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Advanced Drug Delivery Revie

Mouse models of inflammatory bowel disease☆

Stefan Wirtz ⁎, Markus F. Neurath

Laboratory of Immunology, I. Medical Clinic, University of Mainz, Obere Zahlbacher Strasse 63, 55131 Mainz, Germany

Received 12 May 2007; accepted 10 July 2007Available online 16 August 2007

Abstract

Animal models of intestinal inflammation are indispensable for our understanding of the pathogenesis of Crohn disease and Ulcerative colitis, theidiopathic forms of inflammatory bowel disease in humans. The clinical appearance of human IBD is heterogeneous, a fact that is also reflected by thesteadily increasing number of mouse strains displaying IBD like intestinal alterations. The analysis of these models together with genetic studies inhumans greatly enhanced our insights into immunoregulatory processes in the gut and led to the generally accepted hypothesis that a deregulatedimmune response against components of the intestinal microbiota is critically involved in IBD pathophysiology. In this article we provide a briefoverview of selected mouse models of IBD and discuss their contribution to the current understanding of disease mechanisms that contribute to IBD.© 2007 Elsevier B.V. All rights reserved.

Keywords: Inflammatory bowel disease; Crohn's disease; Ulcerative colitis; Animal models; Epithelial barrier; Mucosal immune system

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10742. Animal models of IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10743. Intestinal inflammation due to disturbance of the epithelial integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1074

3.1. DSS colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10753.2. TNBS/Oxazolone colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10753.3. DN N-cadherin transgenic mice/keratin 8−/− mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10753.4. Multiple drug resistant (MDR1) gene deficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10753.5. IKK-γ (NEMO)/IKKαβ deficiency in intestinal epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10763.6. SAMP1/YitFc (Samp) mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076

4. Immune response and inflammatory pathways in intestinal inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10764.1. Defects related to innate immune cell function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1076

4.1.1. STAT3 deficiency in myeloid cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10774.1.2. A20 deficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077

4.2. Defects related to cells of the adaptive immune system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10774.2.1. TNFΔARE MICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10784.2.2. CD45RBHi transfer model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10784.2.3. STAT4 transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10784.2.4. IL-10/CRF2-4 deficient mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10784.2.5. TCRα chain−/− mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Prediction of Therapeutic and Drug Delivery Outcomes Using Animal Models".Abbreviations: TNF, Tumour necrosis factor; IL, Interleukin; IFN-γ, interferon-γ; TGF-β, transforming growth factor-beta; NFκB, nuclear factor kappa b.

⁎ Corresponding author. Lab. of Immunology, I. Medical Clinic University of Mainz Langenbeckstrasse 1, 55101 Mainz, Germany. Tel.: +49 6131 3933472;fax: +49 6131 3933671.

E-mail address: wirtz@uni-mainz.de (S. Wirtz).

0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2007.07.003

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5. Which model to choose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10796. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080

1. Introduction

Crohn's disease (CD) and ulcerative colitis (UC) are chronicinflammatory disorders of the intestine that are also referred toas inflammatory bowel disease [1]. The etiology of IBD stillremains incompletely understood, but it is generally agreed thata complex interplay between genetic, environmental andimmunological factors contributes to disease initiation andprogression. Twin studies and epidemiologic studies showing afamilial and ethnic clustering of the IBDs were the firstindicators for a significant genetic component in the develop-ment of these diseases and led to the search for specific IBDsusceptibility genes. Genome-wide scanning and candidategene-approaches have identified genetic markers which arelinked to IBD. The identification of several genes (NOD2/CARD15, DLG5, SLC22A4/5, MDR1, ATG16L1, IL-23R)with sequence variations that are associated with susceptibilityto CD had a major impact on the understanding of geneticaspects of this disease [2]. Research progress in the recent yearsprovided evidence that an excessive host immune responseagainst antigens of the commensal intestinal microbiota is amain trigger of chronic bowel inflammation and mucosal tissuedestruction in both UC and CD [2]. However, although theyshare some effector pathways, CD and UC are immunologicallyand histopathologically different diseases. Some evidence existthat chronic inflammation in patients with UC is characterizedby a non conventional TH2 immune response that is associatedwith epithelial barrier dysfunction. In contrast, several studieshave demonstrated elevated levels of the cytokines IFN-γ, IL-2,and TNF-α in the gut of CD patients consistent with a TH1 typeresponse [3]. This dominance of a polarized TH1 pathway in CDwas further confirmed by studies demonstrating increasedmucosal expression of IL-12, which is the key T celldifferentiation factor driving cellular immune responses [4].Recent evidence however suggests that the novel IL-23dependent highly proinflammatory TH-17 cell population of Thelper cells, rather than TH1 cells, might be critically involvedin the pathogenesis of CD [5].

2. Animal models of IBD

Much of the recent progress in the understanding of mucosalimmunity has been achieved by the study of new experimentalanimal models of intestinal inflammation [6,7]. Although thesemodels do not represent the complexity of human disease anddo not replace studies with patient material, they are valuabletools for studying many important disease aspects that aredifficult to address in humans, such as the pathophysiologicalmechanisms in early phases of colitis and the effect of emergingtherapeutic strategies. The clinical appearance of human IBD is

heterogeneous, a fact that is also reflected by the steadilyincreasing number of transgenic or gene targeted mouse strainsdisplaying IBD like intestinal alterations. Most of these modelsare based either on chemical induction, immune cell transferor gene targeting, only in some models disease occurs withoutany exogenous manipulation. For the purpose of this review,selected mouse models are broadly categorized into 3categories according to the defect in mucosal immunity thatis believed to be most important for the onset of the disease: (1)defects in epithelial integrity/permeability, (2) defects in innateimmune cells and (3) defects in cells of the adaptive immunesystem.

3. Intestinal inflammation due to disturbance of theepithelial integrity

The single layered intestinal epithelium is a physical andimmunological barrier that prevents direct contact of theintestinal mucosa with the luminal microbiota. A compromisedintestinal barrier may play a crucial role in the development ofIBD, by allowing the entry of luminal antigens and micro-organisms into the mucosa and initiating overwhelminginflammatory responses [8]. Interestingly, in otherwise clini-cally asymptomatic CD patients, increased intestinal epithelialpermeability preceding clinical relapse has been observedsuggesting that a barrier defect may be an early event in diseasereoccurrence. Since first degree relatives of IBD patients,without evidence of disease, also showed abnormal intestinalpermeability, it has been proposed that increased intestinalpermeability may be a primary etiologic factor in IBD [9,10].However, it has also been shown that the inflammatory processitself leads to increased intestinal permeability [11]. Thus atpresent it is not clear if the changes in barrier integrity observedin IBD are particularly involved in the early events of IBDpathogenesis or are rather a secondary phenomenon [12,13].Several IBD associated genes have been shown to be related tothe biological functions of intestinal epithelial cells andmutations are potentially related to increased intestinalpermeability. Variants of the organic cation transportersOCTN1 and OCTN2 have been linked to CD and result indecreased carnitine transport [14]. The DLG5 gene encodes fora membrane-associated guanylate kinase/scaffolding proteinthat is believed to play a role in cell–cell contact formation [15].Another very recently identified CD susceptibility gene isATG16L1, which is highly expressed in intestinal epithelial andimmune cells and is important for autophagy a cellular processinvolved in the degradation and processing of bacteria [16,17].Coding variants of the NOD2/CARD15 gene on Chromosome16 were the first identified disease-predisposing mutations inCD [18,19]. NOD2 is an intracellular pattern recognition

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receptor recognizing bacterial muramyl dipeptide (MDP) that isproduced by macrophages, dendritic cells and epithelial Panethcells. Nod2 variants found in CD were shown to cause alteredresponses to MDP and NFκB activation [20]. Despite thesubstantial relevance of altered transport and barrier function tothe generation of disease in IBD patients, relatively little isunderstood regarding the mechanisms of these changes. Overthe past several years, several animal models have beendeveloped that clearly helped to improve our understanding ofintestinal epithelial cell function.

3.1. DSS colitis

Feeding mice for several days with DSS polymers in thedrinking water induces a very reproducible acute colitischaracterized by bloody diarrhea, ulcerations and infiltrationswith granulocytes [21–23]. It is believed that DSS is directlytoxic to gut epithelial cells of the basal crypts and thereforeaffects the integrity of the mucosal barrier. As T- and B-cell-deficient C.B-17scid or Rag1−/− mice also develop severe colitis,the adaptive immune system obviously does not play a majorpart (at least in the acute phase) in this model [24]. Hence, theacute DSS colitis model is particularly useful to study thecontribution of innate immune mechanisms of colitis. Inaddition, the DSS model has been shown to be suitable tostudy epithelial repair mechanisms [25]. Studies with TLR4−/−

and MyD88−/− mice suggest that TLR signaling is required tolimit bacterial translocation after DSS induced intestinalepithelial injury suggesting that TLR signaling is importantfor the maintenance of the epithelial barrier [26]. In susceptiblestrains, the administration of DSS for several cycles (e.g., 7 daysDSS, 14 days water) results in chronic colitis and if combinedwith a single initial dose of the genotoxic colon carcinogenazoxymethane (AOM), in inflammation-associated colorectalcancer [27]. Patients with UC have an increased risk for thedevelopment of colon cancer [28]. As colonic inflammation issuggested to play a key role in IBD-related colorectal cancer,the AOM/DSS model is a very useful tool to study mechanismslinking inflammation to colon carcinogenesis.

3.2. TNBS/Oxazolone colitis

Colitis can be induced in susceptible mouse strains byintrarectal instillation of the haptenating substances TNBS/DNBS or Oxazolone dissolved in ethanol with or without a skinpresensitation step [23,29]. Ethanol is required to break themucosal barrier, whereas TNBS/Oxazolone is believed tohaptenize colonic autologous or microbiota proteins renderingthem immunogenic to the host immune system. As CD4+ Tcellshave been shown to play a central role in chronic TNBS colitis,this model is useful to study T helper cell-dependent mucosalimmune responses [30]. The TNBS colitis model has been veryuseful in studying many important aspects of gut inflammation,including cytokine secretion patterns, mechanisms of oraltolerance, cell adhesion and immunotherapy. Murine TNBScolitis has been initially described in SJL/J mice, a mouse strainwith high susceptibility that develops chronic TNBS colitis

characterized by a predominant TH1-mediated immune re-sponse with dense infiltrations of lymphocytes/macrophagesand thickening of the colon wall [30]. However, studies withIFN-γ−/− mice on a Balb/c background showed that in thesemice TNBS colitis may be associated with a TH2-mediatedcolonic patch hypertrophy [31,32].

In SJL/J mice, oxazolone colitis has been shown to affectonly the distal colon and particularly mucosal layers.Histological features and an elevated production of TH2cytokines (IL-4, IL-5 and IL-13) of unstimulated and αCD3/αCD28-stimulated lamina propria T cells are in these mice, insome aspects, similar to characteristics that have been observedin human UC. In contrast to several other murine colitismodels, treatment with neutralizing anti-IL-4 antibodies or adecoy IL-13Rα2-Fc protein ameliorates disease [33]. Thisgroup has shown that Oxazolone colitis depends on thepresence of IL-13 producing invariant natural killer (NK) Tcells [34]. Consistently, mice deficient for Epstein Barr virusinduced gene 3 (EBI3) that have a defect in iNKT cellgeneration, are resistant to Oxazolone colitis [35]. ThusOxazolone colitis is one of the few models suitable to studythe contribution of the TH2-dependent immune response tointestinal inflammation. As in the TNBS model, oxazolonecolitis is strain dependent and requires individual optimization.This is exemplified by the fact that oxazolone at lower dosescan induce a mixed TH1/TH2-dependent colitis [36].

3.3. DN N-cadherin transgenic mice/keratin 8−/− mice

Another interesting model providing direct evidence for theimportance of an intact epithelial barrier for mucosal homeo-stasis, are transgenic/chimeric mice expressing a dominantnegative mutant of the cell adhesion molecule N-cadherin inintestinal epithelial cells along the crypt villus axis [37]. Thesemice develop in areas of chimeric/leaky epithelium chronicinflammatory bowel disease and at later time points intestinalneoplasia. The observation that inflammation occurs only in thevicinity of porous epithelial cells is a direct demonstration of thefact that entry of antigens of the normal mucosal microflora issufficient for the induction of responses that lead to colitis.

Recently, missense mutations in the gene for Keratin 8, oneof the major intermediate filament proteins present in intestinalenterocytes, were shown in a subset of patients with IBD [38].Consistently, mice deficient for Keratin 8 develop colonichyperplasia and colitis due to a primary epithelial rather thanimmune cell defect [39]. Treatment with antibiotics markedlydecreased intestinal inflammation indicating that luminalbacteria play an important role in the development of thedisease.

3.4. Multiple drug resistant (MDR1) gene deficient mice

The multidrug resistance (MDR) gene 1, which is respon-sible for drug resistance to chemotherapy in certain types ofcancer, is expressed in the intestinal epithelium and subsets ofhematopoietic cells [40]. The MDR1 encodes P-glycoprotein170, a transport protein involved in the removal of drugs/

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xenobiotic substances from cells. Mdr1α−/− mice displayspontaneous bowel inflammation triggered by the bacterialflora [41]. Adoptive transfer of mdr1α−/− bone marrow toirradiated wild-type mice does not induce intestinal pathology,in striking contrast to chimera of mdr1α−/− mice reconstitutedwith wild-type bone marrow. Thus, mucosal inflammation inthese mice is most likely caused by dysfunction of intestinalepithelial cells and not by alterations in lymphocyte function.Interestingly, an MDR1 polymorphisms leading to decreased P-glycoprotein expression and function has been associated withhuman UC demonstrating the clinical relevance of this model[42].

3.5. IKK-γ (NEMO)/IKKαβ deficiency in intestinal epithelialcells

Aberrant activation of the transcription factor NFκB controlsthe inducible expression of most of the genes of inflammatorycytokines involved in the pathogenesis of IBD and recently anassociation of a promoter polymorphism within the NFκB1gene with UC has been described [43]. Different strategies toblock NFκB mucosal inflammation in animal models abrogateddisease suggesting that inhibition of NFκB activation haspotential as an anti-inflammatory treatment strategy in inflam-matory bowel disease [44,45].

However, the complete shutdown of canonical NFκBsignaling in intestinal epithelial cells by conditional targetingof NEMO, the regulatory unit of the IKK complex or both IKK-α and β, the catalytic subunits, causes severe chronic pancolitisstarting at 1–2 weeks of age [46]. In 2-week-old mice,inflammatory infiltrates were dominated by large numbers ofDC and granulocytes, and only some CD4+ T cells. However,colitis in 12 and 36 week old mice is characterized by thepresence of abundant lymphoid follicles, a massive infiltrationwith DC, CD4+ T cells and granulocytes and increasedexpression of inflammatory cytokines and chemokines. Histo-logical examination of colon sections from these mice revealedthe presence of crypts showing extensive epithelial destructionsuggesting that a localized disruption of epithelial integrityfacilitates the translocation of bacteria from the lumen into themucosa. TLR-mediated bacterial recognition has a critical role ininducing intestinal inflammation in NEMOIEC−/− mice. Mostnotably, these mice exhibit a reduced expression of the inducibleantimicrobial peptide defensin-3 in the small bowel. Recent dataprovided evidence for a role of several defensins in thepathogenesis and disease progression of CD [47]. A lowhuman β-defensin 2 – the homolog of mouse defensins 3 – copynumber has been reported to predispose to colonic CD [48].Defensins are primary produced in Paneth cells, a specializedileal cell type that also produces the NOD2 gene product.Stimulation of Paneth cells with the NOD2 ligand MDP elicitssecretion of these antimicrobial factors. CD associated poly-morphisms of NOD2 have shown a decreased ability toubiquitinylate NEMO leading to decreased NFκB activation[49]. Thus, the stunning phenotype of the NEMOIEC−/− micepotentially supports the recently proposed concept of a “defensindeficiency” defect in subgroups of patients with IBD. Despite

the fact that NFκB activation in inflammatory cells mediatesdetrimental proinflammatory cytokine production, NFκB sig-naling in IEC emerges as a crucial factor for maintaininghomeostasis between commensal microflora and the immunesystem of the host. In addition, Zaph et al. showed that micedeficient for IKK-β in IEC are unable to eliminate an infectionwith the nematode parasite Trichuris muris suggesting thatNFκB in IEC is also important for protective immune responsesagainst pathogens [50]. This dichotomy of NFκB function indifferent cell types of the gut has certainly to be taken intoaccount when considering NFκB as a therapeutical target inIBD.

3.6. SAMP1/YitFc (Samp) mice

This model is an excellent experimental system to studydisease mechanisms of CD, because the inflammation occursspontaneously and it is one of the few models with severeinflammation in the terminal ileum, the primary location of CDlesions [51]. These lesions are characterized by transmuralinflammation, granulomtas and alterations in epithelial mor-phology. Different from other models germ-free housed SAMPmice do develop intestinal inflammation, albeit to a lesserextend. Increased epithelial permeability precedes the onset ofinflammation and it has been suggested that epithelial celldysfunction may be primarily responsible for the diseaseprogression. However, it remains to be determined whetherthe epithelial permeability defect observed in these mice is acause or a consequence of ileitis in this model. Interestingly,peroxisome proliferator-activated receptor gamma (PPAR-γ)has been identified as a susceptibility gene in both the SAMPmouse and in human CD demonstrating the unique value of thismodel for the study of disease mechanisms in CD [52].

4. Immune response and inflammatory pathways inintestinal inflammation

In both MC and UC incompletely understood diseasemechanisms finally result in a persistent activation of themucosal immune system. However, it is still an open question,whether the immune system is activated because of continuedstimulation resulting from defects in epithelial mucosal barrierfunction or as a result of an inherent abnormality of immunecells. Intestinal immune cell populations and their solublemediators have been eminently characterized in patients withIBD. Studies in animal models accumulated a large body ofevidence supporting the assumption that excessive immune cellactivation or inappropriate downregulatory responses arecritically involved in the induction of mucosal inflammation.

4.1. Defects related to innate immune cell function

Innate immune cells recognize foreign organisms throughreceptors that recognize pathogen-associated molecular patterns(PAMPs), such as cell wall components and nucleic acids.Several families of mammalian pattern recognition receptors(PRRs) that recognize PAMPs have been identified including

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Toll like receptors (TLR), nucleotide-binding oligomerizationdomain-like (NOD-like) receptors (NLRs), C-type lectinreceptors (CLRs), and triggering receptors expressed onmyeloid cells (TREMs) [53–55]. In general, the interactionbetween PAMPs and PRRs results in the activation of innateimmune cells leading to a cascade of events whose majorpurpose is to eliminate the infectious agents. Innate immunecells, in particular dendritic cells and macrophages, in thegastrointestinal tract are significantly involved in both homeo-stasis in the normal gut and dysregulated immune responsesduring intestinal inflammation. Activated innate immune cellsare able to stimulate proliferation of primary antigen specificresponses and moreover direct via the production of regulatorycytokines (e.g. IL-6, IL-12, IL-23, IL-27) the differentiation ofnaïve T cells [56–58].

It has been shown recently that resident lamina propriamacrophages in the healthy gut express reduced innate responsereceptors and have a downregulated production of proinflam-matory cytokines, suggesting that this status of “inflammatoryanergy” could be a potential mechanism for the absence ofinflammation in the normal intestinal mucosa despite the closeproximity of the commensal bacteria flora [59]. Furthermore,intestinal DCs constantly collect bacteria and other antigensfrom the gut lumen by extending transepithelial dendrites intothe lumen and intestinal DCs located below specializedintestinal epithelial cells, called M cells, detect commensaland pathogenic bacteria and regulate immunological toleranceto the microbiota and food antigens, whereas effector immuneresponses are initiated only to pathogens [59,60]. In IBDmyeloid cells produce large amounts of proinflammatorymolecules including cytokines and chemokines. This popula-tion of inflammatory cells consists mainly of infiltrating cellsand not of resident tissue macrophages.

4.1.1. STAT3 deficiency in myeloid cellsMice with specific disruption of the signal transducer and

activator of transcription 3 (STAT3) gene in macrophages andneutrophils develop a spontaneous enterocolitis [61]. Diseasewith transmural lesions and a high incidence of colorectaladenocarcinomas develops in mice that are several months old.Interestingly, when these mice were crossed with TLR4−/− orMyD88−/− mice the double deficient mice developed signifi-cantly less colitis indicating the critical requirement of thebacterial microflora [62]. STAT3 in macrophages and neutro-phils is a critical factor within the signal transduction pathwayof IL-10 suggesting that the absence of an IL-10 mediatedcounterregulatory effect on colonic macrophages continuouslysubjected to stimulation by luminal bacterial or food antigen issufficient for the development of chronic intestinal inflamma-tion. Enterocolitis in these mice, is dependent on the presence ofadaptive immune cells and the production of IL-12p40, whereasthe presence of TNF-α is dispensable [62].

4.1.2. A20 deficient miceA20 is an inducible and broadly expressed cytoplasmic

protein that inhibits TNF-induced NFκB activity. A20-de-ficient mice develop spontaneous inflammation, cachexia and

premature death at least in part due to failure of A20 deficientcells to terminate TNF-induced NFκB responses [63].Interestingly, different from STAT3−/− mice (see above)A20−/−/Rag1−/− mice lacking T and B cells developed similardisease indicating that lymphocytes are not required forintestinal inflammation in this model.

4.2. Defects related to cells of the adaptive immune system

It is now generally accepted that T lymphocytes, in particularCD4+ cells, play a key role in both normal and pathophysiologicalimmune regulatory processes in the gastrointestinal tract [64].However, whereas activation of innate immune cells is a commonfeature of IBD, different Tcell subsets are present and activated inCD versus UC indicating that failure of regulation by T helpercells could be a major contributing factor to the pathogenesis ofIBD [65]. In the inflamed mucosa of patients with CD excessiveproduction of IFN-γ IL-18, TNF-α and IL-12p40 has beenobserved consistent with a TH1 response [64,66]. In addition,mRNA and protein levels for T-bet the master transcriptionalregulator of TH1 effector cells were upregulated in the in CD [67].An exaggerated TH1-type response has also been described as akey feature in the induction of inflammation inmost experimentalanimal models [6,68]. For instance, in an adoptive transfer modelT-bet deficient T cells failed to induce colitis, whereas T cellsgenetically modified to overexpress T-bet induce increaseddisease progression [67]. The results obtained in animal studiesfinally resulted in promising clinical trials aimed to reduce IL-12levels in patients with active CD [69,70]. However, recently IL-23, a heterodimeric cytokine sharing the p40 subunit with IL-12,has been discovered [71]. With the identification of the IL-23R asCD susceptibility gene and the analysis of the important role ofIL-23 (rather than IL-12) in several animal models a rethinkingabout the role of IL-12 in CD launched [72,73]. IL-23 togetherwith IL-6 and TGF-β induces the differentiation of naive CD4+ Tcells into highly pathogenic helper Tcells subset – so called TH17cells - that produce high amounts of IL-17, IL-22, and TNF-α, butnot IFN-γ and IL-4 [74–76]. IL-17, a proinflammatory cytokinepredominantly produced by activated T cells, enhances T cellpriming and stimulates many different cell types e.g. endothelialcells, macrophages, and epithelial cells to produce multipleproinflammatory mediators [77]. Several recent studies knockingout IL-12 and IL-23 subunits in IBD animal models suggest thatIL-23, rather than IL-12 drives chronic inflammation in the gut.Yen et al. used IL-10−/− mice (see below) as a model of T cell-ediated IBD and clearly showed that the incidence of chroniccolitis was suppressed by deficiency of the p19 subunit of IL-23but not by IL-12p35 deficiency [78]. In addition, Hue et al.showed that IL-23p19 is important for the onset of chronicintestinal inflammation in amodel of IBD,without the presence ofT cells [79]. Although these data showed that IL-23 initiates andperpetuates both innate and T cell mediated intestinal inflamma-tion, at least in the DSS and TNBS models p19−/− deficient micehave increased disease [80]. In addition, IL-23 has been shown tobe required for host protection against a intestinal bacterialpathogen indicating that further research effort is required for abetter understanding of the precise role of IL-23 in the gut [76].

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In the healthy gut several immune mechanisms exist thattightly control the persistent stimulation of immune cells by thecommensal microbiota. Tolerance against mucosal antigensincludes induction of T cell anergy or generation of antigenspecific regulatory T cells, which secrete immunosuppressivecytokines such as IL-10 and TGF-β [81]. As one possiblemechanism to induce antigen specific tolerance is discussed thatmucosal dendritic cell population present antigen in a way to Tcells that induces suppressor cells or functional anergy [82].Maintenance of immunological tolerance towards specificantigens in the gut is in part achieved during maturation of theimmune system by specific deletion of self reactive clonesthrough negative selection in the thymus [77]. In addition tothymus derived CD4+CD25+ Tregs Tr1 cells have been shown tosuppress colitis in vivo in adoptive transfer models [83]. TheseTr1 cells can be generated in vitro in the presence of interleukinIL-10 and their regulatory effect is mediated by secretion of IL-10itself. Moreover, genetically engineered Tr1 cells obtained byretroviral transduction of the IL-10 gene have been recently usedfor prevention of experimental colitis suggesting the therapeuticpotential of cytokine induced regulatory T cells [84]. Recently, ithas been demonstrated that FoxP3+ Tcells with regulatory capacitycan be generated in vitro by activation of CD 4+CD25−FoxP3− Tcells in the presence of TGF-β suggesting that Tregs can also begenerated thymus independently in the periphery [85,86]. Theseinduced Treg cells suppressed TH1 mediated colitis induced by theadoptive transfer of CD4+ CD62L+ T cells [87,88].

4.2.1. TNFΔARE MICEAnti-TNF-α neutralizing antibodies provide marked clinical

benefits in patients with CD [89]. However, the specificmolecular and cellular mechanisms of the pathogenic action ofTNF are still incompletely understood. Gene targeting of AU-rich elements (ARE) in the untranslated region of the TNF-αmRNA in mice is associated with increased constitutive andinducible levels of TNF due to dysregulated processing ofTNF-α mRNA. Overproduction of TNF leads to polyarthritisand chronic intestinal inflammation with infiltrating inflam-matory cells and transmural inflammation. Interestingly, like inhuman CD mucosal inflammation is mainly located in theterminal ileum and is characterized by granulomata [90].Studies with TNFΔARE/Rag1 double deficient mice showedthat ileitis but not arthritis depends on the presence of lym-phocytes. Notably, the adoptive transfer of T cells from thesemice can transfer disease to recipient mice emphasizing theimportance of TNF production in T cells and not innate immunecells for disease development. The importance of TH1 associatedcytokines in TNF dependant intestinal inflammation has beendemonstrated by crossing the TNFΔARE mice to IL-12p40−/− orIFN-γ−/− strains. These mice develop much milder disease [91].

4.2.2. CD45RBHi transfer modelCD4+CD45RBHi T cells isolated via fluorescence activated

cell sorting from the spleens of donor mice transferred tosyngenic immunodeficient SCID or RAG1/2−/− recipient micecause a wasting syndrome with transmural intestinal inflam-mation primarily in the colon starting (depending on the local

bacterial flora of the animal facility) 5–10weeks after cell transfer[92–95]. Recipientmice repopulatedwith the CD4+CD45RBLo Tcell subset or both populations do not develop colitis, althoughthese cells also colonize the host gut. CD25+FoxP3+ cells withinthe CD4+CD45RBLo population account for the prevention ofcolitis since depletion of CD25+ cells from the CD45RBlo cellsabrogates their colitis prevention potential [95]. Thus, adoptivetransfer of CD4+CD25- T cells is also a suitable method toinduce experimental colitis. This approach has the advantagesthat no flow-cytometric sorting is required and that diseasestarts much earlier than in the classical CD4+CD45RBHi model[96]. Several studies identified IL-10 and TGF-β as central anti-inflammatory factors in this model. Regulatory T cells (Tr1cells) which produce mainly IL-10 due to co culture with IL-10,prevent onset of gut inflammation and antigen-specific immuneresponses when transferred together with pathogenic CD4+-

CD45RBHi T cells as systemic administration of recombinantIL-10 or TGF-β do. The role of IL-10 in this model was furtheremphasized by the fact that SCID mice administered bothCD45RBhi and regulatory T cells together with anti-IL-10receptor antibodies develop colitis [97]. As in many othermodels of experimental colitis, bacterial antigens play a crucialrole for pathology since treatment with antibiotics or germ-freebreeding of recipient SCID mice is associated with significantlyless severe bowel inflammation.

4.2.3. STAT4 transgenic miceSTAT4 is a regulatory transcription factor specifically

associated with IL-12/IL-23 receptor signaling [98]. Mice overexpressing STAT4 under control of a cytomegaloviruspromoter system, which express highly elevated nuclearSTAT4 levels in spleen and lamina propria CD4+ T cellsafter systemic administration of the antigenic stimulus DNP-KLH, have been shown to develop severe transmural colitis[99]. Infiltrating lamina propria CD4+ T cells from these miceproduce after stimulation with αCD3/αCD28 in vivo and invitro predominantly TNF-α and IFN-γ, but not IL-4,consistent with a TH1-type cell response. This demonstratesthat an abnormal activation of the TH1 effector pathway can besufficient to destroy the mucosal immune balance. Thissuggestion is supported by the finding that colitis in thesemice can be adoptively transferred to immunocompromizedSCID mice by CD4+ T cells that have been primed withvantigens from the autologous bacterial flora. Further support forthe pivotal role of STAT4 dependent TH1 responses for colitisdevelopment have been provided in the BMC→Cd3ɛTg26 andthe CD45RBHi models where cells from STAT4 deficient miceonly induced very mild disease [100].

4.2.4. IL-10/CRF2-4 deficient miceIL-10 is a well-known suppressor of TH1 cells and

macrophage effectors functions. Several, in vitro studieshave shown that IL-10 inhibits IL-12 and TNF-α production,suppresses costimulatory B.7.1 and B.7.2 molecule expres-sion, inhibits T cell proliferation and may also promote theformation of antigen-specific regulatory T cells [83]. Micewith targeted deletion of the IL-10 gene spontaneously

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develop chronic enterocolitis with massive infiltration oflymphocytes, activated macrophages, and neutrophils [101].The disease is accompanied by a TH1 cytokine response,which can be ameliorated by neutralizing antibodies to IL-12p40 and to a lesser extent IFN-γ or systemic administra-tion of recombinant IL-10 [102]. In a further study the samegroup observed a similar intestinal phenotype in genetargeted mice with a T cell specific IL-10 deficiency [103].These data highlight the importance of T cell derived IL-10in the regulation of mucosal T cell responses, whereas IL-10expression from other cell types seems to be more importantin the context of acute septic shock. IBD like disease is alsopresent in mice deficient for CRF2-4, the IL-10 receptor betachain, and in mice with myeloid cell specific STAT3 deficiencythat reveal a defect in IL-10 signaling [104,105]. As in manyother models, the colitis in IL-10−/− mice does not develop ingerm-free bred mice and is thus driven by antigens of themucosal microflora. Interestingly, increased intestinal perme-ability prior to the development of established colitis has beendescribed in IL-10−/− mice. Although administration ofrecombinant IL-10 or IL-10 gene therapy proved therapeuticefficacy in several animal models [106–108] of colitissystemic injection of recombinant IL-10 showed in phase IIIclinical trials only a modest potency in patients with activeCD and surprisingly at high doses, IL-10 even induced theproduction of the proinflammatory cytokine IFN-γ [109,110].It has been speculated that the lack of clinical efficacy mayresult from limited bioavailability of IL-10 in the intestinalmucosa. Therefore alternative approaches for localized IL-10delivery e.g. by ex vivo retrovirus based gene therapy or bythe use of IL-10 secreting transgenic bacteria are underconsideration [111–113].

4.2.5. TCRα chain−/− miceMice deficient for the TCR-α chain (TCR-α−/−) spontaneously

develop mucosal inflammation at 12–16 weeks of age with somecharacteristics similar to UC in humans [114]. In contrast, TCR-βchain−/− exhibit only mild disease, whereas TCR-γδ−/− do notdevelop spontaneous disease [115]. Colitis in TCR-α−/− mice isassociated with increased numbers of aberrant TH2- type CD4+-

TCRα–β+ T cells producing predominantly IL-4 and non-T cellsproducing IFN-γ. However, these elevated IFN-γ levels are notcritical for development of colitis because TCR-α/IFN-γ double-knockout mice display similar pathology as TCR-α−/− mice. Incontrast, both anti-IL-4 neutralizing antibody treated TCR-α−/−

mice and TCR-α−/−/IL-4−/− mice exhibited no or much milderclinical or histological signs of inflammation, indicating thepredominance of a pathological TH2 type immune response in thegastrointestinal tract of these mice. TCR-α deficient micemaintained germ free or colonized with a limited number ofdefined intestinal bacteria do not develop intestinal inflamma-tion. Although a pathophysiological role of TH2 effector me-chanisms remains unclear in IBD patients, the polarized TH2responses, and disease amelioration by anti-IL-4 strategies inthe inflamed colon of TCR-α−/− mice demonstrate thatintestinal inflammation can be associated with TH2 mediatedpathology.

5. Which model to choose

As more and more sophisticated IBD models becomeavailable researchers can exploit the unique potential of eachmodel to ask specific questions. Although no single animalmodel recapitulates all of the pathogenic and clinical features ofhuman IBD, each animal model contributed to our improvedunderstanding of the mechanisms underlying initiation andperpetuation of chronic intestinal inflammation. In the majorityof the models only the large bowel rather than the smallintestine – the location of preferential clinical manifestation andimportant characteristics of CD – is affected. However, in mostof these models, as in patients with CD, TH1/TH17 type T cellsactivated by IL-12/IL-23 can be found in the inflammatorylesions, where they are suggested to drive local inflammatoryresponses by producing IFN-γ, TNF-α and IL-17. At present itis not clear why many models do not develop ileitis and in someaspects these models may have limited value for investigatingthe precise etiopathogenesis of CD in the terminal ileum.Probably the best mouse model resembling Crohn's ileitis is theSAMP1/YitFc because inflammation is most severe in theterminal ileum and as in humans the phenotype developsspontaneously without exogenous manipulation. Unfortunately,the onset and severity of disease in most of the spontaneousgene targeted or transgenic models are highly variable andgenerally depend largely on environmental factors such as theenteric flora of the animal facility. In some models (e.g. IL-10−/−

mice) it takes several months for manifestation of colitis makingthem unsuitable for large scale drug screening studies. Thereforefor the purpose of evaluating novel therapeutic agentsexperimentally induced acute colitis models (e.g. DSS- orTNBS colitis) are widely used although they resemble only insome aspects human disease. These inducible models offer theadditional advantage of cost-efficiency, high reproducibility andthey can be used for characterization of novel gene targetedmouse strains. However, in almost all of the inducible andspontaneous models the inbred strain background is of greatimportance and has to be taken into consideration in theexperimental design of the studies.

6. Conclusion

Studies with animal models have improved our understand-ing of the complex field of human IBD and allowed themolecular dissection of pathophysiological mechanisms that arepresumably responsible for disease initiation and progression.There is now convincing evidence supporting a concept that ingenetically susceptible hosts aberrant immune responses andloss of tolerance to environmental factors are major factorscontributing to the mucosal inflammation. Functional defects inthe epithelial component of the barrier are believed to allowinappropriate access of bacterial and other microbial antigens tothe lamina propria. In the healthy homeostatic gut commensalsencountering the lamina propria would mediate a poorinflammatory response due to induction of tolerance. In IBD,however, defects in innate and/or adaptive immune cells finallyresult in a non self-limiting chronic inflammation. Although the

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etiology of IBD is still unclear, promising biological therapeuticstrategies (e.g. blocking IL-12/IL-23 in CD) on the basis of thisimproved mechanistic understanding of the gut immune systemare emerging.

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