Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues.

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Review

Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues Vassilis Valatas a,c,n, Giorgos Bamias b,c, George Kolios b,c a

Laboratory of Gastroenterology, Faculty of Medicine, University of Crete, Greece Academic Department of Gastroenterology, Laikon Hospital, Kapodistriakon University of Athens, Athens c Laboratory of Pharmacology, School of Medicine, Democritus University of Thrace, Alexandroupolis, Greece b

art ic l e i nf o

a b s t r a c t

Article history: Received 19 October 2014 Received in revised form 3 February 2015 Accepted 12 March 2015

Inflammatory bowel diseases, ulcerative colitis and Crohn's disease are characterized by chronic relapsing inflammation of the gastrointestinal tract of unknown etiology that seems to be the consequence of a genetically driven dysregulated immune response against various local and environmental triggers through a defective epithelial barrier. During the last decades, a large number of animal experimental models of intestinal inflammation have been generated and provided valuable insights into the mechanisms that either maintain mucosal homeostasis or drive intestinal inflammation. Their study enabled the identification of various treatment targets and the development a large pipeline of new drugs, mostly biologics. Safety and therapeutic efficacy of these agents have been evaluated in a large number of clinical trials but only a minority has reached the clinic so far. Translational successes but mostly translational failures have prompted to re-evaluate results of efficacy and safety generated by pre-clinical testing and to re-examine the way to interpret experimental in vivo data. This review examines the contribution of the most popular experimental colitis models to our understanding of the pathogenesis of human inflammatory bowel diseases and their translational input in drug development and discusses ways to improve translational outcome. & 2015 Published by Elsevier B.V.

Keywords: Inflammatory bowel disease Crohn's disease Ulcerative colitis Experimental colitis Animal models Translation

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental colitis models with epithelial barrier defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental colitis models with innate immunity defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mice with NOD and autophagy defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Experimental models of colitis characterized by aberrant adaptive T-cell responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Excessive effector cell responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Regulatory and effector T cell imbalance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Findings in Human IBD and translational outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Strategies to increase the translational value of experimental colitis models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

n Correspondence to: Department of Gastroenterology, University Hospital of Heraklion, PO Box 1352, Voutes, Heraklion GR-71100, Crete, Greece. Tel.: þ 30 2810392356; fax: þ30 2810542085. E-mail addresses: [email protected] (V. Valatas), [email protected] (G. Bamias), [email protected] (G. Kolios).

Inflammatory bowel disease (IBD) comprises of two separate clinical entities ulcerative colitis (UC) and Crohn's disease (CD). Both are characterized by chronic relapsing inflammation of the gastrointestinal tract of unknown etiology. The principal hypothesis for their pathogenesis is that intestinal inflammation represents the end result of a genetically driven dysregulated immune response against antigens of the intestinal microflora though a

http://dx.doi.org/10.1016/j.ejphar.2015.03.017 0014-2999/& 2015 Published by Elsevier B.V.

Please cite this article as: Valatas, V., et al., Experimental colitis models: Insights into the pathogenesis of inflammatory bowel disease and translational issues. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.03.017i

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Table 1 Experimental models of IBD. Epithelial barrier defects DSS (Okayasu et al., 1990) N-cadherin dominant negative (Hermiston and Gordon, 1995) Muc1/2 / (Nishida et al., 2012; Van der Sluis et al., 2006) Mdr1a / (Panwala et al., 1998)

Aberrant adaptive T cell responses Excessive effector cell responses TNBSa (Neurath et al., 1995) Oxazolonea (Boirivant et al., 1998) TNFΔARE (Kontoyiannis et al., 1999)

Innate immunity defects A20 / (Lee et al., 2000) STAT3 / (Takeda et al., 1999) CD40 mAb-RAG / (Uhlig et al., 2006) Helicobacter hepaticus-SCID/RAG / (Li et al., 1998) IEC/IKK-γ / (Nenci et al., 2007) TRUC (Garrett et al., 2007)

STAT4 tg (Wirtz et al., 1999) TCRα / (Mizoguchi et al., 1996) Regulatory and effector T cell imbalance CD45RBhigh adoptive transfer (Powrie et al., 1993) IL-2 / (Willerford et al., 1995) TGFβ1 / (Shull et al., 1992) IL-10 / (Berg et al., 1996)

Spontaneous colitis models C3H/HeJBir (Mahler et al., 1998) SAMP1/Yit(Fc) (Kosiewicz et al., 2001)

Gαi2 / (Rudolph et al., 1995) Tgε26 (Hollander et al., 1995) TGFβRII DN (Gorelik and Flavell, 2000)

a Can also be categorized to experimental colitis associated with epithelial barrier defects as disruption of the epithelial barrier with ethanol is necessary for the development of intestinal inflammation.

Fig. 1. Experimental colitis models with epithelial barrier and innate immunity defects. Chemical disruption of the epithelial barrier, deletion of genes related to the formation of adherent junctions and inadequate production of mucin, defensins and lysozyme by the epithelium result to bacterial invasion. Loss of immunoregulation, aberrant microbial sensing by pattern recognition receptors and poor microbial handling due to defective autophagy lead to up-regulation of pro-inflammatory mediators by innate immune cells and chronic intestinal inflammation. APC: Antigen Presenting Cell, Atg16l1: Autophagy related 16l1, DN: Dominant negative, DSS: Dextran Sulfate Sodium, IEC: Intestinal Epithelial Cell, IFN-γ: Interferon-γ, Ikk-γ: IκB kinase-γ, IL-1β: Interleukin 1β, Mdr1a: Multiple Drug Resistance 1a, Nod2: Nucleotide-Binding Oligomerization Domain 2, Stat3: Signal Transducer and Activator of Transcription, TLR: Toll-like Receptor, TNF-α: Tumor Necrosis Factor-a.

defective epithelial barrier and under the influence of various environmental triggers.(Abraham and Cho, 2009) A significant amount of data to support this hypothesis has been generated by studies in experimental models of intestinal inflammation. Since the description of the first such model by Kirsner and Elchlepp (1957) almost 60 years ago, more than 50 different murine models have been generated by genetic engineering guided partly by human genome wide association studies in IBD (Kirsner and Elchlepp, 1957). In the following years, murine experimental colitis models have helped to identify the key elements of mucosal immune homeostasis such as the integrity of the epithelial barrier, the dynamic innate immune responses and the tight regulation of adaptive immune responses. The purpose of this review is to

present the most popular experimental colitis models (Table 1), to describe their contribution to our understanding of human IBD pathogenesis, and to discuss their translational implication for the treatment of the human condition.

2. Experimental colitis models with epithelial barrier defects There is accumulating evidence that epithelial barrier dysfunction participates in IBD pathogenesis (Fig. 1). Strong support for such a mechanism comes from animal models of experimental colitis as chemical disruption or genetic defects in epithelial barrier components result to intestinal inflammation. Administration of


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Table 2 Effectiveness of various successful treatment strategies for human IBD in most widely used experimental models. Drug/Target

Model

Outcome

References

Sulfasalazine or 5-ASA

TNBS Acute DSS Oxazolone IL-10 / TNBS Acute DSS Chronic DSS Oxazolone SAMP1/YitFc Acute and Chronic DSS Chronic DSS Acute DSS Acute DSS Chronic DSS Acute DSS TNBS IL-10 / SAMP1/YitFc TNBS IL-10 / Acute DSS Acute DSS Chronic DSS Adoptive Transfer SAMP1/YitFc SAMP1/YitFc Gαi2 /

Effective Effective Effective Effective Effective Ineffective Effective Effective Effective Effective Effective Effective Ineffective Effective Ineffective Effective Ineffective Effective Effective Effective Effective Ineffective Effective Effective Effective Ineffective Effective

(Selve and Wohrmann, 1992) (Axelsson et al., 1998; Gillberg et al., 2013) (Kojima et al., 2004) (Brown et al., 2010) (Daniel et al., 2008; Fiorucci et al., 2004; Yamamoto et al., 2013) (Kojouharoff et al., 1997; Yamamoto et al., 2013) (Kojouharoff et al., 1997) (Kojima et al., 2004) (Burns et al., 2001) (Gillberg et al., 2013; Kverka et al., 2011) (Kverka et al., 2011) (Gillberg et al., 2013) (Melgar et al., 2008) (Kojouharoff et al., 1997) (Kojouharoff et al., 1997) (Neurath et al., 1997) (Kullberg et al., 2001; Rennick et al., 1997) (Marini et al., 2003) (Davidson et al., 1998) (Davidson et al., 1998) (Kato et al., 2000) (Soriano et al., 2000) (Farkas et al., 2006; Teramoto et al., 2005) (Picarella et al., 1997) (Matsuzaki et al., 2005) (Rivera-Nieves et al., 2005) (Bjursten et al., 2005)

Prednisolone or Dexamethasone

Thiopurines Methotrexate TNF-α blockade

IL-12/23 (p40) blockade MAdCAM- 1/ a4β7 blockade

For all studies with antibody blockade the outcome refers to treatment of established disease.

the chelating agent dextran sulfate sodium (DSS) in the drinking water of mice causes acute colonic inflammation manifested by weight loss, diarrhea, and rectal bleeding (Okayasu et al., 1990). Acute DSS colitis shares several pathological features with ulcerative colitis including more severe distal colonic involvement, mucin depletion, epithelial degeneration, neutrophilic infiltration of the mucosa resulting to crypt abscess formation and superficial mucosal erosions. Loss of zonula occludens due to chelation of divalent cations (e.g., Ca þ þ , Mg þ þ ) by dextran sulfate sodium causes increased epithelial permeability that precedes development of intestinal inflammation (Poritz et al., 2007). The inflammatory cascade launched by acute DSS-injury includes upregulation of various cytokines involved in innate immunity and T helper (Th)1 adaptive responses, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, IL-10, and IL-12. Repeated administration of lower doses of dextran sulfate sodium for longer periods generates chronic intestinal inflammation with mixed Th1 and Th2-associated inflammatory responses (Alex et al., 2009). Technically, the DSS colitis model is characterized by a highly reproducible phenotype, a short and stable time frame for disease development, and minimal technical requirements. The self-limiting nature of the disease together with the ability to produce acute or chronic colitis depending on the protocol used allows for the study of mechanisms of epithelial regeneration and fibrosis. These features have rendered DSS colitis a popular model to use for the preclinical evaluation of the efficacy of novel therapeutic strategies in IBD (Table 2). However its chemical nature, its self-limited course and its relative independence of the presence of conventional intestinal microflora represent important differences from human IBD that have to be taken into account. In all, acute DSS colitis is more suitable for the study of the events that follow the temporary failure of mucosal homeostasis after epithelial destruction and loss of barrier integrity as well as of the mechanisms that lead to mucosal healing after initial injury. The relevance of this model to the chronic recurring inflammation that is characteristic of IBD remains questionable.

N-cadherin is a transmembrane protein that plays important role in cell adhesion through the formation of adherens junctions in a calcium-dependent manner (Radice, 2013). Mice expressing a dominant negative N-cadherin mutant protein under the control of the I-FABP promoter active in small intestinal epithelial cells developed small intestinal inflammation with patchy distribution reminiscent of CD (Hermiston and Gordon, 1995). Other phenotypic similarities between N-cadherin dominant negative mice and CD include the transmural involvement and development of aphthoid ulcers. The model is technically demanding and probably this is the reason that it has not been used since its development. However, it provides evidence that a defective epithelial barrier can drive inappropriate intestinal inflammatory responses. This concept is further supported by studies in mice with conditional deletion of p120-catenin, a regulator of cadherin stability, that rapidly develop lethal intestinal inflammation (Smalley-Freed et al., 2010). Another integral part of the intestinal barrier is surface mucus. It is composed mostly of gel-forming mucins which are large, highly glycosylated proteins produced by goblet cells. Aside from its lubricating function the inner intestinal mucus layer serves as an immunological barrier impermeable to microorganisms and enriched with a variety of anti-microbial peptides and secretory IgA (Johansson et al., 2013). The importance of mucus integrity for intestinal homeostasis is highlighted by the development of intestinal inflammation in Muc2 / mice which have defective mucin production. Colitis in this strain is phenotypically similar to UC (Van der Sluis et al., 2006). The murine multiple drug resistance 1 a (mdr1a) gene, participates in the regulation of epithelial transcellular permeability. Mdr1a / mice, develop spontaneous, flora-dependent colonic inflammation reminiscent of UC (Panwala et al., 1998). Inflammation has been attributed to increased epithelial permeability accompanied by decreased phosphorylation of tight junction proteins and increased bacterial translocation, as well as significant transcriptomic changes of intestinal enterocytes associated


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with higher basal chemokine secretion and increased responsiveness to bacterial lipopolysaccharide (Collett et al., 2008). Animal studies are further corroborated by genome wide association studies reporting IBD-susceptibility genes that encode for proteins involved in adherent junction and tight junction assembly, epithelial transport and mucin production (Pastorelli et al., 2013). However, whether an intrinsic defect of the intestinal barrier is the primary abnormality underlying the pathogenesis of IBD remains unknown. Although increased intestinal permeability has been found in CD patients and unaffected relatives (Peeters et al., 1997), junctional molecules are only affected during active intestinal inflammation (Gassler et al., 2001). Furthermore, it is debated whether mucin reduction that is observed in patients with UC represents a causative defect, rather than an epiphenomenon secondary to mucosal inflammation (Einerhand et al., 2002). Nevertheless, the concept of reinforcing intestinal barrier as a therapeutic strategy in IBD has been successfully translated in the case of phosphatidylcholine administration in UC. Phosphatidylcholine is an essential component of protective colonic mucus and alterations of phospholipid content have been observed in UC patients (Ehehalt et al., 2010). The administration of phosphatidylcholine has been found safe and induced significant clinical responses in patients with active UC including patients with inadequate response to mesalazine (Karner et al., 2014).

3. Experimental colitis models with innate immunity defects The development of colitis in mice devoid of B cells and T cells has proved that adaptive immunity is not an absolute prerequisite for intestinal inflammation. In fact, the presence of innate immunity defects alone is sufficient to compromise intestinal homeostasis. Such “innate” models of experimental colitis can be generated using nude mice (severe combined immunodeficiency-SCID or recombination-activation gene-RAG deficient mice), either by their colonization with Helicobacter species or via administration of an agonistic anti-CD40 antibody (Li et al., 1998; Uhlig et al., 2006). These models have provided evidence for the significant contribution of IL-23 in the innate mechanisms of IBD pathogenesis (Hue et al., 2006; Uhlig et al., 2006). Defects of immunoregulation in myeloid cells also result in colitis (Fig. 1). Conditional deletion of the signal transducer and activator of transcription 3 (STAT3) gene in neutrophils and macrophages in mice resulted in TLR-dependent, enhanced IL-12p40 production due to loss of the counter-regulatory effects of IL-10 (Kobayashi et al., 2003; Takeda et al., 1999). Disruption of the gene encoding for A20, a cytoplasmic protein inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), resulted in failure to terminate TNF-induced NFκB-mediated responses (Lee et al., 2000). Loss of immunoregulation also occurs by ablation of T-bet, a transcription factor that mediates Th1 responses, in recombination-activation gene 2 (RAG2) deficient mice. T-bet / X RAG2 / (TRUC) mice develop a UC-like severe colitis due to TNF-α over-production by colonic dendritic cells (Garrett et al., 2007). Interestingly, colitis is flora-dependent and communicable via fecal transplantation to wild type and RAG2 / mice, indicating that innate immunity defects can drive the selection of a colitogenic flora. Increased intestinal permeability has been detected in TRUC mice before the onset of inflammation, which makes TRUC mice a valuable model to study the relationship between innate immunity defect, mucosal barrier dysfunction and dysbiosis. The epithelium represents the frontline of innate defense mechanisms of the mucosal immune system. The recognition of pathogenassociated molecular patterns through Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NODs) receptors and subsequent NF-κB activation in epithelial cells activates a

variety of defense mechanisms such as release of anti-microbial peptides, secretion of proinflammatory cytokines and the production of trophic factors that limit bacterial penetration and maintain an intact epithelial barrier (Pastorelli et al., 2013). Mice that lack TLR-2 and TLR-4 receptors or TLR-signaling molecules (myeloid differentiation factor 88, MyD88 / ) have been found more susceptible to DSS colitis (Rakoff-Nahoum et al., 2004). Furthermore, shutdown of NFκB signaling in intestinal epithelial (IEC) cells by conditional deletion of NFκB essential modulator (NEMO), also called IκB kinase (IKK)-γ, resulted in a chronic cecum-sparing colitis. Defective TLR signaling contributed to the pathogenesis as crossing of the IKK-γ / mice to MyD88 / mice prevented colitis development. IEC/IKK-γ / mice also exhibited increased TNF-α-mediated epithelial cell apoptosis and impaired expression of defensins that resulted to increased bacterial translocation (Nenci et al., 2007). The C3H/HeJBir model is another model that highlights the pivotal role of abnormal innate responses and TLR signaling in the development of experimental intestinal inflammation (Mahler et al., 1998). The C3H/HeJ mice spontaneously develop selflimiting colitis of the cecum and proximal colon (Sundberg et al., 1994). Inflammation develops due to aberrant B-cell and Th1 T-cell responses triggered by a limited number of bacterial antigens (Fig. 1). Flagellins have been identified as the dominant antigens in C3H/HeJBir mice, and serum IgG antibody responses against flagellin epitopes have been also identified in a significant proportion of CD patients (Lodes et al., 2004). Defective TLR5 signaling, the receptor for the recognition of flagellins, has been proposed as the underlying abnormality (Beckwith et al., 2005).

3.1. Mice with NOD and autophagy defects Innate immunity defects merge in the case of NucleotideBinding Oligomerization Domain 2 (NOD2) and autophagy related genes. NOD2 is an intra-cellular sensor of muramyl dipeptide (MDP), a component of bacterial cell wall. MDP recognition by NOD2 leads to NF-κB and mitogen-activated protein kinase activation which then results to the up-regulation of various proinflammatory genes. Genetic variants of the NOD2/CARD15 gene are frequent among patients of European ancestry and polymorphisms in this gene confer the highest relative risk for developing CD, among all currently known genetic associations (Hugot et al., 2001). Three murine strains with functional NOD2 defects have been generated so far. Two of them are NOD2deficient mice and one (Nod22939insC) is transgenic and express the murine homolog of the human NOD23020insC mutated allele that has been associated with human CD (Maeda et al., 2005). Although none of these mice develop colitis spontaneously, their study has provides valuable information for the various functions of NOD2 (Mizoguchi and Mizoguchi, 2008). These studies have associated NOD2 polymorphisms or deletion with inadequate innate immune responses. These include reduced defensin production by small intestinal Paneth cells (Kobayashi et al., 2005), defective autophagic responses to invasive bacteria (Cooney et al., 2010), loss of TCRγδ þ and CD8αα þ TCRαβ þ intraepithelial lymphocytes (Jiang et al., 2013) and dysbiotic microflora (CouturierMaillard et al., 2013). Another set of mechanisms proposed by animal studies to be involved in colitis pathogenesis refers to defective down-regulation of immune responses. These include loss of NOD2-mediated regulation of TLR-2 signaling in dendritic cells (Watanabe et al., 2004), loss of the control of IL-12p40 release from dendritic cells (Brain et al., 2013) and up-regulation of IL-1β production by macrophages (Fig. 1) (Maeda et al., 2005). Among the autophagy-related genes implicated in the pathogenesis of IBD, atg16l1 is the most prominent. Chimeric mice lacking ATG16L1 in haematopoietic cells were highly susceptible


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to DSS colitis, partly due to exacerbated production of IL-1β and IL18 by ATG16L1-deficient macrophages (Saitoh et al., 2008). Paneth cells of mice that are hypomorphic for ATG16L1 protein expression (ATG16L1HM), displayed striking structural abnormalities and decreased lysozyme secretion to the ileal lumen. These abnormalities were coupled with transcriptional up-regulation of genes involved in peroxisome proliferator-activated receptor pathways, adipocytokine signaling, lipid metabolism and the acute phase reaction (Cadwell et al., 2008). Exposure of these mice to DSS resulted in pathology resembling human CD, that included transmural inflammation, subserosal fibrosis and ileal involvement as opposed to the UC-like phenotype produced with the wild-type mice (Cadwell et al., 2010). Investigation of the functional consequences of autophagy-related gene polymorphisms in the context of experimental colitis has provided evidence that defective macroautophagy, can result in inefficient innate immunity and intensified pro-inflammatory responses that disturb mucosal homeostasis (Fig. 1). The existence of such a vicious cycle where incomplete bacterial clearance due to NOD2 and autophagy defects triggers and sustains aberrant inflammatory responses has been speculated by studies in CD patients. Impaired bacterial handling has been suggested by early studies that found high concentration of bacteria attached to intestinal mucosa, and multiple bacterial inclusions within solitary enterocytes of CD patients irrespective of the presence of active inflammation (Swidsinski et al., 2002). Reduced α-defensin secretion has been reported for human CD but it occurred independently of NOD2 status (Simms et al., 2008). Paneth cells from patients with CD who are homozygous for the ATG16L1 risk allele displayed comparable pathology to that reported for the ATG16L1HM mice (Cadwell et al., 2008). Similar, but less severe abnormalities have been reported for Paneth cells from CD patients independently of disease-associated variants of ATG16L1 (Thachil et al., 2012). Moreover, peripheral blood mononuclear cells and dendritic cells from individuals expressing CDassociated NOD2 or ATG16L1variants exhibit increased proinflammatory cytokine production in response to NOD2 ligands and defective intracellullar bacterial killing, respectively (Cooney et al., 2010; Plantinga et al., 2011). However, the theory of defective autophagy has not yet been translated to treatment strategies. Sirolimus and everolimus are drugs approved for clinical use in solid organ transplantation to prevent graft rejection in the basis of their ability to block T- and B-cell activation (Tsang et al., 2007). These agents inhibit the mamalian target of rapamycin pathway, which is a major inhibitor of autophagy, hence they induce autophagy (Sudarsanam and Johnson, 2010). Both have been used successfully to treat refractory CD cases (Dumortier et al., 2008; Massey et al., 2008). In contrast, a proof-of-concept study that compared efficacy of everolimus to azathioprine in adults with moderate-to-severe active CD was prematurely terminated due to lack of efficacy (Reinisch et al., 2008).

4. Experimental models of colitis characterized by aberrant adaptive T-cell responses Mucosal T-cells bear a phenotype of activated effector cells. Dominant effector T-cell responses in intestinal mucosa, mainly Th1 and Th17, are pivotal for coordination of neutrophilic inflammation, restitution of epithelial barrier integrity, henceforth leading to symbiosis with commensal microbiota and effective control of invading pathogens. Tight regulation of such effector cell responses is required in order to maintain mucosal homeostasis. Animal studies have shown that loss of this control either by enhanced effector cell responses or by defective regulatory responses can induce intestinal inflammation.

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4.1. Excessive effector cell responses Haptenization of colonic proteins by treatment with the chemical Trinitrobenzene Sulfonic acid (TNBS) can drive CD4 þ effector T cell responses that mediate intestinal inflammation. Intrarectal administration of TNBS, in combination with ethanol to disrupt the epithelial barrier, induces colitis of variable severity depending on the mouse strain, the concentration of TNBS and the administration protocol. In the “classic” model, a single administration of TNBS to BALB/c and SJL/J mice produces a CD-like acute self-limited colitis characterized by transmural granulocytic and lymphocytic infiltrates and dominant Th1 responses (Neurath et al., 1995). Repetitive administration of lower doses induced a sequential Th1/Th17 cytokine pattern followed by Th2-associated cytokines latter on in the disease course that drives the development of fibrosis in some strains (Alex et al., 2009; Fichtner-Feigl et al., 2007). Therefore, this model is considered valuable for studying epithelial restitution and proliferation as well as mechanisms of T-cell mediated autoimmune intestinal inflammation during different stages of disease evolution. Similarly to the DSS colitis model it has been widely used for the preclinical evaluation of various treatment strategies for human IBD (Table 2). Oxazolone is another haptenizating agent that is administered rectally in combination with ethanol and causes left-sided colitis with high mortality. The acute colitis phase is characterized by destruction of epithelial cells resulting in ulcerated mucosa and dense infiltrates by lymphocytes and granulocytes. The model has many similarities to UC including goblet cell depletion, lack of transmural inflammation and the predominance of Th2-associated cytokines. Most importantly, inflammation in this model is mediated by T cells and natural killer T cells through secretion of IL-4 and IL-13. Colitis is prevented by treatment with anti-IL-4 neutralizing antibodies, or the decoy protein IL-13Rα2-Fc (that binds IL-13), IL-13 deficiency in CD4 T cells, and natural killer T cell depletion (Boirivant et al., 1998; Heller et al., 2002; Hoving et al., 2012). Τhe mice that survive the acute phase exhibit a self-limited disease course as the acute inflammation is counterbalanced by significant up-regulation of transforming growth factor (TGF)-β (Boirivant et al., 1998). Genetic modifications that drive overexpression of Th1 effector cytokines in mice result to intestinal inflammation with features relating to human CD. In the TNFΔARE mice deletion of AU-rich elements (AREs) in the 30 UTR untranslated region of TNF mRNA increases TNF-α mRNA stability and protein production. Mice develop chronic ileitis and proximal colitis with CD-associated features such as transmural inflammation and granulomas. An interesting feature is the development of extra-intestinal pathology such as arthritis and alopecia (Kontoyiannis et al., 1999). Inflammation in this model is mediated by CD8 T cells and monocytes and depends on Th1-asociated cytokines as crossing mice to IL-12p40 / or IFN-γ / strains ameliorated disease (Kontoyiannis et al., 2002). The TNFΔARE model has been useful for the study of the effects of TNF-α overproduction to various elements of the mucosal immune system. Restricting TNF overexpression in myeloid cells, T lymphocytes or intestinal epithelial cells has been found sufficient for the induction of intestinal pathology (Roulis et al., 2011). Recently, the iTNFΔARE strain was also reported which expressed the ΔARE deletion only in epithelial cells. Overproduction of TNF by epithelial cells was sufficient to induce intestinal inflammation; nevertheless these mice did not demonstrate extraintestinal manifestations (Bamias et al., 2013). STAT4 overexpression in CD4 þ T cells also produced a CD-like, chronic, transmural ileitis and colitis. Loss of STAT4 transcriptional regulation of IL-12/IL-23 receptor signaling in STAT4tg mice resulted in intestinal pathology mediated by enhanced Th1 responses against intestinal microflora (Wirtz et al., 1999). In contrast,


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excessive Th2 responses can induce experimental intestinal inflammation with features of human UC. Lack of T cell receptor (TCR)-α chains in the TCRα / mice, results in a population of T cells with restricted diversity whose TCR receptors are formed by β chains only. CD4 þ ββ T cells produce increased amounts of IL-4 that induces a continuous, superficial Th2-mediated colitis resembling human UC. Increased IL-4 is responsible for the production of autoantibodies (anti-tropomyosin antibodies and anti-neutrophil cytoplasmic antibodies) by B cells that are detected in TCRα / mice albeit with no pathologic significance (Mizoguchi et al., 1996).

4.2. Regulatory and effector T cell imbalance Defective immunoregulation leading to excessive effector T cell responses is the primary mechanism leading to inflammation in a variety of animal models of colitis. Regulatory CD4 þ CD25 þ T cells, expressing the transcriptional repressor FOXP3 and Tr1 CD4 þ T cells producing IL-10 are the most prominent among the various T cells destined to restrain aberrant T cell activation in the periphery (Barnes and Powrie, 2009). Colitis in the CD45RBhigh adoptive transfer model develops due to the relative absence of both. The model is generated by the transfer of small numbers of naive CD45RBhigh CD4 þ T cells, that do not contain regulatory T cells and Tr1 cells, in nude mice that are deficient of T and B lymphocytes (Asseman et al., 2000; Powrie et al., 1993). Inflammation develops within 6–8 weeks after transfer, following a combination of homeostatic expansion and polyclonal T cell activation in response to microbial antigens of the gut microflora. Colitis is characterized by transmural inflammation, ulcerations, loss of mucosal architecture and dense polymorphonuclear and mononuclear leukocyte infiltrations, features partly resembling CD. On the other hand, cotransfer of CD45RBlow cells, which act as regulatory lymphocytes, prevents adoptive transfer colitis induced by the effector CD45RBhigh population. Initial studies have classified the adoptive transfer model as IL12 dependent, Th1-mediated (Powrie et al., 1994). Subsequent studies however have identified a mixed Th1/Th17 response (Hue et al., 2006; Kullberg et al., 2006). Conceptually, the adoptive transfer model emphasizes the importance of integral immunoregulation and especially the role of regulatory T cells, for the maintenance of intestinal immune homeostasis. Moreover, using innate immune cells and T cells from different sources, it enables to study the differential effects of gene overexpression and/or deletion on T cells of the donor or/and innate immune cells of the recipient. Therefore, the adoptive transfer model has been extensively used for the study of various aspects of mucosal T cell activation and one of its major contributions was the recognition of the pivotal role of the IL-23-dependent Th17 pathway in intestinal inflammation (Hue et al., 2006; Kullberg et al., 2006). One of the reasons for the popularity of this model is that T cell activation and subsequent inflammation occurs in known time frames. This provides the opportunity to study events that occur early, during initiation of pathogenic T cell responses and prior to the onset of intestinal inflammation, such as the regulation of T cell priming in the mesenteric lymph nodes (Valatas et al., 2013a). There are a number of genetically modified mice that develop chronic intestinal inflammation through mechanisms that involve lack of regulatory T cells or defects in their function. Deletion of the IL-2 or IL-2 receptor a genes results to loss of regulatory T cells in the periphery, massive enlargement of peripheral lymphoid organs associated with T and B cell expansion and fatal autoimmune multiorgan inflammation, including colitis (Fontenot et al., 2005; Willerford et al., 1995). Similarly, deletion of TGF-β or defective TGF-β signaling on CD4 þ T cells results in loss of

autocrine regulation in innate immune cells, lack of regulatory T cells in the periphery and resistance of effector cells to TGFβmediated suppression by regulatory T cells. Accordingly, both TGFβ / and T cell–specific dominant-negative TGF-β receptor II mice develop multiorgan inflammation due to excessive effector T cell responses (Gorelik and Flavell, 2000; Shull et al., 1992). Disruption of the IL-10 gene has generated another widely used model of colitis. IL-10 is a pluripotent cytokine acting in various cells of innate and adaptive immunity to suppress inflammatory responses (Moore et al., 1993). IL-10 / mice develop a progressive, discontinuous, transmural inflammation that starts from the right colon and extends distally as disease progresses. Small intestinal lesions have been reported but less often. As the severity of disease worsens, a large number of animals also develop colorectal adenocarcinomas (Berg et al., 1996). Disease could be reproduced by transfer of CD4 þ T cells from IL-10 / mice to RAG / recipients suggesting that aberrant adaptive T cell responses due to defective IL-10 production by regulatory populations of CD4 T-helper cells is the principal mechanism of colitis development (Rennick et al., 1997). Similarly to the adoptive transfer model the type of pathogenic responses were originally characterized as Th1-related because disease could be significantly attenuated by neutralization of IFN-γ (Berg et al., 1996). Subsequent studies demonstrated enhanced Th17 responses and disease attenuation using anti-IL-17 and anti-IL-6 antibodies (Yen et al., 2006). Furthermore, disease could be prevented in IL-10 / x p19–/– but not in IL-10 / x p35–/– double knockout mice and only anti-p40 antibodies were found able to reverse established disease, suggesting a critical role of IL-23 signaling in disease development (Davidson et al., 1998; Yen et al., 2006). Collectively, the studies with the IL-10 / mice have highlighted the indispensable role of IL-10 in the regulation of pathogenic T cell responses initiated and maintained by IL-23 probably in response to ordinary enteral antigens. The gradual mode of disease progression that allows studies of early intervention, the ability to study individual components of IBD pathogenetic pathways by creating double knock-out mice combined with the fact that IL-10 / mice is one of the few models to be commercially available partly explain the popularity of this model. The Gαi2 / model is one of the most interesting experimental colitis models due to the multiplicity of immune pathways involved and the severe penetrating phenotype that develops (Rudolph et al., 1995). G protein–coupled receptors are seven transmembrane domain proteins that deliver intracellular signals affecting multiple biological pathways (Braun et al., 2000). It has been shown that Gi protein signaling plays a major role for in the regulation of proinflammatory cytokine production including IL-12 and IL-23 secretion from mouse dendritic cells and human monocytes (He et al., 2000). Furthermore, Gαi2 signaling plays a significant role in B cell development, and Gαi2 / mice exhibited reduction in multiple B cell populations. This was coupled with defective IL-10 production by B-cells in response to endotoxin or antigen challenge (Dalwadi et al., 2003; Ohman et al., 2005). Moreover, a relative resistance of Gαi2 / T cells to TGF-β- and regulatory T cellmediated suppression has been reported (Gotlind et al., 2011; Wu et al., 2005). Gαi2–/– mice on a 129 or a mixed B6/129 background develop severe transmural colitis with dense inflammatory infiltrates by lymphoytes, neutrophils, and plasma cells, destruction of mucosal architecture, crypt abscesses and loss of goblet cells. Inflammation is mediated by excessive Th1/ Th17 responses in combination with defective immunoregulation due to relative IL-10 insufficiency and resistance to regulatory T cell and B-cell populations. The drawback of this model is that it is demanding to maintain by crossbreeding of susceptible and resistant mice strains.


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The Tgε26 mouse model is associated with defects in intrathymic development of T cells resulting to regulatory and effector T cell imbalance. Tgε26 mice are transgenic for the human CD3ε gene. This results in very early arrest of T cell development which prevents the induction of a normal thymic microenvironment. BM transplantation from wild-type mice restores the T cell compartment but abnormal T cell development and selection in the thymus results in failure to negatively select autoreactive T cells and/or to positively select Tregs (Hollander et al., 1995; Veltkamp et al., 2005). Consequently, colitogenic CD4 þ T cells develop, which are characterized by increased expression and secretion of IFN-γ and TNF-α. Tgε26 mice develop chronic transmural colitis than can be rescued by transfer of CD4 þ CD25 þ regulatory T cells (Hollander et al., 1995; Veltkamp et al., 2005). 4.3. Findings in Human IBD and translational outcomes There has been accumulating evidence indicative of excessive effector T cell responses in human IBD. Specifically, up-regulated Th1/Th17 response has been proposed as a major pathogenic drive for CD (Siakavellas and Bamias, 2012). Direct evidence of the implication of both Th1 and Th17 have been provided by the study of Annunziato et al who showed that isolated lymphocytes from the lamina propria of CD patients produced IL-17 and exhibited features related to Th17 polarization that is selective expression of IL-23R, CCR6, and the transcription factor retinoic acid receptorrelated orphan nuclear receptor (ROR)-γt. Isolated lymphocytes also expressed T-bet and stimulation with IL-12 resulted to increased production of IFN-γ and down-regulation of ROR-γt and IL-17 (Annunziato et al., 2007). On the other hand, UC has been associated primarily with Th2-related pathways. Specifically, lamina propria mononuclear cells isolated from UC patients exhibited increased secretion of IL-13 upon stimulation than those from healthy controls or patients with CD. The critical cell population for IL-13 secretion was a “non-classical” natural killer T (NKT) cell expressing surface CD161 but lacking expression of an invariant T cell receptor (Fuss et al., 2004). Therefore, data from experimental colitis studies, showing that aberrant adaptive T cell responses of Th1/Th17 or Th2 type generated CD-like or UC-like phenotypes respectively, are complementary to T cell immunophenotypes that are detected in human IBD. This has created a dichotomy in the pathogenetic models for each disease, which, in turn, led to biological therapies that targeted specific effector cytokines for CD or UC. This concept has been challenged by the results of human trials of antibody therapies against signature effector cytokines of the Th1/Th17 or the Th2 response. Fontolizumab, an antibody directed against IFN-γ, was not able to induce statistically significant response over placebo in a phase II study in CD (Reinisch et al., 2010). Phase II trials of Secukinumab, an anti-IL-17 antibody, and AMG827, an anti-IL17 receptor antibody, both resulted in a disproportionate number of cases of worsening CD in subjects with active CD (Hueber et al., 2012; Targan et al., 2012). Similarly, a phase II clinical trial for tralokinumab, an anti-IL-13 antibody that has been developed primarily for asthma, has failed to meet its primary endpoint which was a statistically significant difference over placebo in clinical response at week 8 in patients with moderate to severe UC (Danese et al., 2014). There have been various suggestions attempting to explain these translational failures. In the case of IFN-γ and IL-17, the mutual antagonism reported between the two cytokines may have resulted to enhancement of Th17-induced inflammation by administration of anti- IFN-γ antibodies and stimulation of IFN-γ production by anti-IL-17 blocking antibodies (Harrington et al., 2005; Nakae et al., 2007). Additionally, data from animal models such as the SAMP/Yit that will be subsequently discussed, have demonstrated

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a shift of the immunophenotype of the pathogenic adaptive responses with disease evolution. Initial phases are characterized by high levels IFN-γ while established lesions are primarily associated with mixed responses (Bamias et al., 2005). Concerning the lack of effectiveness of IL-13 neutralization in UC there is yet no probable explanation, however some novel data suggest a critical role for activation of STAT6 by IL-13 in the resolution of experimental intestinal inflammation (Fichtner-Feigl et al., 2014). An alternative explanation would be that specific targeting of adaptive immune responses may not be adequate to restrain inflammation in human IBD. This argument is supported by the successful application of anti-TNF agents in the treatment of both CD and UC (Nielsen and Ainsworth, 2013). Although the primary proposed mechanism of action of anti-TNF-α agents in IBD is the induction of apoptosis of activated T-cell, TNF-α neutralization may restrain inflammatory responses from a variety of immune and stromal cell populations (Atreya et al., 2011). The ability of anti-TNF-α agents to induce regulatory populations of macrophages that inhibit proliferation of activated T cells, and produce anti-inflammatory cytokines is an example of the multiplicity of pathways, including innate immune mechanisms, which can be modulated by ΤNF-α blockage (Vos et al., 2011). Furthermore, phase II clinical trials in CD patients reported that administration of ustekinumab, an anti-p40 antibody that targets both IL-12 and IL-23, can induce significant clinical responses especially for patients that have failed anti-TNF-α therapy. Despite the relative absence of supporting evidence it can be speculated that the favorable outcome of anti-p40 administration may not only be related to the simultaneous targeting of Th1/Th17 adaptive responses but to additional inhibitory effects on IL-23R-dependent innate pathogenic pathways and pro-apoptotic effects on activated T lymphocytes (Buonocore et al., 2010; Fuss et al., 1999; Uhlig et al., 2006). Another hypothesis that has not yet been translated to human IBD is that defective immunoregulation contributes to disease pathogenesis. Although a number of animal models can be generated by single defects in immunoregulatory pathways, as previously described, human studies have failed to consistently identify defective immunoregulation for patients with IBD. Specifically, no functional or numerical defects of regulatory T cells have been detected in patients with CD or UC with the exception of inadequate expansion of intestinal regulatory T cell populations that has been reported during active disease (Maul et al., 2005; Saruta et al., 2007; Yu et al., 2007). However, a single open-label phase I/II study in a small number of patients with refractory CD that received autologous transplantation of in vitro expanded Tr1 regulatory T cell clones selected by specificity to ovalbumin showed promising results (Desreumaux et al., 2012). It is also possible that the immunoregulatory defects lie within the effector cell environment and not the regulatory T cells themselves. Enhanced resistance of effector cells to regulatory T cell-mediated suppression has been recently suggested by studies in IBD (Annunziato et al., 2007; Himmel et al., 2012). In the case of IL-10, genome-wide association studies have shown significant associations between poymorphisms of the IL-10 gene and IBD and especially UC (Vermeire et al., 2010). However, the majority of patients with UC and CD do not exhibit reduced mucosal expression of IL-10, with the exception of small subsets of patients including patients with CD that carry the NOD23020insC mutated allele (Akagi et al., 2000; Melgar et al., 2003; Noguchi et al., 2009; Schreiber et al., 1995). More importantly, the hypothesis that IL-10 supplementation might benefit patients with IBD, supported by results from animal studies, was not confirmed by clinical trials (Fedorak et al., 2000; Schreiber et al., 2000; van Deventer et al., 1997). Administration of recombinant human IL-10 (Tenovil) in patients with active CD did not


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result in overall significant clinical benefit over placebo as shown by a Cochrane meta-analysis (Buruiana et al., 2010). Attempts to increase IL-10 bioavailability by administering transgenic Lactococcus lactis that secrete human IL-10 showed no significant benefit in a phase II trial in active UC, whereas some promising results were reported in an initial open-label trial in CD (Braat et al., 2006; Vermeire et al., 2010). Although a benefit for subgroups of patients with low IL-10 levels cannot be excluded, the potential of IL-10 supplementation strategies for the treatment of active intestinal inflammation remains questionable (Marlow et al., 2013). The reported examples of inadequate translation do not devaluate the ability of animal models of IBD to predict success of therapeutic strategies in humans (Valatas et al., 2013b). Most of the aforementioned outcomes in clinical trials might have been predicted if data generated by animal studies were properly interpreted. Specifically, INF-γ blockage has been found to protect from the development of intestinal inflammation in many different models only when neutralizing antibodies were administered before the onset of inflammation (Bamias et al., 2005; Berg et al., 1996; Davidson et al., 1998; Obermeier et al., 1999; Powrie et al., 1994). In contrast, IFN-γ blockage has failed to ameliorate established inflammation when administered later in the course of experimental colitis, a situation that better simulates patients with active IBD participating in clinical trials (Berg et al., 1996; Davidson et al., 1998; Kullberg et al., 2001). Similarly, the modest therapeutic benefit of IL-10 supplementation in human IBD can be traced back to the relevant studies in animal models that reported limited efficacy of administration of IL-10 in established experimental colitis (Barbara et al., 2000; Berg et al., 1996). Moreover, recent studies with animal models of colitis using genetically modified mice that could not mount efficient IL-17 responses have shown the importance of IL-17 in the regulation rather than the propagation of experimental intestinal inflammation. Administration of IL-17 / or IL-17R / donor T cells to immunodeficient mice, elicited an accelerated, aggressive wasting disease in comparison with mice that receive wild-type T cells (O’Connor et al., 2009). IL-23p19 / mice were more susceptible to DSS and TNBSinduced colitis than wild type mice (Becker et al., 2006; Yang et al., 2008). Collectively these studies suggested a significant role of IL-17R signaling in the suppression of T-bet expression and the regulation of Th1 responses and have efficiently predicted the exacerbation of disease observed in subsequent clinical trials that tested the IL-17 or IL-17R neutralization antibodies in human IBD (Strober and Fuss, 2011). Finally, findings in another murine model of IBD, the SAMP1/ YitFc mouse, have highlighted the limitations of the Th1/Th2 dichotomy applied for the characterization of pathogenic effector T cell responses in both experimental and human IBD. The SAMP1/ YitFc mouse is a subline of the senescence accelerated mouse (SAM)P1 strain mouse, that spontaneously develop ileitis with many features similar to human CD. These include localization of the inflammation to terminal ileum, discontinuous and transmural bowel involvement, formation of granulomas, perianal disease and development of strictures (Rivera-Nieves et al., 2003). The primary immunological defects are largely unknown but epithelial barrier defects due to altered ileal expression of the tight junction proteins claudin-2 and occluding seem to participate in disease pathogenesis (Olson et al., 2006). Inflammation in this model has been initially characterized as Th1-related, with high levels of high mucosal levels of IFN-γ and TNF and responsive to TNFneutralization (Kosiewicz et al., 2001). However subsequent studies by Bamias et al that analyzed mucosal immunophenotype in the course of disease reported that Th1 and Th2 mechanisms participate in different stages of the natural history of ileitis with increased expression of IL-5 and IL-13 in later stages of the disease

as well as dominant Th2 polarization of lamina propria lymphocytes isolated from chronic lesions (Bamias et al., 2005). Similar shifts from Th1 to Th2 or from Th1 to Th1/Th17 mixed type of responses have been reported by studies on the IL-10 / , TNBS and DSS colitis models (Alex et al., 2009; Fichtner-Feigl et al., 2007; Spencer et al., 2002). Results from animal studies are further supported by a study of the immunophenotype of lymphocytes isolated from the lamina propria in children with CD. This study reported dominant Th1 responses characterized by enhanced IFN-γ secretion during the first attack of CD and mixed Th1/Th2 responses with additional increases of IL-4 and IL-10 secretion in long standing disease (Kugathasan et al., 2007). These observations may possibly explain some of the heterogeneity of clinical responses observed for IBD patients that participate in interventional clinical trials.

5. Strategies to increase the translational value of experimental colitis models There are numerous models of experimental intestinal inflammation that suffer from a variety of defects in many different components of the intestinal immune system. This suggests that multiple immunological pathways are involved in human IBD. This hypothesis is corroborated by genome wide association studies in IBD patients that identified more than 150 genetic risk loci so far associated with increased risk for IBD development (Khor et al., 2011). Experimental animal models can mimic various aspects of human IBD, and have been successfully applied for the preclinical evaluation of agents used in the treatment of IBD (Table 2). However, experimental colitis models cannot fully recapitulate IBD and this partly explains the small likehood of translation of interventional amimal studies into effective drugs in the clinic (Hackam and Redelmeier, 2006). There are vast differences between murine experimental colitis and human disease in terms of genetics. In contrast to the polygenic nature of human IBD, experimental IBD models are generated by deletion or transgenic overexpression of a single gene, which limits operating pathogenic pathways. Furthermore, genetic heterogeneity of IBD patients participating in interventional clinical trials cannot be compared to homogeneous populations of genetically identical mice. Accordingly, a single experimental colitis model with favorable outcome to an investigational drug should be regarded as a single responding patient. Therefore, an agent in test should be considered a promising candidate for human clinical trials only when successfully tested in multiple models of experimental colitis. In fact many strategies that have failed in clinical trials have been inadequately tested in pre-clinical animal studies (Valatas et al., 2013b; van der Worp et al., 2010). Obviously there are also vast differences between IBD and experimental colitis in the environmental influences that may interact with the mucosal immune system. The magnitude of this interaction has been underscored by concordance rates reported by studies in families and twins with IBD. The concordance rates for monozygotic twins has been estimated at between 20% and 50% for CD and about 16% for UC, indicating that the likelihood of developing IBD strongly depends on environmental influences (Halme et al., 2006). The complexity of interactions of gut microflora with the intestinal mucosal system is only recently beginning to be explored (Kamada et al., 2013). Although the mouse and human intestinal microbiota(s) are similar at the division level, with Firmicutes and Bacteroidetes dominating, most bacterial genera and species are different (Ley et al., 2005). Mice raised in germ-free environments and then colonized with specific microbial communities, referred to as gnotobiotic animals, provide alternative experimental systems to study the impact of different microbial communities in the development of experimental IBD.


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Furthermore, transplantation of adult human fecal microbiota into germ-free mice has been found to establish a relatively stable gut community in the recipient mice similar to that of the donor that can be partly transferred from generation to generation (Turnbaugh et al., 2009). Experimental colitis protocols with such humanized gnotobiotic mice fed a western diet may represent a valuable tool that better simulates IBD. Finally, participants in human IBD trials are heterogeneous patient populations, with different disease phenotypes and severity, in different stages of disease evolution and often resistant to multiple therapeutic agents. This situation can hardly be modeled in the pre-clinical settings. Still, preclinical studies have correctly predicted success or failure in human trials in the majority of cases were adequate data from multiple experimental colitis models have been generated. 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with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin Gastroenterol Hepatol 4, 754–759. Brain, O., Owens, B.M., Pichulik, T., Allan, P., Khatamzas, E., Leslie, A., Steevels, T., Sharma, S., Mayer, A., Catuneanu, A.M., Morton, V., Sun, M.Y., Jewell, D., Coccia, M., Harrison, O., Maloy, K., Schonefeldt, S., Bornschein, S., Liston, A., Simmons, A., 2013. The intracellular sensor NOD2 induces microRNA-29 expression in human dendritic cells to limit IL-23 release. Immunity 39, 521–536. Braun, M.C., Lahey, E., Kelsall, B.L., 2000. Selective suppression of IL-12 production by chemoattractants. J. Immunol. 164, 3009–3017. Brown, J.B., Lee, G., Managlia, E., Grimm, G.R., Dirisina, R., Goretsky, T., Cheresh, P., Blatner, N.R., Khazaie, K., Yang, G.Y., Li, L., Barrett, T.A., 2010. Mesalamine inhibits epithelial beta-catenin activation in chronic ulcerative colitis. Gastroenterology 138, 595–605 605.e591-593. 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