i*, Akira AndohyMedical School, Boston, MA, USA
Anti-Integrin-a4 Therapy 506Anti-Integrin-b7 Therapy 507
Acknowledgments 5182relapsing 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. IBD is classified into two major forms, Crohns
is 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 profileAnti-Interleukin-12p40 and Anti-Interleukin-23Therapy 507
Inflammatory bowel disease (IBD) is a chronic
disease (CD) and ulcerative colitis (UC), which aremediated by both common and different mechanismsand have distinct clinical features.36 For example, CDA
hBiological Therapy and IBD Models 505Anti-TNF-a Therapy 505
Conclusion 5185-Aminosalicylic Acid 505Cyclosporine/TacrolimusNonsteroidal Anti-Inflammatory Drugs504504Corticosteroids 503nima
ttp://assical Therapy and IBD Models 5Cl 03Cigarette Smoking 503
The Hygiene Hypothesis 501
iology and Mouse IBD Models 5Et 01assification of Mouse IBD Models 5Cl 01troduction 4In 99O U T*Department of Pathology, HayDepartment of Mucosal Immunology, Srvard499l Models for the Study of Human Disease
dx.doi.org/10.1016/B978-0-12-415894-8.00022-1Recombinant Keratinocyte Growth Factor Therapy 5Anti-CD20 Therapy 51414Recombinant Interleukin 11 Therapy 514
Recombinant GM-CSF Therapy 513
Anti-Interferon-Gamma Therapy 513
Anti-CD25 Therapy 513
Anti-CD3 Therapy 511
Anti-CTLA4 Therapy 511
Stem Cell Therapy 510
Recombinant Interleukin-10 Therapy 509
Anti-CCR9 Therapy 509
Anti-Interleukin-6R Therapy 509
Anti-Interleukin-17A Therapy 508I N Eniversity School of Medicine, Shiga, JapanAtsushi MizoguchDisease for Drug DiscoveryC H A
Animal Models of IT E R
flammatory BowelCopyright 2013 Elsevier Inc. All rights reserved.
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, theileitis 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
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.3,1113 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 proposed10 mysteries with IBD, some of which are associatedwith the etiological factors.16 Among them, the hygienehypothesis (probiotics), appendectomy, and cigarette
IL-2 KO CD25 KOCD122 KO
Dominant negative TGFR II Tg
Ubiquitin-editing enzyme A20 KO
Autophagy-related gene (Atg) 5- deficient thymus-implanted nu/nu mice
C3H/HeJBir CD3eTg model
( T-cell-specific suppressor of cytokine signaling (SOCS) 1 Tg
Epithelial-specific core 11,3-galactosyltransferase KO
IL-7 Tg CD45RB
Mucin 2 KO IL-10 KO
Wiskott-Aldrich syndrome protein (WASP) KO
Phosphatidyl inositol 3 kinase (PI3K) p110 KI
CD4-specific phosphoinositide-dependent kinase (PDK) 1 KO
G protein Gi2 KO
Liver-specific soluble CD86 Tg
CD11c-specific integin 8 KOInducible STAT4 Tg
Myeloid-specific STAT3 KO
Inducible STAT3 KO
24 TCR Tg x CD1d Tg Epithelium polarized sorting factor AP1B KO Epithelial-specific IB kinase NEMO KO
Keratin 8 KO
RAG KO with deficiency of nuclear factor of activated T cells (NFAT)c2
RAG KO with deficiency of T-box transcription factor T-bet
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE500SAMP/YitEpithelial-specific N-cadherin KI
TNFR1-deficient, epithelial-specific TGF activation kinas
Src-homology-2-containing inositol 5 phosphatase (SHIP)1 KO
Runt-related transcription factor RUNX3
Inducible enteric glia KO
Epithelial-specific X-box-binding protein (XBP
Glutathione peroxidase (GPX
Janus family tyrosine kinase
RAG KO treated with anti-CD40T cell-specific B-lymphocyte-induced maturation protei
IL-6 receptor gp130 KI
Multiple drug resistan
FIGURE 22.1 The different types of IBD models. These include chspontaneous models (black), congenital models (brown), and geneticallyinto conventional transgenic models (light blue), conventional knockoknockout models (pink), knock-in models (orange), and innate models(colitis, red square), small intestine (ileitis, blue square), or multiple orVI. URINARY TRACT, K1-deficient DKO
F15 (TL1A) Tg
ecific IL-15 Tg
Casitas B-lineage lymphoma protein cb1-b KO
CD2-specific TNFSF14 (LIGHT) Tg
B-cell-specific CD40L TgEndothelial-specific integrin V KO
CD4-specific TGF KO/2 DKO
JAK) 3 KO (Blimp1) KO
e (Mdr) 1a KO
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).IDNEY AND BOWEL
smoking may have potential for IBD therapy. In addi-
engineered models are further classified into sixsubgroups.8 Conventional transgenic or conven-
At least 66 kinds of IBD model had been established
with high humidity) for microbes in the southern parts,these northsouth gradients may support the hygiene
ETIOLOGY AND MOUSE IBD MODELS 501tional 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.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)dchemically 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 ofCD4CD45RBhigh nave 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, geneticallyVI. URINARY TRACT, Khypothesis.Analysis of the recent evolutionary history of human
interleukin (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 ofby 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, TCRVa3.2 Vb9 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 Northsouth gradientsexist within individual countriesdCD is less common inthe southern US and in the south of France.24 Givena more favorable environment (warm temperatureIDNEY AND BOWEL
helminths on colitis may be mediated by suppression of
cial effect of Trichuris suis ova on UC that has significantcontributions from Th2 responses.35 On the other hand,
in the development of UC. A second possibility is that
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE502pre-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
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.4244 Speculations have been raisedto explain the mysterious but consistent finding.A possibility is that the appendix is actively involvedinterferon gamma (IFN-g) and IL-17 production,enhancement of IL-4, IL-10, and transforming growthfactor beta (TGF-b) production, induction of regulatoryCD8 T cells, and/or inhibition of macrophageactivation.3033 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-VI. URINARY TRACT, Kpatients 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 CD4 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 CD4 T cells were shown to possess colito-genic ability.49 Indeed, colitogenic CD4 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 CD4 T cells. The primedCD4 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.5254
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 appendectomyIDNEY AND BOWEL
transiently improved endoscopically recognized inflam-
spinal cord. The ability of nicotine to suppress thehyperexcitability of colonic DRG was recently shown
heme oxygenase (HO)-1-dependent inhibition of IFN-
and vitamin D3) were also reported to possess suppres-sive ability against this colitis.77
CLASSICAL THERAPY AND IBD MODELS 503in 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 bymation 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.
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 CD4 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 CD4 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 CD4T 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 toVI. URINARY TRACT, KGlucocorticoid-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 CD4CD25 T cell population thatcontains both recently activated CD25 effector T cellsg-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 byCD11b cells and of IL-22 by CD11b cells.
CLASSICAL THERAPY AND IBD MODELS
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 nave T cells to an IL-10-producing CD4 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 CD4 T cells.76 Dendritic cells(DCs) generated in vitro by the same regimen (DexIDNEY AND BOWEL
and Foxp3 regulatory Tcells (Treg).79 Interestingly, the
Cyclosporinedwhich serves as an inhibitor ofcalcineurindis effective in hospitalized patients
to recruit the transcription factor GATA3 to Th2-type
(LRRK2)dwhich is encoded by a major susceptibilitygene for CD3,11,12dwas shown recently to possess an
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE504with 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 abilityauthors 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 GITR, but not GITR, CD4T-cell population was shown to suppress the colitis ofa CD45RB model,82 raising the possibility that a GITRCD4 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 CD103 PDCA(plasmacytoid marker) 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/TacrolimusVI. URINARY TRACT, Kability 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 CD4 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.14,101gene 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 2IDNEY AND BOWEL
Since the incidence of spontaneous colitis development
a selective Cox-1 inhibitor (SC-560) or a selective Cox-2
Therapy with 500 mg/kg 5-ASA (mesalamine) in
at inducing remission in moderate to severely activeUC outpatients.1
BIOLOGICAL THERAPY AND IBD MODELS 505inhibitor (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-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.1in 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 46 weeks after termina-tion of piroxicam treatment.100 Administration of eitherVI. URINARY TRACT, KTNF-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 CD3 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 developmentmouse 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
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 effectiveIDNEY AND BOWEL
of colitis in A20 KO mice.115 Interestingly, administra- may exacerbate colitis of CD45RB models.129 Taken
was shown to have therapeutic potential in
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE506tion 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 3040%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 CD4 T cellsVI. URINARY TRACT, KDSS-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.134together, 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.
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 integrinIDNEY AND BOWEL
In addition to anti-a4-integrin therapy, therapeutic
most common serious adverse effect.Administration of an Act-1 mAbdwhich was initially
anti-aE-integrin mAb suppressed an antigen (OVA)-
accumulation of b7 integrin cells in the inflamedcolon after challenge with DSS.146 Treatment with anti-
BIOLOGICAL THERAPY AND IBD MODELS 507reported 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 nave CD4 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 asmeasures 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
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 the
140VI. URINARY TRACT, Kb7-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 disappointinginduced 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 integrin 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 specificIDNEY AND BOWEL
outcome.17,150 Ustekinumab (human IgG1 against p40) Mouse IBD models have suggested differing roles
the development of colitis in a CD45RB model, a patho-
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE508has 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 CD3 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-specificCD4 T-cell subset.162 In addition, deficiency of p19dramatically improved the colitis of CD4 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.166VI. URINARY TRACT, Kgenic 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 byCD4 T cells.180 Indeed, adoptive transfer of CD4 Tcells from IL-17F KO mice or from IL-17A-deficient(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.
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. CD4 T cells expressing both IL-17Aand IFN-g were detectable in the inflamed colon of CDpatients,169 and CD4 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 CD4 T cells was demonstratedwhen compared to IFN-g-producing effector CD4 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 cellsdcausedIDNEY AND BOWEL
and IL-17F-deficient double KO mice still induced
improved Th2-mediated chronic colitis of TCRa KO
Consistent with these findings, gp130 knock-in (KI)
types (e.g., adaptive versus innate) targeted.
BIOLOGICAL THERAPY AND IBD MODELS 509mice.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 CD4 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.190severe colitis in recipient RAG KO mice.181,182 In addi-tion, a protective role of CD4 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 CD4 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.
IL-6 activates various cell types carrying themembrane-bound IL-6R/gp130 heterodimer receptors(classical IL-6 signaling) as well as IL-6R gp130 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 CD62Lnave CD8 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-6VI. URINARY TRACT, KApathogenic 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
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.3,11,12mice, 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 cellIDNEY AND BOWEL
Polymorphisms within IL-10 receptor genes (IL-10R1 IBD therapy have recently been reported. Allogeneic
to clarify this possibility, delivery of IL-10 into the intes-
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE510and 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 Crohns 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 Foxp3 Treg to cause thecolitis of a CD45RBmodel.207 Impaired regulatory functionof Foxp3 Treg by absence of WiskottAldrich 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 futureVI. URINARY TRACT, Ktinal 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 humantransplantation 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 orderIDNEY AND BOWEL
umbilical-cord-derived MSCs was reported to improve inconsistencies in the data published. An initial pilot
patients and 490 UC patients showed that abatacept is
BIOLOGICAL THERAPY AND IBD MODELS 511a 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 CD11b cells resultedin the loss of the beneficial effect of MSC therapy onthis colitis, suggesting that interactions between MSCsand CD11b 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 someVI. URINARY TRACT, Knot efficacious for the treatment of moderate to severeCD or UC.9,233
Constitutive expression of CTLA4 among CD4Tcells 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,237239 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 (ICOS) 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.
CD3 is a surface molecule expressed by T-cell popula-tions, including CD4 T cells, CD8 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 CD4 T cells for the induction ofcolitis has been well documented using CD4 T-cell-transfer systems. For example, development ofTh1-mediated colitis in a CD45RB model was inducedby adoptive transfer of CD4 CD45RBhigh nave CD4study in human IBD showed an unfavorable outcome,suggesting the existence of many steps to be overcometo successfully apply stem cells to IBD 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 CDIDNEY AND BOWEL
T cells from WT mice.244 Adoptive transfer of CD4
IBD patients.170,171 This CD4 T-cell subset, although itproduced IL-17A, had immune suppressive abilityin vitro.170 T-follicular-helper CD4 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 CD4 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 CD4 T cells, someCD8 T cells also possess suppressive ability in colitis.CD8 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
inflamed intestine, including Foxp3 IL-17 CD4 (CD4 T cellssharing Treg and Th17 phenotype), IL-17 IFN-g CD4 (CD4 Tcells sharing Th17 and Th1 phenotype), and IFN-g IL-21 CD4(CD4 T cells sharing Th1 and Tfh phenotype) T-cell subsets.
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE512T cells from WASP KO mice induced Th2-mediatedcolitis in immunodeficient hosts,245 and flagellin-specific CD4 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 ofCD4 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 CD4TCRab T cells, but not of CD8 TCRab T cells orTCRgd T cells.248,249 Deficiency of CD4 T cells due toabsence of major histocompatibility complex (MHC)class II abolished the development of Th2-mediatedchronic colitis of TCRaKOmice.49 In addition, deficiencyof CD8 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. CD4T-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, theCD4 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 CD4 T-cell population, CD25Foxp3 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 CD4 T-cell subsets have beendemonstrated to develop specifically in the inflamedmucosa of IBD patients. A CD4 T-cell subset sharingboth Th1 (IFN-g production) and Th17 (IL-17A produc-tion) functions appeared in the inflamed colon of CDpatients.169 Another CD4 T-cell subset expressing bothIL-17A and Foxp3 also appeared in the inflamed colon ofVI. URINARY TRACT, KTreg
CD4+ T cells
FIGURE 22.2 There are functionally different CD4D T-cellsubsets, depending on their cytokine profiles. Some subsets maydifferentiate from other subsets as indicated by the blue lines. UniqueCD4 T-cell subsets have been identified to specifically appear in theIDNEY AND BOWEL
many IBD models, while they have also identified the
placebo-controlled double-blind, single- and multiple-
dosesof fontolizumabexhibited adoubling in the response
BIOLOGICAL THERAPY AND IBD MODELS 513IL-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.
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-escalatingexistence 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.VI. URINARY TRACT, Krate 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, CD4 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 toasTh1-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 CD3 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,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 twoIDNEY AND BOWEL
maturation, and survival. The therapeutic potential of
of DSS-induced colitis by GM-CSF treatment was also
fromgoblet cells.288 Interestingly, the colonwith improved
DSS,291293 and KGF2 therapy also enhanced the
inducing remission,296 although no severe adverse
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE514seen 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 mucusrecombinant 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 CD11c myeloid cells.282 An improvementVI. URINARY TRACT, Keffects 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 beenwound 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.
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-Hodgkins 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 ininflammation 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 (150 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 byIDNEY AND BOWEL
reported.298 Among ten patients with Graves disease age.309 B cells in this colitismodelmay control the homeo-
BIOLOGICAL THERAPY AND IBD MODELS 515who 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 CD4 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 ofVI. URINARY TRACT, Kstasis 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 CD4 CD8a intraepithelial T cells andCD3 CD4 NKT cells with immune regulatoryability.314 MHC-class-I-mediated antigen presentationwas required for this B-cell-mediated induction of a regu-latory CD8 T-cell subset capable of controlling colitisthrough production of perforin.315 CD4 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 CD4T 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,IDNEY AND BOWEL
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
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 IBD5-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
SUP SUP 4 In clinical development
Anti-IL12p40 SUP SUP WOR SUP X WOR SUP(1) 1 In clinical development
Anti-IL17A SUP ? SUP X ? SUP(1) 0 In clinical development
Anti-CCR9z X ? WOR 4 In clinical developmentAnti-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
suggesting that the pathogenic function of B cells in this
B-cell depletion may be a more favorable therapy for
Further accumulation of data from both IBD models
14. Sartor RB. Microbial influences in inflammatory bowel diseases.Gastroenterology 2008;134:57794.
17. Plevy SE, Targan SR. Future therapeutic approaches forinflammatory bowel diseases. Gastroenterology 2011;140:
22. ANIMAL MODELS OF INFLAMMATORY BOWEL DISEASE518CD (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.
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 unfavorablepattern 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.model 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 thatVI. URINARY TRACT, K183846.18. Elson CO, Sartor RB, Tennyson GS, Riddell RH. Experimental
models of inflammatory bowel disease. Gastroenterology1995;109:134467.
19. Strober W, Fuss IJ, Blumberg RS. The immunology of mucosalmodels of inflammation. Annu Rev Immunol 2002;20:495549.15. Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C,et al. A human gut microbial gene catalogue established bymetagenomic sequencing. Nature 2010;464:5965.
16. Colombel JF, Watson AJ, Neurath MF. The 10 remainingmysteries of inflammatory bowel disease. Gut 2008;57:42933.and 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.
The authors would like to thank Emiko Mizoguchi and HirotsuguImaeda for helpful advice and Cindy W. Lau for nice editorialassistance.
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22. Animal Models of Inflammatory Bowel Disease for Drug DiscoveryIntroductionClassification of Mouse IBD ModelsEtiology and Mouse IBD ModelsThe Hygiene HypothesisAppendectomyCigarette Smoking
Classical Therapy and IBD ModelsCorticosteroidsCyclosporine/TacrolimusNonsteroidal Anti-Inflammatory Drugs5-Aminosalicylic Acid
Biological Therapy and IBD ModelsAnti-TNF- TherapyAnti-Integrin-4 TherapyAnti-Integrin-7 TherapyAnti-Interleukin-12p40 and Anti-Interleukin-23 TherapyAnti-Interleukin-17A TherapyAnti-Interleukin-6R TherapyAnti-CCR9 TherapyRecombinant Interleukin-10 TherapyStem Cell TherapyAnti-CTLA4 TherapyAnti-CD3 TherapyAnti-CD25 TherapyAnti-Interferon-Gamma TherapyRecombinant GM-CSF TherapyRecombinant Interleukin 11 TherapyRecombinant Keratinocyte Growth Factor TherapyAnti-CD20 Therapy