downregulationof the Th1 response, upregulation of IL-10 expression,and induction of Treg expansion through interactionwith macrophages.220 In addition, infusion of human

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umbilical-cord-derived MSCs was reported to improvea Th2-mediated acute colitis that was induced inBALB/c mice by TNBS.221 In Th1-mediated acute colitisthat was induced in C57BL/6 mice by TNBS, infusion ofBM-derived MSCs before TNBS challenge had a preven-tive effect.222 Removal of splenic CD11bþ cells resultedin the loss of the beneficial effect of MSC therapy onthis colitis, suggesting that interactions between MSCsand CD11bþ cells may be necessary to fully elicit thebeneficial effect of MSCs.222

A dose-dependent therapeutic effect of human ASCinfusion was proposed in acute colitis that was inducedinmice byDSS.223 In contrast, ASCdidnot have apreven-tive effect on this colitis when ASC infusion was per-formed before challenge with DSS, suggestinga transient effect of ASC therapy. A transient effect isalso supported by a study using a near-infrared in vivoimaging system. MSCs accumulated in the inflamedcolon rapidly after the infusion, but they disappearedfrom all tissues (colon, lung, spleen, and lymphnodes) within 3 days after infusion.222 Interestingly,MSCs that were coated with anti-VCAM-1 mAb throughpalmitated protein G (PPG) showed a more enhancedability to home into the inflamed colon of aDSSmodel.224

Repeated injections of BM-derivedMSCs (days 0, 2, and 4after DSS challenge)were proposed to allow the accumu-lation of MSCs in the inflamed colon and have a thera-peutic effect on DSS-induced acute colitis in rats.225

Busulphan-induced hypoplasia of bone marrow exacer-bated DSS-induced acute colitis, and MSC transplanta-tion ameliorated the disease exacerbation.226 Theauthors proposed that this beneficial effect may bemedi-ated by restoration of epithelial barrier integrity, but notfrom immune response. The ability of MSC therapy toenhance healing of wounds from DSS-induced acutecolitis in rats was also proposed.227 In contrast, a recentstudy suggested no significant effect of MSCs on theprevention of DSS-induced or TNBS-induced acutecolitis.228 The authors proposed that pretreatment ofMSCs with IFN-g is required to elicit their immunesuppressive ability to effectively prevent these forms ofcolitis. A clinically useful observation has also beenmadedMSC phenotype and function were not alteredby drugs that have been used for IBD therapy, includingazathioprine, methotrexate, 5-ASA, and anti-TNFmAb.229

In addition to MSCs, a novel method has recentlybeen established to allow a long-term expansionof leucine-rich-repeat-containing G-protein-coupledreceptor (Lgr)-5-expressing colonic epithelial stemcells.230 The authors demonstrated that intrarectal injec-tion of ex vivo-generated epithelial stem cells was able tocover the colonic ulcerations in a DSS model.

The therapeutic effect ofMSCs on colitis has been sup-ported only by acute colitis models, and there are some


inconsistencies in the data published. An initial pilotstudy in human IBD showed an unfavorable outcome,suggesting the existence of many steps to be overcometo successfully apply stem cells to IBD therapy.

Anti-CTLA4 Therapy

Cytotoxic T-lymphocyte-associated molecule 4(CTLA4) is a co-stimulatory molecule that interactswith CD80 and CD86. Anti-CTLA4 antibody (ipilimu-mab) was approved in 2011 by the FDA for treatmentof stage IV melanoma. Development of colitis has beendocumented as one of the serious adverse effects ofanti-CTLA4 therapy.231,232 Abatacept is a geneticallyengineered fusion protein that chimerizes CTLA4 tothe Fc portion of IgG to block the co-stimulatorysignaling. Four placebo-controlled trials in 451 CDpatients and 490 UC patients showed that abatacept isnot efficacious for the treatment of moderate to severeCD or UC.9,233

Constitutive expression of CTLA4 among CD4þTcells is restricted primarily to Treg cells.234 The protec-tive role of CTLA4 in colitis has beenwell explored usingTh1-mediatedcolitis of aCD45RBmodel, andaconsistentoutcome has been reported separately by independentgroups. CTLA4-deficient Treg failed to suppress colitisin a CD45RB model,235 and also in Helicobacter-inducedinnate colitis.236 In addition, administration of anti-CTLA4 mAbs exacerbated the colitis in a CD45RBmodel,237–239 and treatment with a CTLA4 agonist sup-pressed this colitis.240 In contrast, anti-CTLA4 therapysuppressed Th1-mediated acute colitis of a TNBS modelby inducing IL-10-producing inducible T-cell co-stimulator (ICOSþ) regulatory Tcells.241

Different effects of CTLA4 on colitis have beenproposed in CD45RB versus TNBS models. In humans,anti-CTLA4 therapy induced the development of colitisin a subset of patients.

Anti-CD3 Therapy

CD3 is a surface molecule expressed by T-cell popula-tions, including CD4þ T cells, CD8þ T cells, TCRgdT cells, and NKT cells. A randomized double-blindplacebo-controlled clinical trial on 127 corticosteroid-refractoryUCpatients showed that anti-CD3 therapy (vis-ilizumab) was not effective and was associated withincreased cardiac and vascular adverse effects.242Anotherphase I clinical trialwithanti-CD3 therapy (NI-0401) failedto improve moderate to severe CD.243

A requirement for CD4þ T cells for the induction ofcolitis has been well documented using CD4þ T-cell-transfer systems. For example, development ofTh1-mediated colitis in a CD45RB model was inducedby adoptive transfer of CD4þ CD45RBhigh naıve CD4þ

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Treg

CD4+ T cells

TTreegggggFoxp3

Th2

Tfh

Tr1

Th9

Th3

IFN-γIL-10

IL-9

IL-4

?Th17/Treg

TTre

1IL-10

T

reggg

1

Th17Th22

Th1

?Th1/Tr1

IL-17

IL-17

IL-22IL-22

IFN-γIL-10

TGF-β

IL-21

?Th17/Th1

1 ?Tfh/Th1

IL-10

IL-4

Foxp3

IFN-γIL-21

IFN-γIL-17

FIGURE 22.2 There are functionally different CD4D T-cell

subsets, depending on their cytokine profiles. Some subsets maydifferentiate from other subsets as indicated by the blue lines. UniqueCD4þ T-cell subsets have been identified to specifically appear in theinflamed intestine, including Foxp3þ IL-17þ CD4þ (CD4þ T cellssharing Treg and Th17 phenotype), IL-17þ IFN-gþ CD4þ (CD4þ Tcells sharing Th17 and Th1 phenotype), and IFN-gþ IL-21þ CD4þ(CD4þ T cells sharing Th1 and Tfh phenotype) T-cell subsets.


T cells from WT mice.244 Adoptive transfer of CD4þT cells from WASP KO mice induced Th2-mediatedcolitis in immunodeficient hosts,245 and flagellin-specific CD4þ T cells also had the ability to induce thedevelopment of colitis.246

Genetic deletion of some T-cell subsets has been repro-ducibly demonstrated to abolish the development ofcolitis and ileitis in many IBD models. Absence ofCD4þ T cells failed to elicit the development of chroniccolitis in IL-10 KO mice.247 Colitis development in IL-2KO mice was abolished by genetic deletion of CD4þTCRabþ T cells, but not of CD8þ TCRabþ T cells orTCRgdþ T cells.248,249 Deficiency of CD4þ T cells due toabsence of major histocompatibility complex (MHC)class II abolished the development of Th2-mediatedchronic colitis of TCRaKOmice.49 In addition, deficiencyof CD8þ T cells improved the ileitis of TNF(ARE)mice.128 Furthermore, deficiency of TCRgd T cells im-proved the colitis in phosphoinositide-dependent kinase(PDK1) KO mice.250 Interestingly, mice that were geneti-cally engineered to develop aberrant type II NKT cellsspontaneously developed colitis.22 Genetic modificationof T-cell functions also caused colitis and ileitis. CD4þT-cell-specific overexpression of dominant negativeTGF-b receptor type II led to spontaneous developmentof multi-organ inflammation, including colitis.251 T-cell-specific depletion of Blimp-1 induced the spontaneousdevelopment of colitis.252 Ectopic overexpression ofTNFSF15 in Tcells also caused the spontaneous develop-ment of transmural inflammation in the small, but notlarge, intestine.253

The data from adoptive T-cell transfer and geneticallyengineered mouse models of IBD clearly indicate thatsomeT-cell subsets arenecessary for the inductionof colitisand ileitis.However, these IBDmodels have also identifiedfunctionally different T-cell subsets. For example, theCD4þ T-cell population can be classified into at least 13different subsets, primarily depending on the profile ofcytokines, which play an inflammatory versus protectiverole in colitis/ileitis (Fig. 22.2). Therefore, a critical concernmay be raised when all T cells are depleted by anti-CD3therapy. Among the CD4þ T-cell population, CD25þFoxp3þ Treg, IL-10-producing Tr1, and TGF-b-producingTh3 subsets have been well confirmed to possess a strongability to suppress colitis and ileitis in many IBDmodels.5,254 Regulatory Treg and effector Th17 T-cellsubsets differentiate reciprocally from the same precursorcells, depending upon the cytokine environment. Interest-ingly, several unique CD4þ T-cell subsets have beendemonstrated to develop specifically in the inflamedmucosa of IBD patients. A CD4þ T-cell subset sharingboth Th1 (IFN-g production) and Th17 (IL-17A produc-tion) functions appeared in the inflamed colon of CDpatients.169 Another CD4þ T-cell subset expressing bothIL-17A and Foxp3 also appeared in the inflamed colon of


IBD patients.170,171 This CD4þ T-cell subset, although itproduced IL-17A, had immune suppressive abilityin vitro.170 T-follicular-helper CD4þ T cells (termedTfh)dwhich produce IL-21 and IL-4dstimulate B cells ingerminal centers to produce antibodies. Interestingly, theTfh subset was proposed to expand in the inflamedmucosa particularly in CD patients and acquire the abilityto produce IFN-g.255 A novel CD4þ T-cell subset (termedTh9)dwhich produces both regulatory cytokine IL-10and effector cytokine IL-9dwas shown to cause colitis ina CD45RB model.256 In addition to CD4þ T cells, someCD8þ T cells also possess suppressive ability in colitis.CD8þ T-cell subsetsdwhich are characterized by expres-sionofCD11cor by lowexpressionofCD28dhada regula-tory ability to suppress colitis in a CD45RBmodel.257,258 Inaddition, an intraepithelial lymphocyte (IEL) subsetdwhich expressed CD8aa homodimer (not CD8ab hetero-dimer)andalsoCD4dwasproposed to suppress the colitisof a CD45RB model through production of IL-10.259

Furthermore, an NKT-cell subset expressing TCR variableregion a14 may have contributed to the improvement ofacute colitis induced by DSS.260

A requirement for some T-cell subsets for the induc-tion of colitis/ileitis has been well demonstrated in

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many IBD models, while they have also identified theexistence of different T-cell subsets, some of whichcontribute to the suppression of colitis/ileitis. Clinicaltrials of anti-CD3 therapy capable of depleting allT-cell subsets had an unfavorable outcome.

Anti-CD25 Therapy

IL-2 is recognized by a heterotrimeric receptorcomposed of IL-2Ra (CD25), IL-2Rb (CD122), andcommon g chain (CD132). CD25 is a potentialCD-susceptibility gene, and the IL-2 gene is locatedwithinan IBD-susceptibility locus.3,11,12Ananti-CD25mAb (basi-liximab) has been approved since 1998 by the FDA forprevention of acute rejection after organ transplantation.Some initial uncontrolled pilot studies demonstrated thepotential effectiveness of anti-CD25 therapy (daclizumab)in fulminatingUC.261However, a subsequent randomizeddouble-blind placebo-controlled dose-ranging trial in 159patients with moderately active UC showed that anti-CD25 therapy rather decreased the remission andresponse, with increased adverse events including naso-pharyngitis and pyrexia.262 Since the ability of IL-2(together with IL-4) to render lymphocytes steroid-resistant has been reported,263 a randomized placebo-controlled trial was also performed on patients withsteroid-refractory UC. However, in the trial, on 149patients with moderate to severe UC despite treatmentfor at least 14 days with oral prednisone, anti-CD25therapy did not increase the effect of steroid on the induc-tion of remission.264

A protective role of IL-2 in IBD has been highlighted bythe spontaneous development of colitis in IL-2KOmice265

and CD25 KOmice.266 A regulatory role of CD25 in colitishas also been supported by the constitutive expression ofCD25 on Treg capable of suppressing colitis and by therequirement of IL-2 for maintaining the regulatory func-tion of Treg.267 Although CD25 can be transientlyexpressed on effector T cells, inherent and continuousexpression of CD25 is seen on the Treg.268 On the otherhand, the therapeutic effect of anti-CD25 mAbs on colitishas been largely unexplored in IBD models. Only onestudy proposed that anti-CD25 therapy had no effect onTh1-mediated acute colitis of a TNBS model.269

A protective role of CD25 in colitis has been sup-ported by the spontaneous development of colitis inCD25-deficient mice, but the effectiveness of anti-CD25therapy has been largely unexplored in IBD models.Clinical trials on active UC had an unfavorable outcome.

Anti-Interferon-Gamma Therapy

CD is generally characterized by an enhanced IFN-g-producing Th1 response, while UC tends to exhibit anenhanced Th2 (e.g., IL-5) response.10 A dose-escalating


placebo-controlled double-blind, single- and multiple-dose safety and tolerability clinical trial of anti-IFN-gtherapy (fontolizumab) in 133 patients with CDAI scoresbetween 250 and 450 showed no statistically significantdifference in theprimaryendpointsbetween fontolizumaband placebo groups.270 However, patients receiving twodosesof fontolizumabexhibited adoubling in the responserate at day 56. A recent randomized double-blind placebo-controlled multiple-dose phase II clinical trial in 201patients with CDAI scores between 250 and 450 showedthat fontolizumab, although reducing C-reactive proteinlevels, induced no strong clinical response.271

A series of studies has indicated that development ofcolitis in CD45RB models depends on the Th1 responseassociated with IFN-g production.244 Development ofcolitis in B7.2 Ig Fc transgenic mice was abolishedwhen they were crossed with IFN-gKOmice.272 In addi-tion, deficiency of IFN-g improved the ileitis inTNF(ARE) mice.128 Similarly, CD4þ T cells producingIFN-g were responsible for the development of ileitisin SAMP1/Yit mice.273 Interestingly, deficiency of IFN-g improved a Th2-mediated acute colitis of an oxazolonemodel.274 Taken together, these mouse models supportthe inflammatory role of IFN-g in IBD.

Colitis of IL-10 KOmice has generally been referred toas“Th1-mediated” colitis.However, IFN-gmayplaymorea complicated role in this colitis model. Anti-IFN-gadministration improved Helicobacter hepaticus-inducedcolitis in IL-10 KO mice.275 In contrast, IFN-g may playno role, or a protective role, in the inherent colitis of IL-10 KO mice. The lack of a role of IFN-g in sustaining thecolitis of IL-10 KO mice was initially proposed in 1998by Rennick and colleagues.153 In addition, IFN-g-deficientand IL-10-deficient double KO mice developed a moreexacerbated form of colitis as compared to IL-10 KOmice.276 Similarly, the lack of an obvious role of IFN-g incolitis has been found in some IBD models. Absence ofIFN-g still induced the development of severe colitis ina CD3ε transgenic mouse model.154 WT mice and IFN-g-receptor KO mice exhibited a comparable susceptibilityto TNBS colitis,156 and administration of anti-IFN-gmAbs also had no effect on this colitis.277 Deficiency ofIFN-g did not affect the severity of Th2-mediated chroniccolitis of TCRa KOmice.216

Some IBDmodels have supported a proinflammatoryrole of IFN-g in colitis/ileitis, but other IBDmodels havefound no role, or a protective role, of IFN-g in colitis.Clinical trials of anti-IFN-g therapy did not achieve theprimary end point.

Recombinant GM-CSF Therapy

Granulocyte macrophage colony-stimulating factor(GM-CSF) is a hematopoietic growth factor that contrib-utes in promoting myeloid cell development,

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maturation, and survival. The therapeutic potential ofrecombinant GM-CSF (sargramostim) has been testedin CD patients in several human trials.17,278 Initial find-ings from a small number of patients suggested a benefi-cial effect of GM-CSF therapy on moderate to severeCD,279 but a subsequent randomized double-blindplacebo-controlled trial in 129 patients with moderateto severe CD did not achieve the primary end point.280

Interestingly, a recent study found that expression ofCD116 (receptor of GM-CSF) on granulocytes andmono-cytes was decreased in the context of intestinal inflam-mation, and that the reduced expression was moreprominent in UC,281 raising the possibility that the lowlevel of CD116 expression makes GM-CSF therapyinsufficient.

In a preclinical setting, the therapeutic effect ofGM-CSF on colitis has only been assessed using aDSS-induced acute colitis model. Daily administrationof pegylated GM-CSF promoted wound healing inDSS-induced acute colitis, presumably by enhancingBM-derived CD11cþ myeloid cells.282 An improvementof DSS-induced colitis by GM-CSF treatment was alsoseen in RAG1 KO mice, lacking both T and B cells, sug-gesting that the therapeutic effect is independent of theadaptive immune response.283 A specific plasmacytoidDC subset expressing 440c (a sialic-acid-binding Ig-likelectin) was shown to mediate the improvement of thiscolitis. In addition, GM-CSF KO mice developeda more exacerbated form of colitis after challenge withDSS as compared to WT mice.284

A beneficial effect of GM-CSF therapy on IBD hasonly been supported using a DSS-induced acute colitismodel, and clinical trials have had an unfavorableoutcome.

Recombinant Interleukin 11 Therapy

IL-11 is a thrombopoietic growth factor that stimu-lates the proliferation of megakaryocyte progenitors.Recombinant IL-11 has been used to restore the numberof platelets in patients after certain kinds of chemo-therapy. A randomized double-blind controlled trial in51 patients with CD showed that subcutaneous injec-tions (once weekly for 12 weeks) of recombinant IL-11were significantly inferior when compared to predniso-lone therapy for the induction of remission in activeCD.285

Subcutaneous injection of recombinant IL-11 hasbeen shown to improve inflammation in the jejunumand colon of a rat UCmodel (humanHLA-B27 transgenicrats), suggesting an anti-inflammatory effect of IL-11on colitis.286,287 A preventive and therapeutic effect ofrecombinant IL-11 on acute colitis was also shown usinga TNBS colitis model in rats.288,289 The preventive effectmay have a contribution from amassive release of mucus


fromgoblet cells.288 Interestingly, the colonwith improvedinflammation still exhibited high levels of arachidonicacid derivatives, such as PGE2 and LTB4.289

A therapeutic effect of recombinant IL-11 has beensupported only by rat models of IBD, and clinical trialson CD have had an unfavorable outcome.

Recombinant Keratinocyte Growth FactorTherapy

Keratinocyte growth factor 1 (KGF1, also known asFGF7) and KGF2 (FGF10)dwhich belong to the familyof fibroblast growth factors (FGFs)dinteract with oneof the FGF receptors expressed particularly by epithelialcells, and may serve as a protective factor for epithelialtissues. A randomized double-blind placebo-controlleddose-escalation (1–50 mg/kg) clinical trial of intravenousinjection of a truncated recombinant KGF2 in 88 patientswith active UC showed no evidence of therapeuticeffectiveness.290

A preventive and therapeutic effect of recombinantKGF2 was shown in acute colitis induced byDSS,291–293 and KGF2 therapy also enhanced thewound healing in this acute colitis.293 In addition, KGFKO mice were more susceptible to DSS-induced acutecolitis, and recovery from this colitis was impaired inKGF KO mice.294 TCRgd T cells, which located in closeproximity to the area of intestinal damage, primarilyproduced the KGF.

The therapeutic potential of KGF2 in IBD has onlybeen supported by an acute colitis model, and clinicaltrials on UC patients have had an unfavorable outcome.

Anti-CD20 Therapy

IBD, and particularly UC, are characterized byincreased production of autoantibodies such as anti-neutrophil cytoplasmic antigens with a perinuclearstaining pattern (pANCA) and anti-tropomyosin, bothof which are produced in response to entericmicrobes.295 B-cell-depletion therapy through rituximab(a chimeric monoclonal antibody against CD20) hasbeen approved since 1997 by the FDA for the treatmentof non-Hodgkin’s B-cell lymphoma, and has also beensuccessfully applied for the treatment of some autoim-mune diseases. A double-blind randomized controlledtrial in 24 patients with steroid-resistant moderatelyactive UC showed no significant effect of rituximab ininducing remission,296 although no severe adverseeffects were reported. A case report showed an improve-ment of colitis associated with active systemiclupus erythematosus after rituximab therapy.297 Incontrast, an exacerbation of UC with decreased IL-10production after rituximab therapy has been

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reported.298 Among ten patients with Graves’ diseasewho were treated with rituximab, one patient rapidlydeveloped UC by 68 days after treatment and anotheracquired a low-grade colonic inflammation.299 A pedi-atric patient with nephritic syndrome developed severeUC at 42 days after treatment with rituximab.300 Rituxi-mab treatment also induced the development of UC ina rheumatoid arthritis patient within 10 weeks aftertreatment.301 Interestingly, a patient with bullous lupusdeveloped appendicitis and then UC-like colitis after rit-uximab therapy.302

Since the specific function of B cells is the productionof immunoglobulin (including autoantibodies), themajor function of B cells has generally been believed tobe mediated by the immunoglobulin. However, it isbecoming increasingly apparent that B cells havea much broader spectrum of immune functions; theycan produce a broad array of cytokines, including the re-gulatory cytokine IL-10, and can serve as a second lineof antigen-presenting cells to induce the expansionof Treg.303,304 Therefore, functions of B cells may bemuch more complicated than previously and evencurrently predicted. Indeed, a growing body of evidencefrom IBD models has unveiled different roles (protec-tive, inflammatory, or no role) of B cells, dependingupon several factors such as the fundamental mecha-nism and the location of inflammation.

Autoantibodies were initially thought to cause theTh2-mediated colitis in TCRa KO mice because of theclose correlation between colitis severity and productionof autoantibodies.305 However, subsequent studiesfound that deficiency of B cells resulted in the exacerba-tion of colitis in TCRa KO mice, suggesting a protectiverole of B cells in this colitis model.306 Three major mech-anisms for the B-cell-mediated protective function havebeen proposed. One mechanism depends on immuno-globulin that may enhance the clearance of apoptoticepithelial cells to prevent the consequent activation ofeffector CD4þ T cells.306 This possibility may be sup-ported by a finding that intake of infected orTLR-ligand-carrying apoptotic epithelial cells by macro-phages stimulated the effector immune response.307 Asecond mechanism is operated by an inducible regula-tory B-cell population (called “Breg”) that appears inthe mesenteric lymph nodes (MLN) only in the contextof inflammation and improves the ongoing colitisthrough production of IL-10.209,304 A third mechanismmay be mediated by another inducible B-cell population(called “B12”) that appears in the inflamed colon,produces IL-12 but not IL-10 in response to bacterialDNA CpG, and consequently improves Th2-mediatedchronic colitis of TCRa KO mice.165,308

Deletion of B cells by a single administration of anti-CD20 mAb induced the rapid development of colitiswithin 15 days in young IL-10 KO mice at 6 weeks of


age.309 B cells in this colitismodelmay control the homeo-stasis of Treg tomaintain the immune tolerance.Aprotec-tive role of B cells in Th1-mediated chronic colitis ofa CD45RBmodel has been demonstrated,310 and a recentstudy also found a protective ability of B cells carryingagalactosyl IgG in this colitis model.311 B10 is anotherregulatory B-cell subset that expands initially in thespleen, expresses CD5 and high levels of CD1d, andproduces large amounts of IL-10.312 B10 cells that werestimulated with enterobacterial extract were shown tosuppress Th1-mediated chronic colitis of a CD45RBmodel.313 In another colitis model, Gai2 KOmice, B cellscontributed to the suppression of colitis by facilitatingexpansion of CD4þ CD8aþ intraepithelial T cells andCD3þ CD4� NKT cells with immune regulatoryability.314 MHC-class-I-mediated antigen presentationwas required for this B-cell-mediated induction of a regu-latory CD8þ T-cell subset capable of controlling colitisthrough production of perforin.315 CD4þ T-cell-specificoverexpression (under control of the CD4 promoter) ofdominant negative TGF-b receptor type II led to sponta-neous development of multi-organ inflammation,including colitis. B cells played a protective role in thiscolitis as indicated by the exacerbation of colitis afterdepletion of B cells by anti-CD20 administration.316

NFATc2 KO mice did not develop colitis, but doubleKO mice deficient in both NFATc2 and RAG2 (lacking Tand B cells) spontaneously developed colitis. Adoptivetransfer of B cells, but not Tcells, fromWTmice improvedthis colitis in an IL-10-independent manner.317 Thesemouse IBD models support the protective role of B cellsin colitis.

The role of B cells per seversus bacterial-specific IgG inDSS-induced acute colitis may vary. Deficiency of CD19(B cells) made mice more susceptible to DSS-inducedacute colitis, and this exacerbation was improved byadoptive transfer of regulatory B10 B cells.312 Similarly,CD19-specific deletion of MyD88 rendered mice highlysusceptible to DSS-induced acute colitis.318 In contrast,a subset of IgG contributed to the exacerbation of thisacute colitis. Administration of IgG specific for a colito-genic bacterial antigen (flagellin) exacerbatedDSS-induced colitis by enhancing FcRn-dependentantigen presentation.319 IgE was also proposed to serveas a causative factor in Th2-mediated acute colitisinduced by oxazolone.320

An inflammatory or dispensable role of B cells hasbeen demonstrated in some IBD models, particularlyin ileitis and systemic inflammatory models. B cellswere required for the exacerbation of ileitis in SAMP/YitFc mice by cooperating with colitogenic CD4þT cells,321 and expression of b7 integrin on B cells wasindispensable for eliciting their inflammatory ability.322

Interestingly, development of IL-10-producing regula-tory B cells was impaired in the SAMP1/Yit mice,

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TABLE 22.1 Effects of Therapeutic Agents on Mouse IBD Models

CD models UC models

Colitis Ileitis Colitis Colitis

Acute Chronic Chronic Acute Chronic Acute

Therapy TNBS CD45 IL10 SAMP TNF OXA TCR

DSS Injury

model

Other

models Mouse model score

Hygiene hypothesis SUP SUP X SUP 4 In clinical development

Cigarette smoking SUP SUP SUP SUP SUP 32 Suppressive effect onUC but not CD

Steroid SUP SUP SUP SUP SUP SUP ? 62 Conventional therapyfor IBD

Cyclosporine SUP X X SUP SUP 2 Effective in severe UC,not CD, patients

Tacrolimus SUP X ? SUP SUP(2) 8 Effective in steroid-refractory UC

NSAID* WOR WOR WOR WOR �81 Alert in the use for IBD

5-ASA SUP SUP SUP SUP 16 Effective in IBD

Anti-TNFa SUP SUP SUP SUP SUP WOR SUP WOR SUP(1) 55 Approved by FDA forIBD therapy

Anti-a4 SUP SUP SUP SUP SUP(1) 32 Approved by FDA forCD therapy

Anti-IL-6R SUP SUP SUP SUP SUP WOR SUP(1)WOR(2)

37 In clinical development

Anti-IL23 WOR SUP SUP SUP(2) 13 In clinical development

Anti-b7 ? SUP SUP SUP(1) 9 In clinical development

Mesenchymal stemcellsy

SUP SUP 4 In clinical development

Anti-IL12p40 SUP SUP WOR SUP X WOR SUP(1) 1 In clinical development

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22.ANIM

ALMODELSOFIN

FLAMMATORYBOWELDISEASE

516

Anti-IL17A SUP ? SUP X ? SUP(1) 0 In clinical development

Anti-CCR9z X ? WOR �4 In clinical development

Anti-CD20* WOR WOR SUP X SUP WOR WOR SUP(2)WOR(3)

�56 In clinical development

rIL-10 SUP SUP SUP SUP SUP SUP(3) 128 No longer in clinicaldevelopment

Anti-IFNg X SUP WOR SUP SUP SUP X SUP SUP(1)X(1)

5 No longer in clinicaldevelopment

rIL-11y SUP(2) 4 No longer in clinicaldevelopment

rGM-CSFy SUP 2 No longer in clinicaldevelopment

rKGFy SUP 2 No longer in clinicaldevelopment

Anti-CD3 ? ? ? ? ? ? 0 No longer in clinicaldevelopment

Anti-CD25y X ? WOR(1) �3 No longer in clinicaldevelopment

Anti-CTLA4* SUP WOR WOR(2) �25 No longer in clinicaldevelopment

* Alert pattern,y insufficient pattern,z unfavorable pattern.The models include TNBS (TNBS), CD45RB (CD45), IL-10 KO (IL10), SAMP/Yit (SAMP), TNF(ARE) (TNF), oxazolone (OXA), TCRa KO (TCR), and DSS (DSS). Data from other IBDmodels are listed in the column

“Other models.” SUP, suppressive effect was observed; WOR, inflammation was worsened; X, no effect was seen; ?, data did not reach a consensus. Numbers in parentheses are the number of mouse models in

other IBD models that exhibited suppressive or exacerbating effect. Score: probability index of likelihood of success of the therapy in mice.

The clinical status is based on review articles.1,17

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BIO

LOGIC

ALTHERAPYAND

IBD

MODELS

517


suggesting that the pathogenic function of B cells in thismodel may be promoted by the absence of regulatory Bcells.323 B cells were also proposed to participate in theinduction of small intestinal inflammation characterizedby villus atrophy.324 In addition, induced ectopic overex-pression of gp39 on B cells caused the development ofcolitis and ileitis.325 On the other hand, absence of B cellsdid not affect the severity of ileitis in TNF(ARE) mice.128

IL-2 KO mice spontaneously develop a systemicautoimmune disease including colitis,265 and a compa-rable severity of colitis was seen when IL-2 KO micewere crossed with B-cell-deficient mice, suggesting norole of B cells in this colitis.248

A functional difference between large and smallintestinal B cells is suggested by a recent meta-analysisof more than one million mouse IgA repertoires. ManyIgA variable region of heavy chains (VH) sequencesabundantly present in the small intestine were infre-quent or undetectable in the colon.326 Interestingly, themajority of “colitis” models, except the oxazolonemodel, suggest a protective role of B cells, whereas themajority of “ileitis” models propose an inflammatoryrole for B cells. These results, if true, suggest thatB-cell depletion may be a more favorable therapy forCD (ileitis) rather than UC (colitis). However, a casereport showed ineffectiveness of rituximab therapy ina patient with CD.327

Coexistence of inflammatory and regulatory B-cellpopulations in IBD has been confirmed in IBD models.B-cell-depletion therapy for UC is currently under clin-ical development, but an alert has been raised by fivecase reports.

CONCLUSION

Although mouse models of IBD cannot fully reflectthe pathogenesis of human IBD, they may be used tohelp the preclinical evaluation of some candidate thera-pies. There are recognizable patterns when comparingthe findings from IBD models to the outcome fromsome clinical trials (Table 22.1). An “alert” pattern ariseswhen a majority of IBD models raise concern on theexacerbation of disease by therapy. An “unfavorable”pattern is expressed when many (but not just one ortwo) IBD models fail to support therapeutic benefits.An “insufficient” pattern is raised when the potentialtherapeutic effect has been evaluated based on onlyone or two IBD models. Clinical trials targeting mole-cules categorized into these three patterns may showa tendency to have an unfavorable outcome. As seenin anti-TNF-a and steroid therapies, a consensus froma majority of IBD models, including “chronic” colitis/ileitis models, may be necessary to provide a sufficientrationale for the consideration of future clinical trials.


Further accumulation of data from both IBD modelsand clinical trials may help generate a platform forcomputational analysis to determine the likelihood ofsuccess of future therapies in IBD and to developpersonalized therapy depending on the genetic patternof IBD-susceptibility genes and environmental factors.

Acknowledgments

The authors would like to thank Emiko Mizoguchi and HirotsuguImaeda for helpful advice and Cindy W. Lau for nice editorialassistance.

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