**Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease**

Dijana Detel1, Lara Batičić Pučar1, Ester Pernjak Pugel2, Natalia Kučić3, Sunčica Buljević1, Brankica Mijandrušić Sinčić4, Mladen Peršić5 and Jadranka Varljen1\* *School of Medicine, University of Rijeka Croatia* 

## **1. Introduction**

58 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

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Inflammatory bowel disease (IBD) comprises two main chronic pathologies of the gastrointestinal tract: ulcerative colitis (UC) and Crohn's disease (CD), both characterized by alternating phases of active inflammation and clinical remission with different complications and extraintestinal manifestations (Colletti, 2004; Hanauer & Hommes, 2010). The ethiopathogenesis of IBD has still not been elucidated, but it has been suggested that inflammatory processes emerge in genetically susceptible individuals as a result of an irregular, over-expressed immunological reaction to some undefined food antigens or some other agents of microbial origin (Baumgart et al., 2011).

Given the complexity of etiological factors in human IBD, a lot of current knowledge regarding IBD pathogenesis has arisen from the study of various animal models. Although no ideal model of IBD has been accomplished so far, they resemble different important clinical, histopathological and immunological aspects of human IBD (Mizoguchi & Mizoguchi, 2010). Chemically induced murine models by oral administration of dextran sulfate sodium (DSS) and intrarectal application of 2,4,6 trinitrobenzene sulfonic acid (TNBS) are the most commonly used ones, due to their onset and duration of colonic inflammation which is immediate, reproducible and shares a lot of similarities with human IBD. TNBS-induced colitis is one of the most accepted and used Crohn-like disease while the DSS-model is clinically and histologically similar to human ulcerative colitis (Wirtz & Neurath, 2007). These models, together with other animal models of IBD, have given insight in different processes at the molecular level and have revealed the importance of different molecules involved in IBD etiology, representing therefore essential tools in investigating different mechanisms underlying acute or chronic inflammation in the IBD (Uhlig & Powrie, 2009).

<sup>\*</sup> Corresponding Author

*<sup>1</sup>Department of Chemistry and Biochemistry,* 

*<sup>2</sup>Department of Histology and Embryology,* 

*<sup>3</sup>Department of Physiology and Immunology,* 

*<sup>4</sup>Department of Internal Medicine,* 

*<sup>5</sup>Department of Pediatrics*

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 61

Based on structural and biochemical features, DPP IV/CD26 is a member of a family of DPP IV activity and/or structure homologue (DASH) proteins, which also includes quiescent cell proline dipeptidase (QPP), DPP8, DPP9, fibroblast activation protein (FAP), attractin and DPP IV-β (Sedo & Malik, 2001). Since it is well known that most DASH proteins have protease activity, having the possibility to modify the activity of biologically active peptides, it could be suggested that they are important regulatory molecules (Gorrell, 2005). However,

DPP IV/CD26 is widely distributed in mammalian tissues, mainly on epithelial and endothelial cell surfaces, as well as on fibroblasts and lymphocytes (Boonacker & Van Noorden, 2003). The expression of DPP IV/CD26 on hematopoietic cells is well regulated according to the activation status. In humans, it is expressed on a fraction of resting lymphocytes at low density, but is strongly up-regulated following T-cell activation (Fleischer, 1987). In resting peripheral blood mononuclear cells, a small subpopulation of T cells expresses CD26 at high density on the surface (CD26-bright cells), which belongs to the CD45RO+ population of T cells (memory cells) (De Meester et al., 1999; Ishii et al., 2001). Moreover, CD26 expression on T cells may correlate with T-helper subsets. High expression is found on Th1

and Th0 cells, whereas Th2 cells display lower CD26 expression (Willheim et al., 1997).

regulation of many processes in human body (Aytac & Dang, 2004; Mentlein, 1999).

Immune regulation is a complex and important process in which DPP IV/CD26 as a costimulatory molecule in T-cell activation and a regulator of the functional effect of selected biological factors through its enzyme activity, certainly has an important function (Boonacker & Van Noorden, 2003). Furthermore, biochemical and immune studies provide evidence that CD26 interacts with many biologically important molecules including CD45, adenosine deaminase protein, chemokine receptor CXCR4 on the surface of human peripheral blood lymphocytes (Herrera et al., 2001) and the mannose-6-phosphate/insulinlike growth factor II receptor (Ikushima et al., 2000). The costimulatory properties of DPP IV/CD26 have been studied extensively, although different experimental settings sometimes provide conflicting results. It is generally accepted that several distinct anti-CD26 mAbs have costimulatory activities in anti-CD3-driven activation of pure T-cell subsets (either CD4+ or CD8+ T cells), and that the extent and kinetics of the response differs between mAbs, recognizing different epitopes. High CD26 surface expression is correlated with the production of Th1-type cytokines such as IFN-γ (Reinhold et al., 1997b).

Soluble DPP IV/CD26 activity was firstly discovered in the serum in 1968 by Nagatsu et al. (Nagatsu et al., 1968). Later, DPP IV/CD26 activity has been shown in other body fluids including plasma, serum, cerebrospinal and synovial fluids, semen and urine. Although soluble DPP IV/CD26 lacks the transmembrane domain and intracellular tail, due to glycosylations processes, its molecular weight is similar to the transmembrane form. The origin of the soluble DPP IV/CD26 is still not elucidated, but it was suggested that it could be released from the surface of all CD26 expressing cells in contact with blood by proteolytic cleavage (Gorrell et al., 2001). The physiological role of soluble DPP IV/CD26 in biological fluids with respect to the transmembrane DPP IV/CD26 remains poorly understood, but according to previous findings it has been proposed that, as an enzyme, it is involved in the

further research is necessary in order to clarify their biological role.

*Distribution and expression* 

*Soluble DPP IV/CD26* 

*Functions in immune regulations* 

Growing body of knowledge proposes proteases as key factors in the occurrence of inflammatory processes due to their ability to metabolize different biologically active molecules implicated in maintaining the integrity of mucosal barrier (Ravi et al., 2007). Dipeptidyl peptidase IV, known also as CD26 molecule (DPP IV/CD26) is one of them (Gorrell et al., 2001). DPP IV/CD26 is also T-cell differentiation antigen, expressed on various cell types, having numerous functions in a variety of biological processes, as well as immunological mechanisms (Fleischer, 1994). It is also present in a soluble form circulating in body fluids in living organisms with specific peptidase function having unique features in substrate processing: it cleaves dipeptides from the N terminus of polypeptides having proline or alanin at the penultimate position. Since Xaa-Pro peptides are not easily metabolized by other proteases, the action of DPP IV/CD26 is an essential step in the degradation of many polypeptides (Gorrell et al., 2001). Numerous biologically important cytokines, chemokines and neuropeptides with potential and/or confirmed role in IBD ethiopathogenesis are effective DPP IV/CD26 substrates (Mentlein, 2004).

Previous studies proposed a role of DPP IV/CD26 in the pathogenesis of IBD, given its involvement in immune regulations via its expression on immune cells and capability to cleave biologically active molecules (Hildebrandt et al., 2001; Varljen et al., 2005). Additionally, DPP IV/CD26 inhibitors have been pointed out as therapeutic agents in ameliorating inflammatory processes in immunologically mediated diseases such as IBD (Yazbeck et al., 2009; Yazbeck et al., 2008).

The aim of this study was to review our previously published results regarding correlation between disease severity and serum DPP IV/CD26 activity in young and adult patients affected with IBD. Furthermore, our aim was to investigate and review does it and in which manner DPP IV/CD26 affect the immune homeostasis during development, progression and resolution of inflammatory events in two animal models of IBD.

## **2. Dipeptidyl peptidase IV/CD26 molecule**

The exoprotease dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as surface antigen CD26, is a transmembrane glycoprotein with molecular mass of 220-240 kDa, expressed constitutively on a variety of cell types (Lambeir et al., 2003). It is also present in a soluble form in serum, saliva, urine and other biological fluids. So far, the role of this molecule has been investigated in different fields of biochemistry, immunology, endocrinology, oncology, pharmacology, physiology and pathophysiology.

#### *Structural and molecular characteristics*

According to the current biochemical and structural data, DPP IV/CD26 is a type II transmembrane, homodimeric glycoprotein. Each monomer consists of a large extracellular part (739 amino acids), a hydrophobic transmembrane segment of 23 amino acids and a short cytoplasmic N-terminal tail. The primary sequence of DPP IV/CD26 is composed of 766 amino acids and it was found to be conserved in different species (85% similarities between rat and human and 92% similarities between rat and mouse), mostly in the Cterminal protease segment (Lambeir et al., 2003). DPP IV/CD26 is a member of the POP (prolyl oligopeptidase) gene family with an α/β hydrolase domain and a N-terminal βpropeller domain that enclose the large cavity (30-40 Å) which contains a small pocket with the active site. The catalytic site, as a part of extracellular domain of the molecule, contains Ser-630, His-740 and Asp-708, which is not common for classical serine-type peptidases, but is characteristic for the previously mentioned α/β hydrolase fold (Gorrell et al., 2006).

Based on structural and biochemical features, DPP IV/CD26 is a member of a family of DPP IV activity and/or structure homologue (DASH) proteins, which also includes quiescent cell proline dipeptidase (QPP), DPP8, DPP9, fibroblast activation protein (FAP), attractin and DPP IV-β (Sedo & Malik, 2001). Since it is well known that most DASH proteins have protease activity, having the possibility to modify the activity of biologically active peptides, it could be suggested that they are important regulatory molecules (Gorrell, 2005). However, further research is necessary in order to clarify their biological role.

#### *Distribution and expression*

60 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Growing body of knowledge proposes proteases as key factors in the occurrence of inflammatory processes due to their ability to metabolize different biologically active molecules implicated in maintaining the integrity of mucosal barrier (Ravi et al., 2007). Dipeptidyl peptidase IV, known also as CD26 molecule (DPP IV/CD26) is one of them (Gorrell et al., 2001). DPP IV/CD26 is also T-cell differentiation antigen, expressed on various cell types, having numerous functions in a variety of biological processes, as well as immunological mechanisms (Fleischer, 1994). It is also present in a soluble form circulating in body fluids in living organisms with specific peptidase function having unique features in substrate processing: it cleaves dipeptides from the N terminus of polypeptides having proline or alanin at the penultimate position. Since Xaa-Pro peptides are not easily metabolized by other proteases, the action of DPP IV/CD26 is an essential step in the degradation of many polypeptides (Gorrell et al., 2001). Numerous biologically important cytokines, chemokines and neuropeptides with potential and/or confirmed role in IBD

Previous studies proposed a role of DPP IV/CD26 in the pathogenesis of IBD, given its involvement in immune regulations via its expression on immune cells and capability to cleave biologically active molecules (Hildebrandt et al., 2001; Varljen et al., 2005). Additionally, DPP IV/CD26 inhibitors have been pointed out as therapeutic agents in ameliorating inflammatory processes in immunologically mediated diseases such as IBD

The aim of this study was to review our previously published results regarding correlation between disease severity and serum DPP IV/CD26 activity in young and adult patients affected with IBD. Furthermore, our aim was to investigate and review does it and in which manner DPP IV/CD26 affect the immune homeostasis during development, progression

The exoprotease dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as surface antigen CD26, is a transmembrane glycoprotein with molecular mass of 220-240 kDa, expressed constitutively on a variety of cell types (Lambeir et al., 2003). It is also present in a soluble form in serum, saliva, urine and other biological fluids. So far, the role of this molecule has been investigated in different fields of biochemistry, immunology,

According to the current biochemical and structural data, DPP IV/CD26 is a type II transmembrane, homodimeric glycoprotein. Each monomer consists of a large extracellular part (739 amino acids), a hydrophobic transmembrane segment of 23 amino acids and a short cytoplasmic N-terminal tail. The primary sequence of DPP IV/CD26 is composed of 766 amino acids and it was found to be conserved in different species (85% similarities between rat and human and 92% similarities between rat and mouse), mostly in the Cterminal protease segment (Lambeir et al., 2003). DPP IV/CD26 is a member of the POP (prolyl oligopeptidase) gene family with an α/β hydrolase domain and a N-terminal βpropeller domain that enclose the large cavity (30-40 Å) which contains a small pocket with the active site. The catalytic site, as a part of extracellular domain of the molecule, contains Ser-630, His-740 and Asp-708, which is not common for classical serine-type peptidases, but is characteristic for the previously mentioned α/β hydrolase fold (Gorrell et al., 2006).

ethiopathogenesis are effective DPP IV/CD26 substrates (Mentlein, 2004).

and resolution of inflammatory events in two animal models of IBD.

endocrinology, oncology, pharmacology, physiology and pathophysiology.

(Yazbeck et al., 2009; Yazbeck et al., 2008).

**2. Dipeptidyl peptidase IV/CD26 molecule** 

*Structural and molecular characteristics* 

DPP IV/CD26 is widely distributed in mammalian tissues, mainly on epithelial and endothelial cell surfaces, as well as on fibroblasts and lymphocytes (Boonacker & Van Noorden, 2003). The expression of DPP IV/CD26 on hematopoietic cells is well regulated according to the activation status. In humans, it is expressed on a fraction of resting lymphocytes at low density, but is strongly up-regulated following T-cell activation (Fleischer, 1987). In resting peripheral blood mononuclear cells, a small subpopulation of T cells expresses CD26 at high density on the surface (CD26-bright cells), which belongs to the CD45RO+ population of T cells (memory cells) (De Meester et al., 1999; Ishii et al., 2001). Moreover, CD26 expression on T cells may correlate with T-helper subsets. High expression is found on Th1 and Th0 cells, whereas Th2 cells display lower CD26 expression (Willheim et al., 1997).

#### *Soluble DPP IV/CD26*

Soluble DPP IV/CD26 activity was firstly discovered in the serum in 1968 by Nagatsu et al. (Nagatsu et al., 1968). Later, DPP IV/CD26 activity has been shown in other body fluids including plasma, serum, cerebrospinal and synovial fluids, semen and urine. Although soluble DPP IV/CD26 lacks the transmembrane domain and intracellular tail, due to glycosylations processes, its molecular weight is similar to the transmembrane form. The origin of the soluble DPP IV/CD26 is still not elucidated, but it was suggested that it could be released from the surface of all CD26 expressing cells in contact with blood by proteolytic cleavage (Gorrell et al., 2001). The physiological role of soluble DPP IV/CD26 in biological fluids with respect to the transmembrane DPP IV/CD26 remains poorly understood, but according to previous findings it has been proposed that, as an enzyme, it is involved in the regulation of many processes in human body (Aytac & Dang, 2004; Mentlein, 1999).

#### *Functions in immune regulations*

Immune regulation is a complex and important process in which DPP IV/CD26 as a costimulatory molecule in T-cell activation and a regulator of the functional effect of selected biological factors through its enzyme activity, certainly has an important function (Boonacker & Van Noorden, 2003). Furthermore, biochemical and immune studies provide evidence that CD26 interacts with many biologically important molecules including CD45, adenosine deaminase protein, chemokine receptor CXCR4 on the surface of human peripheral blood lymphocytes (Herrera et al., 2001) and the mannose-6-phosphate/insulinlike growth factor II receptor (Ikushima et al., 2000). The costimulatory properties of DPP IV/CD26 have been studied extensively, although different experimental settings sometimes provide conflicting results. It is generally accepted that several distinct anti-CD26 mAbs have costimulatory activities in anti-CD3-driven activation of pure T-cell subsets (either CD4+ or CD8+ T cells), and that the extent and kinetics of the response differs between mAbs, recognizing different epitopes. High CD26 surface expression is correlated with the production of Th1-type cytokines such as IFN-γ (Reinhold et al., 1997b).

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 63

substrate could also have a serine, glycine, valine, threonine, leucine, or hydroxyproline at the penultimate position (Lambeir et al., 2001). However, DPP IV/CD26 is unable to hydrolyze substrates with proline, hydroxyproline or N-methyl glycine on the third position from the N-terminus (Puschel et al., 1982) and therefore, these peptides are DPP IV

A large number of various biologically important peptides have been shown to be substrates of DPP IV/CD26. Some of the most important DPP IV/CD26 substrates are presented in

During the past 50 years, IBD affected millions of people worldwide and therefore has become one of the major gastroenterological problems, especially in the Westernized world. It is a disorder of multiple etiologies, with generally accepted definition that occurs in genetically susceptible individuals, under influence of environmental and microbiological factors, as an overexpressed immunological response to antigens of unknown origin, characterized by chronic uncontrolled inflammation of intestinal mucosa, resulting in its destruction and lost of its function (Colletti, 2004). IBD comprises two main chronic inflammatory diseases of humans, namely ulcerative colitis and Crohn's disease, both characterized by alternating phases of active inflammation and clinical remission with diverse complications and extraintestinal manifestations (Hanauer & Hommes, 2010). Although the scientific knowledge increases exponentially, there are still many unanswered questions in several fundamental aspects of the IBD. The most stimulating field of IBD research is the interaction among the three major factors of the pathophysiology, including genetic predisposition, environmental bacteria and immune deregulation. The early inductive phases of these diseases are particularly difficult to study in humans because patients usually come to clinic only after their symptoms have

**3.1 Clinical relevance of serum DPP IV/CD26 activity in adult patients and children** 

Many investigations and reviews have discussed the role of DPP IV/CD26 activity in inflammation and the potential usefulness of this protein in therapeutics and diagnostics purpose (Hildebrandt et al., 2001; Varljen et al., 2005). However, its exact role still remains unclear. In clinical practice, the differential diagnosis of CD and UC is often difficult. Different biochemical, clinical, endoscopic, pathological and histological features should be combined in order to allocate the appropriate diagnosis. However, a precise diagnosis is not possible in about 10% of patients with chronic colitis, which results in the designation

Given the role of DPP IV/CD26 in the modulation of the immune response, we hypothesized that DPP IV/CD26 is altered in patients with CD and UC and that changes in DPP IV/CD26 serum activity could be related to the disease activity together with other inflammatory parameters. Therefore, the aim of this study was to evaluate the clinical relevance of changes in serum DPP IV activity in adult patients IBD (CD and UC). Furthermore, given the different immune background in patients with CD and UC as well as different expression of DPP IV/CD26 on Th1 and Th2 cells, we wanted to evaluate if DPP IV/CD26 serum activity could

**3. Inflammatory bowel disease and DPP IV/CD26** 

been established (Hanauer, 2006).

»indeterminate colitis« (Geboes et al., 2008).

be used as differentiating marker in the diagnosis of these diseases.

inhibitors.

Table 1.

**with IBD** 

Furthermore, CD26+ CD4+ T cells support differentiation of B cells into antibody-producing plasma cells (Dang et al., 1990).

The question whether the DPP IV/CD26 enzyme activity is involved in T cell activation is still controversial (Lambeir et al., 2003; Schon et al., 1985). Upon CD26-mediated costimulation, IL-2 production is higher in cells expressing wild-type CD26, suggesting that the DPP IV enzymatic activity of CD26 might contribute to, but is not essential for signal transduction. On the other hand, studies with inhibitors of DPP IV/CD26 activity have demonstrated that DPP IV/CD26 plays a key role in T cell activation (Munoz et al., 1992). It was shown that antigen-specific T cell proliferation and IL-2 production *in vitro* could be inhibited by application of the chemical inhibitor Pro-boro-Pro (Flentke et al., 1991). In addition, Lys(Z(NO2))-thiazolidide, Lys(Z(NO2))-piperidide, and Lys(Z(NO2))-pyrrolidide, all synthetic competitive DPP IV/CD26 inhibitors, significantly inhibit DNA synthesis and the production of IL-2, IL-10, IL-12 and IFN-γ in pokeweed mitogen-stimulated purified T lymphocytes (Hildebrandt et al., 2000; Thompson et al., 2007). On the other hand, the presence of these inhibitors enhance the secretion of the immune-inhibitory cytokine TGFβ1, suggesting that TGF-β1 helps regulate DPP IV/CD26 effect on T cell function (Reinhold et al., 1997a).


Table 1. Selected DPP IV/CD26 biologically important substrates (Gorrel et al, 2006)

Firstly, DPP IV/CD26 was considered to cut off distinctively after a proline or an alanine on the second position from the N-terminal end of a polypeptide chain. Meanwhile, the list of DPP IV/CD26 substrates has been enlarged as it has been shown that a DPP IV/CD26

Furthermore, CD26+ CD4+ T cells support differentiation of B cells into antibody-producing

The question whether the DPP IV/CD26 enzyme activity is involved in T cell activation is still controversial (Lambeir et al., 2003; Schon et al., 1985). Upon CD26-mediated costimulation, IL-2 production is higher in cells expressing wild-type CD26, suggesting that the DPP IV enzymatic activity of CD26 might contribute to, but is not essential for signal transduction. On the other hand, studies with inhibitors of DPP IV/CD26 activity have demonstrated that DPP IV/CD26 plays a key role in T cell activation (Munoz et al., 1992). It was shown that antigen-specific T cell proliferation and IL-2 production *in vitro* could be inhibited by application of the chemical inhibitor Pro-boro-Pro (Flentke et al., 1991). In addition, Lys(Z(NO2))-thiazolidide, Lys(Z(NO2))-piperidide, and Lys(Z(NO2))-pyrrolidide, all synthetic competitive DPP IV/CD26 inhibitors, significantly inhibit DNA synthesis and the production of IL-2, IL-10, IL-12 and IFN-γ in pokeweed mitogen-stimulated purified T lymphocytes (Hildebrandt et al., 2000; Thompson et al., 2007). On the other hand, the presence of these inhibitors enhance the secretion of the immune-inhibitory cytokine TGFβ1, suggesting that TGF-β1 helps regulate DPP IV/CD26 effect on T cell function (Reinhold

**Neuropeptides Glucose regulators** 

Vasoactive intestinal peptide Glucagon-like peptide 1 (GLP-1) Peptide YY Glucagon-like peptide 2 (GLP-2)

Endomorfin 1 and 2 Gastrin-releasing peptide

**Mediators of inflammation Other bioactive peptides** 

Interleukines: IL-1, IL-2, IL-6, IL-10 Monomeric fibrin (α chain)

Table 1. Selected DPP IV/CD26 biologically important substrates (Gorrel et al, 2006)

Firstly, DPP IV/CD26 was considered to cut off distinctively after a proline or an alanine on the second position from the N-terminal end of a polypeptide chain. Meanwhile, the list of DPP IV/CD26 substrates has been enlarged as it has been shown that a DPP IV/CD26

Tumor necrosis factor α (TNF-α) Melanostatin Macrophage-derived chemokine Tripsinogen

Bradykinin

Prolactin Enterostatin

Growth hormone-releasing factor

Alpha-1-microglobulin

Neuropeptide Y Glucagon

plasma cells (Dang et al., 1990).

et al., 1997a).

*DPP IV/CD26 substrates* 

Beta-casomorphine

(SDF-1α and 1β)

expressed and secreted)

Stromal cell derived factor- 1α and 1β

(regulated on activation, normal T-cell

Interferon-inducible protein 10 (IP-10)

Substance P

RANTES

Eotaxin

substrate could also have a serine, glycine, valine, threonine, leucine, or hydroxyproline at the penultimate position (Lambeir et al., 2001). However, DPP IV/CD26 is unable to hydrolyze substrates with proline, hydroxyproline or N-methyl glycine on the third position from the N-terminus (Puschel et al., 1982) and therefore, these peptides are DPP IV inhibitors.

A large number of various biologically important peptides have been shown to be substrates of DPP IV/CD26. Some of the most important DPP IV/CD26 substrates are presented in Table 1.

## **3. Inflammatory bowel disease and DPP IV/CD26**

During the past 50 years, IBD affected millions of people worldwide and therefore has become one of the major gastroenterological problems, especially in the Westernized world. It is a disorder of multiple etiologies, with generally accepted definition that occurs in genetically susceptible individuals, under influence of environmental and microbiological factors, as an overexpressed immunological response to antigens of unknown origin, characterized by chronic uncontrolled inflammation of intestinal mucosa, resulting in its destruction and lost of its function (Colletti, 2004). IBD comprises two main chronic inflammatory diseases of humans, namely ulcerative colitis and Crohn's disease, both characterized by alternating phases of active inflammation and clinical remission with diverse complications and extraintestinal manifestations (Hanauer & Hommes, 2010).

Although the scientific knowledge increases exponentially, there are still many unanswered questions in several fundamental aspects of the IBD. The most stimulating field of IBD research is the interaction among the three major factors of the pathophysiology, including genetic predisposition, environmental bacteria and immune deregulation. The early inductive phases of these diseases are particularly difficult to study in humans because patients usually come to clinic only after their symptoms have been established (Hanauer, 2006).

#### **3.1 Clinical relevance of serum DPP IV/CD26 activity in adult patients and children with IBD**

Many investigations and reviews have discussed the role of DPP IV/CD26 activity in inflammation and the potential usefulness of this protein in therapeutics and diagnostics purpose (Hildebrandt et al., 2001; Varljen et al., 2005). However, its exact role still remains unclear. In clinical practice, the differential diagnosis of CD and UC is often difficult. Different biochemical, clinical, endoscopic, pathological and histological features should be combined in order to allocate the appropriate diagnosis. However, a precise diagnosis is not possible in about 10% of patients with chronic colitis, which results in the designation »indeterminate colitis« (Geboes et al., 2008).

Given the role of DPP IV/CD26 in the modulation of the immune response, we hypothesized that DPP IV/CD26 is altered in patients with CD and UC and that changes in DPP IV/CD26 serum activity could be related to the disease activity together with other inflammatory parameters. Therefore, the aim of this study was to evaluate the clinical relevance of changes in serum DPP IV activity in adult patients IBD (CD and UC). Furthermore, given the different immune background in patients with CD and UC as well as different expression of DPP IV/CD26 on Th1 and Th2 cells, we wanted to evaluate if DPP IV/CD26 serum activity could be used as differentiating marker in the diagnosis of these diseases.

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 65

**\* \***

\*, statistically significantly different compared to control group (*P* < 0.001)

ulcerative colitis (UC) compared to healthy controls.

CDAI - Crohn's Disease Activity Index

CDAI ≤ 150 – remission CDAI >150 – active disease

having mild UC.

*Children* 

\*, statistically significantly different compared to CDAI<150 (*P* = 0.023).

**Serum DPP IV activity / (μmol min -1 dm-3)** 

 **CD UC Control group**

When analyzing the correlation between serum DPP IV/CD26 activity in patients with CD and UC, it was noticed that patients affected with CD, having CDAI>250 had statistically significantly lower serum DPP IV/CD26 activity compared to patients having CDAI<150 (Fig. 2).

**\***

 **CDAI <150 CDAI 150-250 CDAI >250** 

Fig. 2. Serum DPP IV/CD26 activity in three groups of patients with Crohn's disease.

Likewise, an inverse correlation between serum DPP IV/CD26 activity and disease severity was found in patients affected with UC (Fig. 3). It could be seen that patients with severe UC had statistically significantly (*P* < 0.05) lower DPP IV/CD26 activity compared to patients

In young patients affected with IBD, DPP IV/CD26 activity in serum was also reduced compared to the levels in healthy controls, likewise in adult patients. The serum DPP

Fig. 1. Serum DPP IV/CD26 activity in patients affected with Crohn's disease (CD) and

**0**

**10**

**20**

**30**

**Serum DPP IV activity / (μmol min -1 dm-3) .**

**40**

**50**

**60**

#### **3.2 Material and methods**

#### *Adult patients*

The study was performed on 62 patients, 38 with CD (mean age ± SD: 42.7±14.4; 19 males, 19 females), and 24 with UC (mean age ± SD: 45.6±17.6; 13 males, 11 females). All patients were admitted to the Department of Gastroenterology, Clinical Hospital Centre Rijeka. Diagnoses of CD or UC were established on the basis of clinical history, laboratory, endoscopic and histological data. The control group included 65 healthy donors (mean age ± SD: 41.6±12.1; 32 males, 33 females). The CD activity was evaluated using the Crohn's Disease Activity Index (CDAI), while the UC activity was evaluated according to the Truelove and Witts' (TW) classification (Truelove & Witts, 1955). The localization of the disease was determined according to the Wienna classification for CD while UC was divided into proctosigmoiditis, left-side colitis and pancolitis. Blood samples were obtained after all patients and controls signed informed consents under the protocols approved by the Ethics Committee.

#### *Children*

The study involved also young patients, 31 children with IBD. Diagnoses of CD or UC were established on the basis of clinical history, laboratory, endoscopic and histological data. CD activity was evaluated by using the Paediatric Crohn´s Disease Activity Index (PCDAI) (Hyams et al., 1991). Blood samples were obtained after all children's parents gave their signed informed consent under the protocols approved by the Ethics Committee. The study group comprised 24 patients with CD (12 with (PCDAI)≥15 and 12 with (PCDAI)<15) and 7 with UC. Their mean ± SD age at diagnosis was 13.84±1.72 years. The control group included 46 healthy children (mean age ± SD: 13.80±2.83 years; 22 males and 24 females).

## **3.2.1 DPP IV/CD26 assay**

Sera were separated from fasting blood samples and stored at –80°C until thawed for enzyme activities. Determination of serum DPP IV/CD26 activities was performed as described by Kreisel et al (Kreisel et al., 1982). DPP IV/CD26 activities were determined by measuring the release of 4-nitroaniline from an assay mixture containing 0.1 mol Tris-HCl (pH 8.0), 2 mmol Gly-Pro *p*-nitroanilide (Sigma Chemical, Steinheim, Germany) as the substrate and serum in a total volume of 0.20 mL. After 30 minutes of incubation at 37°C, the reaction was stopped by the addition of 800 μL of 1 mol sodium acetate buffer (pH 4.5). The absorbance at 405 nm was measured by use of a Varian Cary UV/VIS spectrophotometer (Cary, NC). All of the reactions were performed in duplicate. Enzyme activities in serum were expressed as μmol of hydrolyzed substrate in a volume of 1 dm3 per minute under the assay conditions.

#### **3.3 Results and discussion**

Here reviewed results for adult patients were previously published in *Croatica chemica acta,*  (Varljen et al., 2005), while results of investigations that included children were previously published in *Pediatric Gastroenterology* - Reports from the 2nd World Congress of Pediatric Gastroenterology, Hepatology and Nutrition (Varljen et al., 2004).

#### *Adult patients*

Results of serum DPP IV/CD26 activity in adult patients with CD and UC compared to the control group are presented on Fig. 1. It could be seen that both serum DPP IV/CD26 activities in CD as well as UC are statistically significantly (*P* < 0.05) reduced compared to healthy controls.

\*, statistically significantly different compared to control group (*P* < 0.001)

Fig. 1. Serum DPP IV/CD26 activity in patients affected with Crohn's disease (CD) and ulcerative colitis (UC) compared to healthy controls.

When analyzing the correlation between serum DPP IV/CD26 activity in patients with CD and UC, it was noticed that patients affected with CD, having CDAI>250 had statistically significantly lower serum DPP IV/CD26 activity compared to patients having CDAI<150 (Fig. 2).

\*, statistically significantly different compared to CDAI<150 (*P* = 0.023). CDAI - Crohn's Disease Activity Index CDAI ≤ 150 – remission CDAI >150 – active disease

Fig. 2. Serum DPP IV/CD26 activity in three groups of patients with Crohn's disease.

Likewise, an inverse correlation between serum DPP IV/CD26 activity and disease severity was found in patients affected with UC (Fig. 3). It could be seen that patients with severe UC had statistically significantly (*P* < 0.05) lower DPP IV/CD26 activity compared to patients having mild UC.

#### *Children*

64 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

The study was performed on 62 patients, 38 with CD (mean age ± SD: 42.7±14.4; 19 males, 19 females), and 24 with UC (mean age ± SD: 45.6±17.6; 13 males, 11 females). All patients were admitted to the Department of Gastroenterology, Clinical Hospital Centre Rijeka. Diagnoses of CD or UC were established on the basis of clinical history, laboratory, endoscopic and histological data. The control group included 65 healthy donors (mean age ± SD: 41.6±12.1; 32 males, 33 females). The CD activity was evaluated using the Crohn's Disease Activity Index (CDAI), while the UC activity was evaluated according to the Truelove and Witts' (TW) classification (Truelove & Witts, 1955). The localization of the disease was determined according to the Wienna classification for CD while UC was divided into proctosigmoiditis, left-side colitis and pancolitis. Blood samples were obtained after all patients and controls

signed informed consents under the protocols approved by the Ethics Committee.

The study involved also young patients, 31 children with IBD. Diagnoses of CD or UC were established on the basis of clinical history, laboratory, endoscopic and histological data. CD activity was evaluated by using the Paediatric Crohn´s Disease Activity Index (PCDAI) (Hyams et al., 1991). Blood samples were obtained after all children's parents gave their signed informed consent under the protocols approved by the Ethics Committee. The study group comprised 24 patients with CD (12 with (PCDAI)≥15 and 12 with (PCDAI)<15) and 7 with UC. Their mean ± SD age at diagnosis was 13.84±1.72 years. The control group included 46 healthy children (mean age ± SD: 13.80±2.83 years; 22 males and 24 females).

Sera were separated from fasting blood samples and stored at –80°C until thawed for enzyme activities. Determination of serum DPP IV/CD26 activities was performed as described by Kreisel et al (Kreisel et al., 1982). DPP IV/CD26 activities were determined by measuring the release of 4-nitroaniline from an assay mixture containing 0.1 mol Tris-HCl (pH 8.0), 2 mmol Gly-Pro *p*-nitroanilide (Sigma Chemical, Steinheim, Germany) as the substrate and serum in a total volume of 0.20 mL. After 30 minutes of incubation at 37°C, the reaction was stopped by the addition of 800 μL of 1 mol sodium acetate buffer (pH 4.5). The absorbance at 405 nm was measured by use of a Varian Cary UV/VIS spectrophotometer (Cary, NC). All of the reactions were performed in duplicate. Enzyme activities in serum were expressed as μmol of

Here reviewed results for adult patients were previously published in *Croatica chemica acta,*  (Varljen et al., 2005), while results of investigations that included children were previously published in *Pediatric Gastroenterology* - Reports from the 2nd World Congress of Pediatric

Results of serum DPP IV/CD26 activity in adult patients with CD and UC compared to the control group are presented on Fig. 1. It could be seen that both serum DPP IV/CD26 activities in CD as well as UC are statistically significantly (*P* < 0.05) reduced compared to healthy controls.

hydrolyzed substrate in a volume of 1 dm3 per minute under the assay conditions.

Gastroenterology, Hepatology and Nutrition (Varljen et al., 2004).

**3.2 Material and methods** 

**3.2.1 DPP IV/CD26 assay**

**3.3 Results and discussion** 

*Adult patients* 

*Adult patients* 

*Children* 

In young patients affected with IBD, DPP IV/CD26 activity in serum was also reduced compared to the levels in healthy controls, likewise in adult patients. The serum DPP

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 67

impact on the pathogenesis of IBD. Our results can suggest a functional compartmentalization of DPP IV/CD26, which can be interpreted as an adaptive systemic immune response to a local inflammatory reaction. Meanwhile, the obtained results do not corroborate the hypothesis that the serum DPP IV/CD26 enzymatic activity differs between patients with CD and patients with UC, thus reflecting the concept of different cytokine patterns in one or the other subtype of IBD. Consequently, it seems that the serum DPP IV/CD26 activity could not be used as a specific differential diagnostic marker between CD and UC, and further investigations are necessary in order to establish a new parameter for

**Control group CD ACD ICD**

Throughout the last decade, several experimental animal models of IBD have been developed in order to define different components of the pathophysiological processes that characterize these disorders (Mizoguchi & Mizoguchi, 2010; Strober et al., 1998; Wirtz & Neurath, 2007). Experimental animal models have a number of advantages which include allowing the study of specific pathophysiological events occurring before symptoms onset. Furthermore, investigators can perform genetic and immunologic manipulations of relevant

Although no ideal model of IBD has been accomplished so far, they resemble different important clinical, histopathological and immunological aspects of human IBD. The value of the animal models is the insight they allow into the complex, multifaceted processes and mechanisms that can result in acute or chronic intestinal inflammation. Animal models of IBD have given insight in different processes at the molecular level and have revealed the importance of different molecules involved in IBD etiology, representing therefore essential tools in investigating different mechanisms underlying acute or chronic inflammation in IBD. In recent years quite a number of new experimental models of intestinal inflammation

**\***

**\***

differentiation of CD from UC.

0 20

(ACD-Active Crohn's disease, ICD-Inactive Crohn's disease)

, statistically significantly different compared to control group (P<0.05)

Fig. 5. Serum DPP IV/CD26 activity in children affected Crohn's disease (CD),

mouse genes, possibly involved in disease pathogenesis (Bhan et al., 1999).

40 60

**Serum DPP IV activity / (**

**4. Animal models of IBD** 

have been described (Table 2).

\*

**μmol min-1 dm-3)**

80 100

120

TW-mild and TW-severe - Truelove and Witts' classification (Truelove & Witts, 1955) \*, statistically significantly different compared to TW-mild (P=0.035)

Fig. 3. Serum DPP IV/CD26 activity in two groups of patients with ulcerative colitis.

IV/CD26 activity in children with CD was statistically significantly (*P* < 0.05) decreased compared to the levels in healthy controls. The DPP IV/CD26 activity in children with UC was also decreased but not statistically significantly when compared to controls (Fig. 4).

\* , statistically significantly different compared to control group (*P* < 0.05)

Fig. 4. Serum DPP IV/CD26 activity in children affected with Crohn's disease (CD) and ulcerative colitis (UC)

The serum DPP IV/CD26 activity in children with active CD was statistically significantly decreased (*P* < 0.05) compared with the levels in healthy controls, while in patients with inactive CD it was also found to be decreased, but not statistically significantly (Fig. 5). Based on obtained results, it could be concluded that soluble DPP IV/CD26 in serum seems to be involved in the pathophysiology of IBD and appears to be useful as an available noninvasive marker in the diagnosis of disease activity. Changes of DPP IV/CD26 expression and serum activity were found to occur in several clinical and experimental situations of altered immune function (Gorrell et al, 2006). Results of our study accord with previous investigation which confirmed lower serm DPP IV/CD26 activity in patients affected with IBD (Hildebrandt et al., 2001; Rose et al., 2003). Obtained data, together with previously published results, suggest that the persisted immune dysbalance could have a significant

**\***

 **TW MILD TW SEVERE**

IV/CD26 activity in children with CD was statistically significantly (*P* < 0.05) decreased compared to the levels in healthy controls. The DPP IV/CD26 activity in children with UC was also decreased but not statistically significantly when compared to controls (Fig. 4).

**\***

**Control group CD UC**

Fig. 4. Serum DPP IV/CD26 activity in children affected with Crohn's disease (CD) and

The serum DPP IV/CD26 activity in children with active CD was statistically significantly decreased (*P* < 0.05) compared with the levels in healthy controls, while in patients with inactive CD it was also found to be decreased, but not statistically significantly (Fig. 5). Based on obtained results, it could be concluded that soluble DPP IV/CD26 in serum seems to be involved in the pathophysiology of IBD and appears to be useful as an available noninvasive marker in the diagnosis of disease activity. Changes of DPP IV/CD26 expression and serum activity were found to occur in several clinical and experimental situations of altered immune function (Gorrell et al, 2006). Results of our study accord with previous investigation which confirmed lower serm DPP IV/CD26 activity in patients affected with IBD (Hildebrandt et al., 2001; Rose et al., 2003). Obtained data, together with previously published results, suggest that the persisted immune dysbalance could have a significant

TW-mild and TW-severe - Truelove and Witts' classification (Truelove & Witts, 1955)

Fig. 3. Serum DPP IV/CD26 activity in two groups of patients with ulcerative colitis.

\*, statistically significantly different compared to TW-mild (P=0.035)

, statistically significantly different compared to control group (*P* < 0.05)

**Serum DPP IV activity / (μmol min-1 dm-3)**

\*

ulcerative colitis (UC)

**Serum DPP IV activity / (μmol min -1 dm-3)** 

impact on the pathogenesis of IBD. Our results can suggest a functional compartmentalization of DPP IV/CD26, which can be interpreted as an adaptive systemic immune response to a local inflammatory reaction. Meanwhile, the obtained results do not corroborate the hypothesis that the serum DPP IV/CD26 enzymatic activity differs between patients with CD and patients with UC, thus reflecting the concept of different cytokine patterns in one or the other subtype of IBD. Consequently, it seems that the serum DPP IV/CD26 activity could not be used as a specific differential diagnostic marker between CD and UC, and further investigations are necessary in order to establish a new parameter for differentiation of CD from UC.

(ACD-Active Crohn's disease, ICD-Inactive Crohn's disease) \* , statistically significantly different compared to control group (P<0.05)

Fig. 5. Serum DPP IV/CD26 activity in children affected Crohn's disease (CD),

## **4. Animal models of IBD**

Throughout the last decade, several experimental animal models of IBD have been developed in order to define different components of the pathophysiological processes that characterize these disorders (Mizoguchi & Mizoguchi, 2010; Strober et al., 1998; Wirtz & Neurath, 2007). Experimental animal models have a number of advantages which include allowing the study of specific pathophysiological events occurring before symptoms onset. Furthermore, investigators can perform genetic and immunologic manipulations of relevant mouse genes, possibly involved in disease pathogenesis (Bhan et al., 1999).

Although no ideal model of IBD has been accomplished so far, they resemble different important clinical, histopathological and immunological aspects of human IBD. The value of the animal models is the insight they allow into the complex, multifaceted processes and mechanisms that can result in acute or chronic intestinal inflammation. Animal models of IBD have given insight in different processes at the molecular level and have revealed the importance of different molecules involved in IBD etiology, representing therefore essential tools in investigating different mechanisms underlying acute or chronic inflammation in IBD. In recent years quite a number of new experimental models of intestinal inflammation have been described (Table 2).

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 69

of acute or chronic colitis (Dieleman et al., 1998). Inflammation induced by DSS is most frequent and severe in the distal part of the colon (Okayasu et al., 1990) and its severity depends on the concentration and molecular weight of DSS (Kitajima et al., 2000). Concentrations described in literature range between 1% and 7%, while the most commonly

This study was performed using pathogen-free, male, 8-10-week-old (weighting 20±2 g) wild type (C57BL/6) mice and mice with inactivated gene for DPP IV/CD26 molecule (CD26-/-) generated on a C57BL/6 genetic background, as described previously (Marguet et al., 2000). CD26-/- mice were kindly provided by Dr. Didier Marguet, Centre d'Immunologie Marseille-Luminy, France. Animals were housed and bred under standard conditions at the

Colitis was induced in both mice strains using 3% (w/v) sodium dextran sulfate sodium (DSS; MW 50 kDa; MP Biomedicals, USA) during seven days in drinking water *ad libitum*  (Wirtz & Neurath, 2007). Control mice received regular drinking water throughout the

Handling with animals, experimental procedures and anesthesia were performed in accordance with the general principles contained in the Guide for the Care and Use of Laboratory Animals (National Academic Press). The Ethical Committee at the School of

Animals included in the study were randomly divided into four groups as follows: C57BL/6 and CD26-/- mice treated with the 3% DSS solution for 7 days and control C57BL/6 and CD26-/- group treated with tap water. At day 7, in order to compare the colitis severity, treated and control animal of each genotype were anesthetized by intraperitoneal administration of ketamine (2.5 mg/mice) and sacrificed by cervical dislocation. The remaining animals were given normal drinking water until day 15 when they were sacrificed in order to compare the strain difference during colitis resolution. At each time point, 6-8 animals of each group were sacrificed. During the entire experiment, body mass was measured daily and clinical symptoms were assessed using the disease activity score. The colon segments from the ileocecal valve to the anus were excised *post mortem,* washed with ice-cold phosphate-buffered saline (PBS) and their length and weight were measured, as indirect markers of inflammation. After colon length and weight measurements, tissue samples were opened longitudinally, washed in PBS and proceeded for histology, morphometry and biochemical analysis. *Morphometrical measurements* included evaluation of crypt number, crypt depth and crypt width on hematoxilin - eosin stained tissue samples. Analyses were performed using software Issa (VAMS, Zagreb, Croatia), Pulmix camera

The *clinical score* was assessed as described previously (Howarth et al. 2000; Murthy et al. 1993). Briefly, weight loss of >5% was scored as 0 points, weight loss of 5 to 10% as 1 point, 10 to 15% as 2 points, 15 to 20% as 3 points, and more than 20% as 4 points. For stool consistency, 0 points were given for well formed pellets, 2 points for pasty and semiformed stools that did not stick to the anus, and 4 points for liquid stools that remained adhesive to the anus. Bleeding was scored 0 points for no presence of rectal bleeding and 4 points for gross bleeding from the rectum. These scores were added and divided by three, resulting in

a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis).

used molecular weight ranges between 30 kDa and 50 kDa.

Central Animal Facility of the School of Medicine, University of Rijeka.

Medicine, University of Rijeka approved all of the experiments.

(TMC 76S, Japan) and Olympus BX 40 microscope.

**4.1.1 Induction of DSS-colitis in mice** 

experiment (days 1-15).

*Experimental design* 


TNF, Tumor necrosis factor; UTR, untranslated region; STAT, signal transducer and activating transcription; hsp, heat shock protein

Table 2. Selected animal models of IBD (Mizoguchi & Mizoguchi, 2010)

## **4.1 DSS-induced colitis (ulcerative-like model of colitis)**

Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that may affect individuals of any age. It generally begins in the anus and extends at a variable length from the rectum in a continuous fashion. Patients usually present with a constellation of symptoms including diarrhea, lower abdominal cramping and tenesmus (Shah & Feller, 2009). The dextran sulfate sodium (DSS) model of induced colitis is an excellent preclinical animal model that exhibits numerous phenotypic features with human ulcerative colitis. It was originally described by Ohkusa et al. (Ohkusa, 1985) as a hamster model and was adapted to mice subsequently by Okayasu and its coworkers (Okayasu et al., 1990).

DSS is a polyanionic derivate of dextrane produced by esterification with chlorosulphonic acid. The exact mechanism through which DSS initiates colitis is unknown but according to previously published data, it is supposed that DSS alternates the gut permeability. It was shown that administration of DSS reduces the expression of tight junction proteins like zona occludens-1, leading to increased gut permeability. Another suggested mechanism involves direct cytotoxic action of DSS on the colonic mucosa, which leads to the alteration of integrin-α4 and M290 subunit levels on epithelial cells. Through these effects, DSS induces mucosal injury with consequent activation of immune response, leading to the development of acute or chronic colitis (Dieleman et al., 1998). Inflammation induced by DSS is most frequent and severe in the distal part of the colon (Okayasu et al., 1990) and its severity depends on the concentration and molecular weight of DSS (Kitajima et al., 2000). Concentrations described in literature range between 1% and 7%, while the most commonly used molecular weight ranges between 30 kDa and 50 kDa.

## **4.1.1 Induction of DSS-colitis in mice**

This study was performed using pathogen-free, male, 8-10-week-old (weighting 20±2 g) wild type (C57BL/6) mice and mice with inactivated gene for DPP IV/CD26 molecule (CD26-/-) generated on a C57BL/6 genetic background, as described previously (Marguet et al., 2000). CD26-/- mice were kindly provided by Dr. Didier Marguet, Centre d'Immunologie Marseille-Luminy, France. Animals were housed and bred under standard conditions at the Central Animal Facility of the School of Medicine, University of Rijeka.

Colitis was induced in both mice strains using 3% (w/v) sodium dextran sulfate sodium (DSS; MW 50 kDa; MP Biomedicals, USA) during seven days in drinking water *ad libitum*  (Wirtz & Neurath, 2007). Control mice received regular drinking water throughout the experiment (days 1-15).

Handling with animals, experimental procedures and anesthesia were performed in accordance with the general principles contained in the Guide for the Care and Use of Laboratory Animals (National Academic Press). The Ethical Committee at the School of Medicine, University of Rijeka approved all of the experiments.

#### *Experimental design*

68 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

IL-10 knockout Colitis, acute, chronic, transmural, Th1 (early)/Th2 (late)

Dextran sulfate sodium colitis Colitis, superficial, Th1 (acute), Th1/Th2 (chronic)

C3H-HeJBir Colitis, superficial, acute-resolving, Th1 SAMP1/Yit Ileitis, chronic, transmural, granulomatous, Th1

SAMP1/YitFc Perianal disease, early onset of disease

STAT-4 transgenic mice Colitis, acute, chronic, transmural, Th1

Trinitrobenzene sulfonic acid-induced colitis Colitis, acute, chronic, transmural, Th1

IL-2 knockout Spontaneous colitis, Th1

IL-7 transgenic mice Colitis, acute, chronic, Th1 HLA B27 transgenic Spontaneous, entire colon, Th1

Peptidoglycan-polysaccharide colitis Enterocolitis, transmural

CD4/CD45RBhigh T-cell transfer colitis Colitis, chronic transmural, Th1

Table 2. Selected animal models of IBD (Mizoguchi & Mizoguchi, 2010)

TNF, Tumor necrosis factor; UTR, untranslated region; STAT, signal transducer and activating

Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that may affect individuals of any age. It generally begins in the anus and extends at a variable length from the rectum in a continuous fashion. Patients usually present with a constellation of symptoms including diarrhea, lower abdominal cramping and tenesmus (Shah & Feller, 2009). The dextran sulfate sodium (DSS) model of induced colitis is an excellent preclinical animal model that exhibits numerous phenotypic features with human ulcerative colitis. It was originally described by Ohkusa et al. (Ohkusa, 1985) as a hamster model and was adapted to mice subsequently by Okayasu and its coworkers (Okayasu et

DSS is a polyanionic derivate of dextrane produced by esterification with chlorosulphonic acid. The exact mechanism through which DSS initiates colitis is unknown but according to previously published data, it is supposed that DSS alternates the gut permeability. It was shown that administration of DSS reduces the expression of tight junction proteins like zona occludens-1, leading to increased gut permeability. Another suggested mechanism involves direct cytotoxic action of DSS on the colonic mucosa, which leads to the alteration of integrin-α4 and M290 subunit levels on epithelial cells. Through these effects, DSS induces mucosal injury with consequent activation of immune response, leading to the development

T-cell receptor α mutant mice Colitis, chronic, Th2

TNF-3' UTR knockout mice Colitis

Oxazolone colitis Colitis, Th2

Transfer of hsp60-specific CD8 T cells Colitis, Th1

**4.1 DSS-induced colitis (ulcerative-like model of colitis)** 

**Animal model Disease type** 

*Spontaneous* 

*Genetically engineered* 

*Chemically induced* 

*Adoptive transfer* 

al., 1990).

transcription; hsp, heat shock protein

Animals included in the study were randomly divided into four groups as follows: C57BL/6 and CD26-/- mice treated with the 3% DSS solution for 7 days and control C57BL/6 and CD26-/- group treated with tap water. At day 7, in order to compare the colitis severity, treated and control animal of each genotype were anesthetized by intraperitoneal administration of ketamine (2.5 mg/mice) and sacrificed by cervical dislocation. The remaining animals were given normal drinking water until day 15 when they were sacrificed in order to compare the strain difference during colitis resolution. At each time point, 6-8 animals of each group were sacrificed. During the entire experiment, body mass was measured daily and clinical symptoms were assessed using the disease activity score. The colon segments from the ileocecal valve to the anus were excised *post mortem,* washed with ice-cold phosphate-buffered saline (PBS) and their length and weight were measured, as indirect markers of inflammation. After colon length and weight measurements, tissue samples were opened longitudinally, washed in PBS and proceeded for histology, morphometry and biochemical analysis. *Morphometrical measurements* included evaluation of crypt number, crypt depth and crypt width on hematoxilin - eosin stained tissue samples. Analyses were performed using software Issa (VAMS, Zagreb, Croatia), Pulmix camera (TMC 76S, Japan) and Olympus BX 40 microscope.

The *clinical score* was assessed as described previously (Howarth et al. 2000; Murthy et al. 1993). Briefly, weight loss of >5% was scored as 0 points, weight loss of 5 to 10% as 1 point, 10 to 15% as 2 points, 15 to 20% as 3 points, and more than 20% as 4 points. For stool consistency, 0 points were given for well formed pellets, 2 points for pasty and semiformed stools that did not stick to the anus, and 4 points for liquid stools that remained adhesive to the anus. Bleeding was scored 0 points for no presence of rectal bleeding and 4 points for gross bleeding from the rectum. These scores were added and divided by three, resulting in a total clinical score ranging from 0 (healthy) to 4 (maximal activity of colitis).

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 71

and results of previously published studies showed that DSS-induced damage could extend to the small intestine and therefore, further studies are necessary to validate physiological

A

B

Fig. 6. Influence of DSS-induce damage on small intestine and colon mucosa weight during

In compliance with our results, it could be concluded that administration of DSS in drinking water for seven days resulted in a prominent colon inflammation and gastrointestinal dysfunction, followed by regeneration of the colonic epithelium in C57BL/6 and CD26-/ mouse strains. Shortening of the colon and increase of colon weight, as macroscopic measures for the degree of inflammation, correlates with changes of mucosa weight and pathological changes (Okayasu et al., 1990). Given the fact that the symptoms of inflammation were the most prominent between the seventh and tenth day following DSS administration, this period was classified as acute phase, which is in accordance with

colitis development and resolution in C57BL/6 (A) and CD26-/- (B) animals.

impact of this damage.

Data are presented as mean ± SD

*P* < 0.05, statistically significantly different compared to day 0

\*

*Mucosa fractions isolation* from duodenum, jejunum, ileum and colon segments were prepared from mucosal scrapings according to Ahnen et al. (Ahnen et al., 1982).

#### **4.1.2 Establishment and validation of the DSS-induced colitis at systemic and local level**

Oral administration of DSS in rodents induces a colonic inflammation with many similarities to human IBD. Consistent with previous studies, as disease progressed, clinical symptoms, including loss of body mass, changes of stool consistency and appearance of rectal bleeding, were aggravated. Until day 3, no clinical symptoms of colitis were seen. From day 3 and later, both mice strain showed blood in their feces and diarrhea. From the results presented in Table 3, it could be concluded that body mass of healthy animals, control group, CD26-/- mice, in comparison to the control C57BL/6 mice is lower which is in agreement with previously published data (Marguet et al., 2000). Administration of the DSS solution caused a statistically significant decrease (*P* < 0.05) of body mass on day 3 in C57BL/6 mice, while in CD26-/- mice, extensive body mass loss began one day after, with a maximum fall on the ninth day. As the inflammation progressed, the disease activity index (DAI) in each group, increased gradually and reached its maximum on day 7 in both mice strains. Body weight increased gradually in both control groups. Variations in clinical symptoms and body mass during colitis development, established in C57BL/6 and CD26-/ mice, are shown in Table 3.


aData are presented as mean ± SD

bNumber of mice with diarrhea or gross bleeding/total number of mice in each group DSS: dextran sulfat sodium; DAI: Disease activity index

Table 3. Changes of clinical variables during DSS-induced colitis development and resolution in C57BL/6 and CD26-/- mice.

In order to assess the degree of inflammation at the local level, length and weight of each colon sample was measured. Statistically significant shortening of the colon was observed on day 7 of the experiment in both CD26-/- and C57BL/6 mice. Together with colon shortening, statistically significant increase of colon weight was observed on day 7 and 15 in CD26-/- and C57BL/6 mice. It is known that during colitis development, DSS induces colon tissue obliteration, but recent studies in rats showed that changes through the small intestine are also present (Geier et al., 2009; Ohtsuka & Sanderson, 2003). Therefore, in order to provide further evidence, we isolated small intestinal and colonic mucosa and measured the *changes of mucosa weight* during colitis development. A significant decrease of colonic mucosa weight was observed in C57BL/6 mice, while in CD26-/- mice the statistically significant decrease of ileum and colon mucosa weight was observed (Fig. 6). Our results 70 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

*Mucosa fractions isolation* from duodenum, jejunum, ileum and colon segments were

Oral administration of DSS in rodents induces a colonic inflammation with many similarities to human IBD. Consistent with previous studies, as disease progressed, clinical symptoms, including loss of body mass, changes of stool consistency and appearance of rectal bleeding, were aggravated. Until day 3, no clinical symptoms of colitis were seen. From day 3 and later, both mice strain showed blood in their feces and diarrhea. From the results presented in Table 3, it could be concluded that body mass of healthy animals, control group, CD26-/- mice, in comparison to the control C57BL/6 mice is lower which is in agreement with previously published data (Marguet et al., 2000). Administration of the DSS solution caused a statistically significant decrease (*P* < 0.05) of body mass on day 3 in C57BL/6 mice, while in CD26-/- mice, extensive body mass loss began one day after, with a maximum fall on the ninth day. As the inflammation progressed, the disease activity index (DAI) in each group, increased gradually and reached its maximum on day 7 in both mice strains. Body weight increased gradually in both control groups. Variations in clinical symptoms and body mass during colitis development, established in C57BL/6 and CD26-/-

mass (g) DAIa Diarrheab Gross

0 24.17 ± 1.97 0 0/6 0/6 8.5 ± 0.1 7 19.19 ± 3.62 4.00 ± 0.20 6/6 6/6 7.3 ± 0.4 15 23.35 ± 2.79 0 0/6 0/6 8.0 ± 0.2

0 23.63 ± 1.73 0 0/6 0/6 8.5 ± 0.1 7 19.35 ± 2.16 3.66 ± 0.25 6/6 5/6 7.7 ± 0.2 15 21.57 ± 1.55 0.33 ± 0.05 0/6 0/6 8.0 ± 0.3

bleedingb

Colon length (cm)a

prepared from mucosal scrapings according to Ahnen et al. (Ahnen et al., 1982).

**4.1.2 Establishment and validation of the DSS-induced colitis at systemic** 

**and local level**

mice, are shown in Table 3.

aData are presented as mean ± SD

DSS: dextran sulfat sodium; DAI: Disease activity index

resolution in C57BL/6 and CD26-/- mice.

Day of experiment Body

bNumber of mice with diarrhea or gross bleeding/total number of mice in each group

Table 3. Changes of clinical variables during DSS-induced colitis development and

In order to assess the degree of inflammation at the local level, length and weight of each colon sample was measured. Statistically significant shortening of the colon was observed on day 7 of the experiment in both CD26-/- and C57BL/6 mice. Together with colon shortening, statistically significant increase of colon weight was observed on day 7 and 15 in CD26-/- and C57BL/6 mice. It is known that during colitis development, DSS induces colon tissue obliteration, but recent studies in rats showed that changes through the small intestine are also present (Geier et al., 2009; Ohtsuka & Sanderson, 2003). Therefore, in order to provide further evidence, we isolated small intestinal and colonic mucosa and measured the *changes of mucosa weight* during colitis development. A significant decrease of colonic mucosa weight was observed in C57BL/6 mice, while in CD26-/- mice the statistically significant decrease of ileum and colon mucosa weight was observed (Fig. 6). Our results

Mice strain

C57BL/6

CD26-/-

and results of previously published studies showed that DSS-induced damage could extend to the small intestine and therefore, further studies are necessary to validate physiological impact of this damage.

\* *P* < 0.05, statistically significantly different compared to day 0

Fig. 6. Influence of DSS-induce damage on small intestine and colon mucosa weight during colitis development and resolution in C57BL/6 (A) and CD26-/- (B) animals.

In compliance with our results, it could be concluded that administration of DSS in drinking water for seven days resulted in a prominent colon inflammation and gastrointestinal dysfunction, followed by regeneration of the colonic epithelium in C57BL/6 and CD26-/ mouse strains. Shortening of the colon and increase of colon weight, as macroscopic measures for the degree of inflammation, correlates with changes of mucosa weight and pathological changes (Okayasu et al., 1990). Given the fact that the symptoms of inflammation were the most prominent between the seventh and tenth day following DSS administration, this period was classified as acute phase, which is in accordance with

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 73

There is evidence that susceptibility to DSS varies with the animal species and mice strain. Guinea pig is the most susceptible, with inflammation usually fully established in less than 72 h (Iwanaga et al., 1994). In mice, some strains such as C3H/HeJ and C3H/HeJ Bir were found to be highly susceptible, while others such as NON/LtJ were quite resistant to DSS colitis (Mahler et al., 1998). Our results are in agreement with previously published study regarding the intensity of clinical symptoms between C57BL/6 and CD26-/- mice. Given the fact that there is no statistically significant difference in the intensity of clinical symptoms between mice strains it could be suggested that CD26 deficiency does not increase resistance

Functional studies have demonstrated that inhibition of DPP IV/CD26 enzyme activity may lead to changes in chemokine regulation and a subsequent immunological effect, while *in vitro* studies using activated T lymphocytes have shown that inhibition of DPP IV/CD26 activity can result in a decreased secretion of proinflammatory cytokines, including TNF-α and IFN-γ as well as an increase in the anti-inflammatory cytokine TGF-β. This evidence suggests that DPP IV/CD26 enzyme activity plays an essential role in the immune response and therefore, its enzymatic role is being extensively investigated. In our study, a statistically significant decrease in serum DPP IV/CD26 activity was observed in the acute phase in serum of C57BL/6 mice. The results regarding serum DPP IV/CD26 activity established in a DSS mouse model of colitis are consistent with our previous work in patients with IBD (Varljen et al., 2005). Furthermore, a decrease in DSS colitis disease activity was observed in wild type mice treated with inhibitors but on the other hand the inhibitors were not effective in CD26-/- animals (Yazbeck et al., 2010). Concurrently, during colitis development, an increased expression of DPP8 in wild type and CD26-/- animals and

Considering that DPP IV/CD26 is a member of a large S9b family of structurally homologous serine proteases that possess a unique catalytic activity, and since two recent studies have demonstrated a broad tissue distribution of DPP IV-like enzyme activity in both wild type and CD26-/-, a possible explanation of results obtained in our study could be that other DPP IV-like protease are involved in the activation of the inflammatory response in animal model of colitis (Ansorge et al., 2009; Yu et al., 2009). Furthermore, it was recently demonstrated by Yazbeck and its coworkers (Yazbeck et al., 2008) that inhibition of DPP-like activity ameliorates the severity of inflammation in experimental colitis in mice. However, further studies are required to characterize the role of DPP IV-like proteins in the initiation and activation of immune mechanisms leading to intestinal inflammation and development

One of the most widely used and accepted Crohn-like colitis in scientific research is the TNBS-induced colitis. The TNBS-colitis resembles human Crohn's disease in different aspects, from the clinical manifestation, histological appearance and immunological features. TNBS-colitis is induced in experimental animals by rectal application of TNBS in an adequate, experimentally determined dilution of ethanol, usually 30 to 50%. Ethanol serves as a barrier-breaker which allows TNBS molecules, a contact-sensitizing agent, to enter in deeper layers of the colonic mucosa. The mechanism of TNBS-induced

to the development of DSS-induced experimental colitis (Geier et al., 2005).

**4.1.3 DPP IV/CD26 and DPP IV/CD26-like activity in DSS-colitis** 

DPP2 mRNA expression in wild type animals was observed.

**4.2 TNBS-induced colitis (Crohn-like model of colitis)**

of IBD.

previously reported findings. Furthermore, our findings suggest and confirm that the DSS model of colitis, because of similarities to human IBD, represents a good model to study the molecular and immune mechanisms activated during colitis development and resolution.

In accordance with previously published histological data regarding colonic inflammation present in DSS model of colitis, inflammatory changes are superficial, mainly affecting the mucosa, but may extend to the submucosa and the muscularis mucosa as well. The inflammation is characterized by superficial ulcers, mucosal oedema, crypt distortion and mucosal inflammatory cell infiltration with large numbers of neutrophils, macrophages and lymphocytes (Cooper et al., 1993). *Pathohistological* and *morphometrical analyses* of colon tissue sections confirmed the presence of inflammatory changes in both mice strains. During colitis development, a statistically significant decrease in number of crypts of Lieberkühn per milimetar of mucosa followed by its shortening was recorded in both mice strains along with the infiltration of inflammatory cells in lamina propria (Fig. 7). In the acute phase ( day 7), crypt architectural distortion reached its maximum and during this phase, typical sign of disease, patches of totally destroyed epithelial sheet with deep ulcerations, can be seen. The resolution of inflammation and regeneration of crypts started during the second week and finished on the day 15 (Fig. 7D). In this period, mononuclear types of inflammatory cells were predominant.

Fig. 7. Histological changes in colon tissues during dextran sulfate sodium-induced (DSS) colitis development and resolution in CD26-/- animals. Normal colon (A), acute phase of colitis (B) and process of tissue damage resolution (C, D). Colon sections (2 μm) were stained with hematoxylin and eosin and examined for histological properties. Magnification: 20x (A, C, D); 10x (B).

 

previously reported findings. Furthermore, our findings suggest and confirm that the DSS model of colitis, because of similarities to human IBD, represents a good model to study the molecular and immune mechanisms activated during colitis development and resolution. In accordance with previously published histological data regarding colonic inflammation present in DSS model of colitis, inflammatory changes are superficial, mainly affecting the mucosa, but may extend to the submucosa and the muscularis mucosa as well. The inflammation is characterized by superficial ulcers, mucosal oedema, crypt distortion and mucosal inflammatory cell infiltration with large numbers of neutrophils, macrophages and lymphocytes (Cooper et al., 1993). *Pathohistological* and *morphometrical analyses* of colon tissue sections confirmed the presence of inflammatory changes in both mice strains. During colitis development, a statistically significant decrease in number of crypts of Lieberkühn per milimetar of mucosa followed by its shortening was recorded in both mice strains along with the infiltration of inflammatory cells in lamina propria (Fig. 7). In the acute phase ( day 7), crypt architectural distortion reached its maximum and during this phase, typical sign of disease, patches of totally destroyed epithelial sheet with deep ulcerations, can be seen. The resolution of inflammation and regeneration of crypts started during the second week and finished on the day 15 (Fig. 7D). In this period, mononuclear types of inflammatory cells

Fig. 7. Histological changes in colon tissues during dextran sulfate sodium-induced (DSS) colitis development and resolution in CD26-/- animals. Normal colon (A), acute phase of colitis (B) and process of tissue damage resolution (C, D). Colon sections (2 μm) were stained with hematoxylin and eosin and examined for histological properties.

C D

A B

were predominant.

Magnification: 20x (A, C, D); 10x (B).

 

There is evidence that susceptibility to DSS varies with the animal species and mice strain. Guinea pig is the most susceptible, with inflammation usually fully established in less than 72 h (Iwanaga et al., 1994). In mice, some strains such as C3H/HeJ and C3H/HeJ Bir were found to be highly susceptible, while others such as NON/LtJ were quite resistant to DSS colitis (Mahler et al., 1998). Our results are in agreement with previously published study regarding the intensity of clinical symptoms between C57BL/6 and CD26-/- mice. Given the fact that there is no statistically significant difference in the intensity of clinical symptoms between mice strains it could be suggested that CD26 deficiency does not increase resistance to the development of DSS-induced experimental colitis (Geier et al., 2005).

#### **4.1.3 DPP IV/CD26 and DPP IV/CD26-like activity in DSS-colitis**

Functional studies have demonstrated that inhibition of DPP IV/CD26 enzyme activity may lead to changes in chemokine regulation and a subsequent immunological effect, while *in vitro* studies using activated T lymphocytes have shown that inhibition of DPP IV/CD26 activity can result in a decreased secretion of proinflammatory cytokines, including TNF-α and IFN-γ as well as an increase in the anti-inflammatory cytokine TGF-β. This evidence suggests that DPP IV/CD26 enzyme activity plays an essential role in the immune response and therefore, its enzymatic role is being extensively investigated. In our study, a statistically significant decrease in serum DPP IV/CD26 activity was observed in the acute phase in serum of C57BL/6 mice. The results regarding serum DPP IV/CD26 activity established in a DSS mouse model of colitis are consistent with our previous work in patients with IBD (Varljen et al., 2005). Furthermore, a decrease in DSS colitis disease activity was observed in wild type mice treated with inhibitors but on the other hand the inhibitors were not effective in CD26-/- animals (Yazbeck et al., 2010). Concurrently, during colitis development, an increased expression of DPP8 in wild type and CD26-/- animals and DPP2 mRNA expression in wild type animals was observed.

Considering that DPP IV/CD26 is a member of a large S9b family of structurally homologous serine proteases that possess a unique catalytic activity, and since two recent studies have demonstrated a broad tissue distribution of DPP IV-like enzyme activity in both wild type and CD26-/-, a possible explanation of results obtained in our study could be that other DPP IV-like protease are involved in the activation of the inflammatory response in animal model of colitis (Ansorge et al., 2009; Yu et al., 2009). Furthermore, it was recently demonstrated by Yazbeck and its coworkers (Yazbeck et al., 2008) that inhibition of DPP-like activity ameliorates the severity of inflammation in experimental colitis in mice. However, further studies are required to characterize the role of DPP IV-like proteins in the initiation and activation of immune mechanisms leading to intestinal inflammation and development of IBD.

#### **4.2 TNBS-induced colitis (Crohn-like model of colitis)**

One of the most widely used and accepted Crohn-like colitis in scientific research is the TNBS-induced colitis. The TNBS-colitis resembles human Crohn's disease in different aspects, from the clinical manifestation, histological appearance and immunological features. TNBS-colitis is induced in experimental animals by rectal application of TNBS in an adequate, experimentally determined dilution of ethanol, usually 30 to 50%. Ethanol serves as a barrier-breaker which allows TNBS molecules, a contact-sensitizing agent, to enter in deeper layers of the colonic mucosa. The mechanism of TNBS-induced

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 75

treatment allocation scored microscopical changes, which included overall severity of

The DPP IV/CD26 (in C57BL/6) and DPP IV/CD26-like *enzymatic activities* (in CD26-/ mice) in mice serum, brain and colon homogenates were measured according to the protocol

Brain and colon samples for *Western blot analyses* of CD26 molecule expression were homogenized on ice using RIPA lysis buffer including inhibitors of proteases and phosphatases. After that, homogenates were centrifuged at 14000 rpm for 20 minutes at +4°C and resulting supernatants were measured for total protein concentrations according to the method of Bradford (Bradford, 1976). Equal amounts of total proteins were separated by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Proteins were transferred from the gels to polyvinylidenedifluoride membranes by semi-dry electroblotting. Membranes were incubated overnight with primary anti-CD26 (Santa Cruz Biotechnology Inc., CA), followed by 45 min incubation with secondary antibody, horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Santa Cruz Biotechnology Inc., CA). Membranes were incubated with chemiluminescent Amersham ECL-plus Western blotting detection reagents (Amersham, Little Chalfont, UK) and bands revealing protein expression of the CD26 molecule were visualized after exposure to photosensitive films (AGFA Ortho CP-G plus). Equal total protein loading was ensured with use of the primary mouse ß-actin antibody (Chemicon International, USA), and secondary horseradish

mucosal damage, number of crypts of Lieberkühn and their depth and width.

peroxidase-conjugated goat-anti-mouse IgG (Santa Cruz Biotechnology Inc., CA).

All groups of experimental mice were monitored daily for their body weights, stool consistence and presence of blood, eventual occurrence of rectal bleeding and general clinical state. Characteristic findings associated with intrarectal administration of TNBS solution in mice included their poor clinical state, body weight loss up to 15% and a mortality rate of approximately 11% in both CD26-/- and C57BL/6 mice. Disease symptoms

Body mass of individual experimental animals of all groups of both investigated mice strains were determined each day at about the same time, starting from the day of application of TNBS-ethanol solution, ethanol solution or saline, until the day of sacrifice (the second, seventh, fifteenth and thirtieth day). It has been noticed that body weights of CD26-/- animals, as compared to C57BL/6 strain of animals of the same age and gender, are statistically significantly different (*P* < 0.05) and amount (25.29±2.23)g for C57BL/6 and (23.41±2.51)g for CD26-/- animals. Lower body mass of CD26-/- animals in comparison with their genetic background (C57BL/6) mice as well as enhanced insulin secretion and improved glucose tolerance in mice lacking CD26 was reported before (Marguet et al., 2000). The weights of the brain, liver and spleen were measured analytically for each experimental

It was noticed that weights of livers and spleens were slightly higher in CD26-/- animals as compared to the corresponding groups of C57BL/6 animals, nevertheless their lower body mass. The trend of reduction in weight of liver and spleen on the second day of induction of colitis was observed in both strains of experimental animals. The hepatosomatic index (relative ratio of liver weight and body mass) and the relative ratio of spleen weight and body mass of animals were calculated for all animals. It was found that CD26-/- mice have statistically significantly higher (*P* < 0.05) values of the hepatosomatic index compared to

of (Kreisel et al., 1982), as described in section 3.2.1.

**4.2.3 Evaluation of TNBS-colitis assessment** 

animal and presented in Table 4.

were mostly pronounced in the first five days of experiment.

inflammation involves reaction of TNBS, which is a hapten, with tissue host proteins. TNBS is a covalently reactive compound that attachs to autologous proteins and stimulates a delayed-type hypersensitivity response (Camoglio et al., 2000). This generates a variety of new antigens in situ, as well as stimulates the production of proinflammatory molecules and free radicals which initiate a whole cascade of complex immunological interactions (Grisham et al., 1991). The colonic administration of a single dose of TNBS/ethanol solution induces in mice and rats a granulomatous, transmural inflammation with tissue destruction, mainly localized in the distal part of the colon (Scheiffele & Fuss, 2002).

#### **4.2.1 Induction of TNBS-colitis in mice**

Two mice strains were used in our study: wild type mice strain C57BL/6 and mice with inactivated gene for molecule CD26 (C57BL/6 Jbom-ob, CD26-/-), generated on a C57BL/6 genetic background. CD26-/- mice were kindly provided by Dr. Didier Marguet, Centre d'Immunologie Marseille-Luminy, France. Generation of CD26-/- mice has been described previously (Marguet et al., 2000). Male, 8-10-week-old mice were used in the study. Animals were housed and bred under standard conditions at the Central Animal Facility of the School of Medicine, University of Rijeka. Laboratory animals were housed in plastic cages, fed with standard pellet food (MK, Complete Diet for Laboratory Rats and Mice, Slovenia), given tap water *ad libitum* and maintained under a 12/12 hours dark/light cycle at constant temperature (20±1)°C and humidity (50±5)%. Each study group comprised 8-10 experimental animals. Handling with animals, experimental procedure and anesthesia were performed in accordance with the general principles contained in the Guide for the Care and Use of Laboratory Animals (National Academic Press). The Ethical Committee of the School of Medicine, University of Rijeka, approved all experimental procedures.

TNBS-colitis was induced by rectal administration of 5% (w/v) TNBS (Sigma-Aldrich, Germany) dissolved in 50% ethanol (Kemika, Croatia). Each animal received 0.1 mL of TNBS-ethanol solution, using a vinyl catheter that was positioned 4 cm from the anus, according to the protocol of (Scheiffele & Fuss, 2002). Two control groups of mice were used for each mice strain. Control mice underwent identical procedures, but were instilled equal volumes of saline (NaCl 0.9%) or ethanol solution. Mice were anesthetized with ketamine/xylazine while receiving TNBS, saline or ethanol solution.

## **4.2.2 Analytical methods**

Experimental animals were sacrificed by cervical dislocation after 2, 7, 15 and 30 days upon administration of TNBS, saline or ethanol solution. Peripheral blood samples were taken and serum samples were collected by centrifugation at 3000 rpm for 10 minutes. Livers and spleens were isolated and their weights were noted. Colons were freed from adhering tissue and macroscopic changes were noted. The colon lumen was carefully washed with ice-cold saline, its weight and length was measured after which underwent homogenization procedure. Brains were separated immediately after sacrifice, washed in ice-cold saline and then homogenized on ice. Colon and brain homogenates were centrifuged at 14000 rpm for 20 minutes at +4°C. Resulting supernatants were measured for total protein concentrations according to the method of Bradford (Bradford, 1976).

Colon tissues for *histological and histomorphometrical analyses* were collected and xed in 4% formalin for 24 h. Samples were processed and embedded in paraffin wax. Two-micrometer sections were stained with hematoxylin and eosin. An experienced pathologist blinded to

inflammation involves reaction of TNBS, which is a hapten, with tissue host proteins. TNBS is a covalently reactive compound that attachs to autologous proteins and stimulates a delayed-type hypersensitivity response (Camoglio et al., 2000). This generates a variety of new antigens in situ, as well as stimulates the production of proinflammatory molecules and free radicals which initiate a whole cascade of complex immunological interactions (Grisham et al., 1991). The colonic administration of a single dose of TNBS/ethanol solution induces in mice and rats a granulomatous, transmural inflammation with tissue destruction,

Two mice strains were used in our study: wild type mice strain C57BL/6 and mice with inactivated gene for molecule CD26 (C57BL/6 Jbom-ob, CD26-/-), generated on a C57BL/6 genetic background. CD26-/- mice were kindly provided by Dr. Didier Marguet, Centre d'Immunologie Marseille-Luminy, France. Generation of CD26-/- mice has been described previously (Marguet et al., 2000). Male, 8-10-week-old mice were used in the study. Animals were housed and bred under standard conditions at the Central Animal Facility of the School of Medicine, University of Rijeka. Laboratory animals were housed in plastic cages, fed with standard pellet food (MK, Complete Diet for Laboratory Rats and Mice, Slovenia), given tap water *ad libitum* and maintained under a 12/12 hours dark/light cycle at constant temperature (20±1)°C and humidity (50±5)%. Each study group comprised 8-10 experimental animals. Handling with animals, experimental procedure and anesthesia were performed in accordance with the general principles contained in the Guide for the Care and Use of Laboratory Animals (National Academic Press). The Ethical Committee of the School

TNBS-colitis was induced by rectal administration of 5% (w/v) TNBS (Sigma-Aldrich, Germany) dissolved in 50% ethanol (Kemika, Croatia). Each animal received 0.1 mL of TNBS-ethanol solution, using a vinyl catheter that was positioned 4 cm from the anus, according to the protocol of (Scheiffele & Fuss, 2002). Two control groups of mice were used for each mice strain. Control mice underwent identical procedures, but were instilled equal volumes of saline (NaCl 0.9%) or ethanol solution. Mice were anesthetized with

Experimental animals were sacrificed by cervical dislocation after 2, 7, 15 and 30 days upon administration of TNBS, saline or ethanol solution. Peripheral blood samples were taken and serum samples were collected by centrifugation at 3000 rpm for 10 minutes. Livers and spleens were isolated and their weights were noted. Colons were freed from adhering tissue and macroscopic changes were noted. The colon lumen was carefully washed with ice-cold saline, its weight and length was measured after which underwent homogenization procedure. Brains were separated immediately after sacrifice, washed in ice-cold saline and then homogenized on ice. Colon and brain homogenates were centrifuged at 14000 rpm for 20 minutes at +4°C. Resulting supernatants were measured for total protein concentrations

Colon tissues for *histological and histomorphometrical analyses* were collected and xed in 4% formalin for 24 h. Samples were processed and embedded in paraffin wax. Two-micrometer sections were stained with hematoxylin and eosin. An experienced pathologist blinded to

mainly localized in the distal part of the colon (Scheiffele & Fuss, 2002).

of Medicine, University of Rijeka, approved all experimental procedures.

ketamine/xylazine while receiving TNBS, saline or ethanol solution.

according to the method of Bradford (Bradford, 1976).

**4.2.1 Induction of TNBS-colitis in mice** 

**4.2.2 Analytical methods**

treatment allocation scored microscopical changes, which included overall severity of mucosal damage, number of crypts of Lieberkühn and their depth and width.

The DPP IV/CD26 (in C57BL/6) and DPP IV/CD26-like *enzymatic activities* (in CD26-/ mice) in mice serum, brain and colon homogenates were measured according to the protocol of (Kreisel et al., 1982), as described in section 3.2.1.

Brain and colon samples for *Western blot analyses* of CD26 molecule expression were homogenized on ice using RIPA lysis buffer including inhibitors of proteases and phosphatases. After that, homogenates were centrifuged at 14000 rpm for 20 minutes at +4°C and resulting supernatants were measured for total protein concentrations according to the method of Bradford (Bradford, 1976). Equal amounts of total proteins were separated by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Proteins were transferred from the gels to polyvinylidenedifluoride membranes by semi-dry electroblotting. Membranes were incubated overnight with primary anti-CD26 (Santa Cruz Biotechnology Inc., CA), followed by 45 min incubation with secondary antibody, horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Santa Cruz Biotechnology Inc., CA). Membranes were incubated with chemiluminescent Amersham ECL-plus Western blotting detection reagents (Amersham, Little Chalfont, UK) and bands revealing protein expression of the CD26 molecule were visualized after exposure to photosensitive films (AGFA Ortho CP-G plus). Equal total protein loading was ensured with use of the primary mouse ß-actin antibody (Chemicon International, USA), and secondary horseradish peroxidase-conjugated goat-anti-mouse IgG (Santa Cruz Biotechnology Inc., CA).

#### **4.2.3 Evaluation of TNBS-colitis assessment**

All groups of experimental mice were monitored daily for their body weights, stool consistence and presence of blood, eventual occurrence of rectal bleeding and general clinical state. Characteristic findings associated with intrarectal administration of TNBS solution in mice included their poor clinical state, body weight loss up to 15% and a mortality rate of approximately 11% in both CD26-/- and C57BL/6 mice. Disease symptoms were mostly pronounced in the first five days of experiment.

Body mass of individual experimental animals of all groups of both investigated mice strains were determined each day at about the same time, starting from the day of application of TNBS-ethanol solution, ethanol solution or saline, until the day of sacrifice (the second, seventh, fifteenth and thirtieth day). It has been noticed that body weights of CD26-/- animals, as compared to C57BL/6 strain of animals of the same age and gender, are statistically significantly different (*P* < 0.05) and amount (25.29±2.23)g for C57BL/6 and (23.41±2.51)g for CD26-/- animals. Lower body mass of CD26-/- animals in comparison with their genetic background (C57BL/6) mice as well as enhanced insulin secretion and improved glucose tolerance in mice lacking CD26 was reported before (Marguet et al., 2000). The weights of the brain, liver and spleen were measured analytically for each experimental animal and presented in Table 4.

It was noticed that weights of livers and spleens were slightly higher in CD26-/- animals as compared to the corresponding groups of C57BL/6 animals, nevertheless their lower body mass. The trend of reduction in weight of liver and spleen on the second day of induction of colitis was observed in both strains of experimental animals. The hepatosomatic index (relative ratio of liver weight and body mass) and the relative ratio of spleen weight and body mass of animals were calculated for all animals. It was found that CD26-/- mice have statistically significantly higher (*P* < 0.05) values of the hepatosomatic index compared to

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 77

reduction of the weight of the spleen on the second day of induction of colitis in both strains of experimental animals, due to a reduction in body weights no statistically significant reduction in their relative ratios was observed. Statistically significant increase in the relative ratio of spleen weight and body mass was recorded in the group of CD26-/- animals on the fifteenth day of the induction of colitis, compared to the corresponding control group

*Macroscopic examination* of the distal part of the colon discovered localized inflammation with several ulcerations, mucosal erosions and bowel obstruction with enhanced edematous changes. Colon shortening and thickening with marked colonic edema, accompanied by increased colon weight and presence of hemorrhagic changes was most prominent two days following TNBS administration (Fig. 8). Therefore, day 2 of experiment was classified as acute phase of colitis, which is in accordance with previously reported findings (Scheiffele &

Fig. 8. Macroscopic appearance of the distal part of mice colons in the acute phase of colitis,

Results of our histopathological analyses of colon tissue sections in wild type and CD26-/ mice that received TNBS-ethanol solution confirmed the presence of inflammatory processes and accomplishment of colitis induction. *Microscopic changes*, as well as macroscopic, were most conspicuous in the acute phase of colitis. *Pathohistological analyses* confirmed the presence of inflammatory changes very similar to those seen in human Crohn's disease and revealed that under physiological conditions no differences in histological architecture was

Pathohistological analyses of a wider number of colonic section samples discovered some differences in the manifestation of inflammatory processes between CD26-/- and wild type mice: in CD26-/- mice, ulcerations were mainly localized in one part of the mucosal surface, and inflammatory changes did not overtake the entire mucosa. In most analyzed colon samples from CD26-/- mice, a part of the colonic mucosa was preserved with physiological appearance of crypts, but a transmural inflammation was observed in a number of mice (Fig. 10A). On the other hand, no transmural inflammatory changes were observed in wild

two days after administration of TNBS-ethanol solution.

observed between analyzed mice strains (Fig. 9).

and compared to C57BL/6 animals sacrificed on the same day.

Fuss, 2002).


Table 4. Average brain, liver and spleen weights (g) for different groups of experimental animals at scheduled days of experiment.

C57BL/6 animals. Hepatosomatic index decreased the second day of colitis induction in both strains of experimental animals, as a result of reduction in liver weight, despite of body weight loss. On the other hand, analyzed mice strains did not differ statistically significantly in the relative values of the ratio of spleen weight and body weight in physiological conditions, as well as in the control group treated with ethanol solution. Regardless of the

0.02273

0.02117

0.01498

0.03687

0.02342

0.02273

0.01527

0.00435

0.25067

0.03043

0.02409

0.02203

0.02631

0.01740

0.01315

0.02409

0.02964

0.02331

0.03450

0.02402

Table 4. Average brain, liver and spleen weights (g) for different groups of experimental

C57BL/6 animals. Hepatosomatic index decreased the second day of colitis induction in both strains of experimental animals, as a result of reduction in liver weight, despite of body weight loss. On the other hand, analyzed mice strains did not differ statistically significantly in the relative values of the ratio of spleen weight and body weight in physiological conditions, as well as in the control group treated with ethanol solution. Regardless of the

**experiment Brain mass (g) Liver mass (g) Spleen mass (g)** 

1.32092 ± 0.11335

0.89232 ± 0.14574

1.12842 ± 0.19671

1.24861 ± 0.12537

1.36642 ± 0.14692

1.32092 ± 0.11335

1.27890 ± 0.15756

1.23160 ± 0.05487

1.26048 ± 0.05868

1.35711 ± 0.17414

1.43343 ± 0.12470

1.08765 ± 0.12792

1.53770 ± 0.07895

1.47498 ± 0.18033

1.36693 ± 0.10567

1.43343 ± 0.12470

1.33858 ± 0.22979

1.38604 ± 0.07852

1.24388 ± 0.08975

1.34925 ± 0.24189

0.07822 ± 0.00951

0.07596 ± 0.01806

0.08650 ± 0.02623

0.08424 ± 0.02037

0.07707 ± 0.01311

0.07822 ± 0.00951

0.07680 ± 0.01630

0.07462 ± 0.00938

0.07630 ± 0.01327

0.07728 ± 0.00697

0.08170 ± 0.01447

0.06012 ± 0.01982

0.11080 ± 0.04752

0.11300 ± 0.01758

0.09158 ± 0.02720

0.08170 ± 0.01447

0.08222 ± 0.03727

0.08058 ± 0.00259

0.08260 ± 0.00995

0.08338 ± 0.01257

**Day of** 

colitis 2 0.44733 ±

colitis 7 0.44019 ±

colitis 15 0.44037 ±

colitis 30 0.44113 ±

control 2 0.44716 ±

control 7 0.44488 ±

control 15 0.44838 ±

control 30 0.43701 ±

colitis 2 0.41836 ±

colitis 7 0.42448 ±

colitis 15 0.42556 ±

colitis 30 0.41040 ±

**C57BL/6** physiological 0 0.44776 ±

**C57BL/6** physiological 0 0.44776 ±

**CD26-/-** physiological 0 0.41532 ±

**CD26-/-** physiological 0 0.41532 ±

control 2 0.40117 ±

control 7 0.41232 ±

control 15 0.41924 ±

control 30 0.42334 ±

animals at scheduled days of experiment.

**Mice strain**  **Experimental group** 

reduction of the weight of the spleen on the second day of induction of colitis in both strains of experimental animals, due to a reduction in body weights no statistically significant reduction in their relative ratios was observed. Statistically significant increase in the relative ratio of spleen weight and body mass was recorded in the group of CD26-/- animals on the fifteenth day of the induction of colitis, compared to the corresponding control group and compared to C57BL/6 animals sacrificed on the same day.

*Macroscopic examination* of the distal part of the colon discovered localized inflammation with several ulcerations, mucosal erosions and bowel obstruction with enhanced edematous changes. Colon shortening and thickening with marked colonic edema, accompanied by increased colon weight and presence of hemorrhagic changes was most prominent two days following TNBS administration (Fig. 8). Therefore, day 2 of experiment was classified as acute phase of colitis, which is in accordance with previously reported findings (Scheiffele & Fuss, 2002).

Fig. 8. Macroscopic appearance of the distal part of mice colons in the acute phase of colitis, two days after administration of TNBS-ethanol solution.

Results of our histopathological analyses of colon tissue sections in wild type and CD26-/ mice that received TNBS-ethanol solution confirmed the presence of inflammatory processes and accomplishment of colitis induction. *Microscopic changes*, as well as macroscopic, were most conspicuous in the acute phase of colitis. *Pathohistological analyses* confirmed the presence of inflammatory changes very similar to those seen in human Crohn's disease and revealed that under physiological conditions no differences in histological architecture was observed between analyzed mice strains (Fig. 9).

Pathohistological analyses of a wider number of colonic section samples discovered some differences in the manifestation of inflammatory processes between CD26-/- and wild type mice: in CD26-/- mice, ulcerations were mainly localized in one part of the mucosal surface, and inflammatory changes did not overtake the entire mucosa. In most analyzed colon samples from CD26-/- mice, a part of the colonic mucosa was preserved with physiological appearance of crypts, but a transmural inflammation was observed in a number of mice (Fig. 10A). On the other hand, no transmural inflammatory changes were observed in wild

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 79

in observed parameters, nor at different days of sacrifice. In both mice strains with induced colitis, a statistically significant (*P* < 0.05) decrease in number of crypts of Lieberkühn per mm of mucosa was observed in the acute phase of colitis. Changes persisted even during tissue healing in CD26-/- mice. The width of crypts of Lieberkühn was increased in the acute phase of colitis in both mice strains, but it took longer to achieve physiological values in wild type mice. Furthermore, the depth of crypts of Lieberkühn was decreased in acute colitis in both mice strains. All those changes represent consequences of inflammatory processes in the colon which include mucosa thickening and formation of edema due to

Measurements of DPP IV/CD26 activity and protein expression in wild type mice were performed at systemic and local levels, in the serum and within the gut-brain axis respectively. Furthermore, in order to evaluate whether in conditions of DPP IV/CD26 deficiency other DPP IV/CD26-like enzymes could partially undertake its enzymatic function, DPP IV/CD26-like systemic and local activities were determined in CD26-/- mice. Results of investigations concerning DPP IV/CD26 and DPP IV/CD26-like molecules in TNBS-induced colitis in mice reviewed here are accepted for publication in *Croatica Chemica Acta* (*in press*, vol.no.4, 2011). Fig. 11 shows results of serum DPP IV/CD26 activity in wild

TNBS-ethanol-induced tissue damage.

**4.2.4 DPP IV/CD26 and DPP IV/CD26-like activity in TNBS-colitis** 

type mice with induced colitis compared to control groups.

a, statistically significantly different compared to control group (*P* < 0.05)

solution (colitis group) or ethanol solution (control group).

resolution compared to control group.

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol

Fig. 11. Serum DPP IV/CD26 activity in C57BL/6 mice during colitis development and

A statistically significant decrease (*P* < 0.05) in serum DPP IV/CD26 activity, starting in the acute phase of colitis and achieving physiological values after disease healing could be seen. Our results accord with previously published results that included determination of serum DPP IV/CD26 activity in patients with IBD, as described before in this chapter. Furthermore, our results are in accordance with the observation that serum DPP IV/CD26 activity correlates inversely with disease severity in patients with IBD (Varljen et al., 2005), since the lowest DPP IV/CD26 activity in mice was found in the acute phase of disease.

type animals, but in a number of experimental animals, inflammatory processes affected the entire colonic circumference with very little or no areas of preserved mucosa (Fig. 10B).

Fig. 9. Histological appearance of colonic tissue sections of CD26-/- (A) and wild type mice (B) two days after application of saline solution. Colon sections (2 μm) were stained with hematoxylin and eosin and examined for histological properties. Magnification: 10x.

Fig. 10. Pathohistological appearance of colonic tissue sections of CD26-/- (A, C) and wild type mice (B, D) in the acute phase of colitis, two days after application of TNBS-ethanol solution. Colon sections (2 μm) were stained with hematoxylin and eosin and examined for histological properties. Magnification: 4x (A, B), 10x (D) and 20x (C).

Results of *histomorphometrical analyses* also confirmed the presence of inflammatory changes in both mice strains that received TNBS-ethanol solution. Number of crypts of Lieberkühn per mm of mucosa, and their depth and width for different groups of both mice strains at given days of experiment were measured (data not shown). Statistical analyses of obtained results among both control groups of animals did not reveal statistically significant changes

type animals, but in a number of experimental animals, inflammatory processes affected the entire colonic circumference with very little or no areas of preserved mucosa (Fig. 10B).

A B Fig. 9. Histological appearance of colonic tissue sections of CD26-/- (A) and wild type mice (B) two days after application of saline solution. Colon sections (2 μm) were stained with hematoxylin and eosin and examined for histological properties. Magnification: 10x.

Fig. 10. Pathohistological appearance of colonic tissue sections of CD26-/- (A, C) and wild type mice (B, D) in the acute phase of colitis, two days after application of TNBS-ethanol solution. Colon sections (2 μm) were stained with hematoxylin and eosin and examined for

Results of *histomorphometrical analyses* also confirmed the presence of inflammatory changes in both mice strains that received TNBS-ethanol solution. Number of crypts of Lieberkühn per mm of mucosa, and their depth and width for different groups of both mice strains at given days of experiment were measured (data not shown). Statistical analyses of obtained results among both control groups of animals did not reveal statistically significant changes

histological properties. Magnification: 4x (A, B), 10x (D) and 20x (C).

CD26-/- C57BL/6

A B

C D

in observed parameters, nor at different days of sacrifice. In both mice strains with induced colitis, a statistically significant (*P* < 0.05) decrease in number of crypts of Lieberkühn per mm of mucosa was observed in the acute phase of colitis. Changes persisted even during tissue healing in CD26-/- mice. The width of crypts of Lieberkühn was increased in the acute phase of colitis in both mice strains, but it took longer to achieve physiological values in wild type mice. Furthermore, the depth of crypts of Lieberkühn was decreased in acute colitis in both mice strains. All those changes represent consequences of inflammatory processes in the colon which include mucosa thickening and formation of edema due to TNBS-ethanol-induced tissue damage.

#### **4.2.4 DPP IV/CD26 and DPP IV/CD26-like activity in TNBS-colitis**

Measurements of DPP IV/CD26 activity and protein expression in wild type mice were performed at systemic and local levels, in the serum and within the gut-brain axis respectively. Furthermore, in order to evaluate whether in conditions of DPP IV/CD26 deficiency other DPP IV/CD26-like enzymes could partially undertake its enzymatic function, DPP IV/CD26-like systemic and local activities were determined in CD26-/- mice. Results of investigations concerning DPP IV/CD26 and DPP IV/CD26-like molecules in TNBS-induced colitis in mice reviewed here are accepted for publication in *Croatica Chemica Acta* (*in press*, vol.no.4, 2011). Fig. 11 shows results of serum DPP IV/CD26 activity in wild type mice with induced colitis compared to control groups.

a, statistically significantly different compared to control group (*P* < 0.05) 0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol solution (colitis group) or ethanol solution (control group).

Fig. 11. Serum DPP IV/CD26 activity in C57BL/6 mice during colitis development and resolution compared to control group.

A statistically significant decrease (*P* < 0.05) in serum DPP IV/CD26 activity, starting in the acute phase of colitis and achieving physiological values after disease healing could be seen. Our results accord with previously published results that included determination of serum DPP IV/CD26 activity in patients with IBD, as described before in this chapter. Furthermore, our results are in accordance with the observation that serum DPP IV/CD26 activity correlates inversely with disease severity in patients with IBD (Varljen et al., 2005), since the lowest DPP IV/CD26 activity in mice was found in the acute phase of disease.

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 81

(Fig. 13A). On the other hand, an increased CD26 protein expression in the acute phase of

**0 2 7 15 30 Time (day)**

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol

Fig. 13. DPP IV/CD26 activity (A) and protein expression (B) in colon of C57BL/6 mice

Besides a regulatory system at the enzymatic level, this observed fact could also be partly explained as a compensatory mechanism, considering that a part of the decreased DPP IV/CD26 activity in the colon of wild type mice is indeed a consequence of severe mucosal damage induced by TNBS-ethanol solution. Consequently, increased CD26 protein expression in the acute phase of disease could represent an effort to realize a compensatory mechanism. Since damaged DPP IV/CD26 conformation is present in inflamed tissue, with the consequence of an improper enzyme activity, enhanced DPP IV/CD26 protein expression could represent a mechanism of feed-back. Our results are in agreement with previously reported observations regarding enhanced CD26 mRNA production in inflamed

We have determined that, in physiological conditions, CD26-/- mice express less than 2% of total DPP IV/CD26 activity detected in the colon of wild type mice. This enzyme activity was defined as DPP IV/CD26-like activity in the colon and was investigated during colitis development and resolution, as well. Our results showed statistically significantly (*P* < 0.05) decreased DPP IV/CD26-like activity in inflamed colon homogenates in CD26-/- mice,

a

**C57BL/6 colitis C57BL/6 control group**

 **0 2 7 15 30 B**

 **110 kDa**

**A**

 **42 kDa**

a a

disease was revealed by Western blotting technique (Fig. 13B).

**0,00 0,05 0,10 0,15 0,20 0,25 0,30 0,35 0,40**

 **CD26**

**ß-actin**

during colitis development and resolution.

tissue (Nemoto et al., 1999).

compared to their controls (Fig. 14).

solution (colitis group) or ethanol solution (control group).

a, statistically significantly different compared to control group (*P* < 0.05).

**Enzyme activity** 

**(nkat/mg protein)**

Furthermore, we wanted to evaluate possible changes in serum DPP IV/CD26-like activities during colitis development and healing as well. Therefore, CD26-/- mice with induced colitis as well as their control groups were analyzed. Obtained results showed that CD26-/- mice express approximately 10% of total serum DPP IV/CD26 activity detected in wild type mice. Fig. 12 shows results of serum DPP IV/CD26-like activity in CD26-/- mice with induced colitis compared to their control groups.

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol solution (colitis group) or ethanol solution (control group).

Fig. 12. Serum DPP IV/CD26-like activity in CD26-/- mice during colitis development and resolution compared to control group.

Our results indicated that there are no statistically significant differences in serum DPP IV/CD26-like activity between groups of CD26-/- animals with colitis and their control groups. Therefore, the significance of DPP IV/CD26 over DPP IV/CD26-like serum activity is proposed.

#### **4.2.5 DPP IV/CD26, IBD and the gut-brain axis**

Growing scientific evidence emphasizes neuroimmunomodulation as an important factor in the occurrence of inflammatory and autoimmune processes, as described in different investigations in the last few years (Ohman and Simren, 2010). The complex causal connection between central and enteric nervous system caused the introduction of the term **gut-brain axis** (Romijn et al., 2008). DPP IV/CD26 has previously been shown to play a key role in the metabolism of important bioactive neuro- and immunopeptides, as well as in the activation of the immune response (Vanderheyden et al., 2009). Due to its localization on the cell surface of the nervous and digestive system, likewise on the surface of important immune cells (Matteucci & Giampietro, 2009), we aimed to investigate possible changes in DPP IV/CD26 activity and protein expression at sight of inflammation, in the colon, and in which way those changes reflect on examined parameters in the brain.

Results of our research showed an accentuated decrease in DPP IV/CD26 activity at site of inflammation, in the inflamed colon in wild type animals compared to their control groups

Furthermore, we wanted to evaluate possible changes in serum DPP IV/CD26-like activities during colitis development and healing as well. Therefore, CD26-/- mice with induced colitis as well as their control groups were analyzed. Obtained results showed that CD26-/- mice express approximately 10% of total serum DPP IV/CD26 activity detected in wild type mice. Fig. 12 shows results of serum DPP IV/CD26-like activity in CD26-/- mice with induced

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol

Fig. 12. Serum DPP IV/CD26-like activity in CD26-/- mice during colitis development and

Our results indicated that there are no statistically significant differences in serum DPP IV/CD26-like activity between groups of CD26-/- animals with colitis and their control groups. Therefore, the significance of DPP IV/CD26 over DPP IV/CD26-like serum activity

Growing scientific evidence emphasizes neuroimmunomodulation as an important factor in the occurrence of inflammatory and autoimmune processes, as described in different investigations in the last few years (Ohman and Simren, 2010). The complex causal connection between central and enteric nervous system caused the introduction of the term **gut-brain axis** (Romijn et al., 2008). DPP IV/CD26 has previously been shown to play a key role in the metabolism of important bioactive neuro- and immunopeptides, as well as in the activation of the immune response (Vanderheyden et al., 2009). Due to its localization on the cell surface of the nervous and digestive system, likewise on the surface of important immune cells (Matteucci & Giampietro, 2009), we aimed to investigate possible changes in DPP IV/CD26 activity and protein expression at sight of inflammation, in the colon, and in

Results of our research showed an accentuated decrease in DPP IV/CD26 activity at site of inflammation, in the inflamed colon in wild type animals compared to their control groups

which way those changes reflect on examined parameters in the brain.

colitis compared to their control groups.

solution (colitis group) or ethanol solution (control group).

**4.2.5 DPP IV/CD26, IBD and the gut-brain axis** 

resolution compared to control group.

is proposed.

(Fig. 13A). On the other hand, an increased CD26 protein expression in the acute phase of disease was revealed by Western blotting technique (Fig. 13B).

a, statistically significantly different compared to control group (*P* < 0.05).

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol solution (colitis group) or ethanol solution (control group).

Fig. 13. DPP IV/CD26 activity (A) and protein expression (B) in colon of C57BL/6 mice during colitis development and resolution.

Besides a regulatory system at the enzymatic level, this observed fact could also be partly explained as a compensatory mechanism, considering that a part of the decreased DPP IV/CD26 activity in the colon of wild type mice is indeed a consequence of severe mucosal damage induced by TNBS-ethanol solution. Consequently, increased CD26 protein expression in the acute phase of disease could represent an effort to realize a compensatory mechanism. Since damaged DPP IV/CD26 conformation is present in inflamed tissue, with the consequence of an improper enzyme activity, enhanced DPP IV/CD26 protein expression could represent a mechanism of feed-back. Our results are in agreement with previously reported observations regarding enhanced CD26 mRNA production in inflamed tissue (Nemoto et al., 1999).

We have determined that, in physiological conditions, CD26-/- mice express less than 2% of total DPP IV/CD26 activity detected in the colon of wild type mice. This enzyme activity was defined as DPP IV/CD26-like activity in the colon and was investigated during colitis development and resolution, as well. Our results showed statistically significantly (*P* < 0.05) decreased DPP IV/CD26-like activity in inflamed colon homogenates in CD26-/- mice, compared to their controls (Fig. 14).

Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 83

**A**

 **110 kDa**

**B**

**C**

 **42 kDa**

**0 2 7 15 30 Time (day)**

a a

 **0 2 7 15 30**

**C57BL/6 colitis C57BL/6 control group**

**0 2 7 15 30 Time (day)**

**CD26-/- colitis CD26-/- control group**

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol

Fig. 15. Brain DPP IV/CD26 activity and protein expression in C57BL/6 mice (A, B), DPP IV/CD26-like activity in CD26-/- mice (C) during colitis development and resolution.

Results of our studies show that DPP IV/CD26 is involved in the pathogenesis of IBD. In patients, its activity seems to be a good marker of disease activity, given its inverse correlation with disease severity. Given the potential role of DPP IV/CD26 in IBD, animal models of UC and CD have been established in CD26-/- and wild type mice. Our results showed that CD26-/ mice are not protected from two chemically induced colitis (DSS and TNBS colitis), but show specificity in histological damage compared to wild type mice, as well as differences in the

**0,000**

solution (colitis group) or ethanol solution (control group).

a, statistically significantly different compared to control group (*P* < 0.05).

**0,005**

**0,010**

**Enzyme activity** 

**5. Conclusions** 

**(nkat/mg protein)**

**0,015**

**0,020**

 **CD26**

**ß-actin**

**0,00 0,05 0,10 0,15 0,20 0,25 0,30**

**Enzyme activity** 

**(nkat/mg protein)**

Nevertheless, this observation could not entirely be explained as a consequence of an intrinsic regulatory mechanism which downregulates the activity of DPP IV/CD26-like enzymes in inflammatory processes, but could also partially be attributable to tissue damage induced by TNBS/ethanol solution.

a, statistically significantly different compared to control group (*P* < 0.05). 0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol solution (colitis group) or ethanol solution (control group).

Fig. 14. DPP IV/CD26-like activity in colon of CD26-/- mice during colitis development and resolution compared to control group.

DPP IV/CD26 and DPP IV/CD26-like activities were also analyzed in brain homogenates during colitis development and resolution in wild type and CD26-/- mice. Our results showed that DPP IV/CD26 activity in brain is statistically significantly decreased (*P* < 0.05) in the acute phase of colitis compared to control groups (Fig. 15A). On the other hand, CD26 protein expression, as confirmed by Western blotting (Fig. 15B) remains constant. Furthermore, the activity of DPP IV/CD26-like enzymes was found to remain unchanged (Fig. 15C).

It could be seen that changes in DPP IV/CD26 activity in the colon during inflammatory events, reflect on its activity in the central nervous system, which accentuates the importance of the gut-brain axis in IBD pathogenesis. Therefore, a decreased DPP IV/CD26 activity in the brain is most probably causally connected with its accentuated changes in the colon. Furthermore, a regulatory mechanism which regulates DPP IV/CD26 activity in brain, independently of its protein expression is proposed.

This study reveals new data about DPP IV/CD26 activity and protein expression in a model of Crohn-like colitis in mice. Likewise, due to very little available results of colitis investigation under conditions of CD26 deficiency, our study gives new insights in inflammatory manifestations induced by TNBS-ethanol administration in CD26 -/- mice.

Nevertheless, this observation could not entirely be explained as a consequence of an intrinsic regulatory mechanism which downregulates the activity of DPP IV/CD26-like enzymes in inflammatory processes, but could also partially be attributable to tissue damage

> **0 2 7 15 30 Time (day)**

a a

0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol

Fig. 14. DPP IV/CD26-like activity in colon of CD26-/- mice during colitis development and

DPP IV/CD26 and DPP IV/CD26-like activities were also analyzed in brain homogenates during colitis development and resolution in wild type and CD26-/- mice. Our results showed that DPP IV/CD26 activity in brain is statistically significantly decreased (*P* < 0.05) in the acute phase of colitis compared to control groups (Fig. 15A). On the other hand, CD26 protein expression, as confirmed by Western blotting (Fig. 15B) remains constant. Furthermore, the activity of DPP IV/CD26-like enzymes was found to remain unchanged

It could be seen that changes in DPP IV/CD26 activity in the colon during inflammatory events, reflect on its activity in the central nervous system, which accentuates the importance of the gut-brain axis in IBD pathogenesis. Therefore, a decreased DPP IV/CD26 activity in the brain is most probably causally connected with its accentuated changes in the colon. Furthermore, a regulatory mechanism which regulates DPP IV/CD26 activity in

This study reveals new data about DPP IV/CD26 activity and protein expression in a model of Crohn-like colitis in mice. Likewise, due to very little available results of colitis investigation under conditions of CD26 deficiency, our study gives new insights in inflammatory manifestations induced by TNBS-ethanol administration in CD26 -/- mice.

**CD26-/- colitis CD26-/- control group**

a

induced by TNBS/ethanol solution.

**0,000**

resolution compared to control group.

(Fig. 15C).

a, statistically significantly different compared to control group (*P* < 0.05).

solution (colitis group) or ethanol solution (control group).

brain, independently of its protein expression is proposed.

**0,005**

**0,010**

**Enzyme activity** 

**(nkat/mg protein)**

**0,015**

**0,020**

a, statistically significantly different compared to control group (*P* < 0.05). 0 – control group, physiological condition; 2, 7, 15, 30 – days after administration of TNBS-ethanol solution (colitis group) or ethanol solution (control group).

Fig. 15. Brain DPP IV/CD26 activity and protein expression in C57BL/6 mice (A, B), DPP IV/CD26-like activity in CD26-/- mice (C) during colitis development and resolution.

#### **5. Conclusions**

Results of our studies show that DPP IV/CD26 is involved in the pathogenesis of IBD. In patients, its activity seems to be a good marker of disease activity, given its inverse correlation with disease severity. Given the potential role of DPP IV/CD26 in IBD, animal models of UC and CD have been established in CD26-/- and wild type mice. Our results showed that CD26-/ mice are not protected from two chemically induced colitis (DSS and TNBS colitis), but show specificity in histological damage compared to wild type mice, as well as differences in the

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## **6. Acknowledgements**

This study was supported by the Croatian Ministry of Science, Education and Sports (grant No. 062-0061245-0213). We gratefully acknowledge Dr. Didier Marguet (Centre d'Immunologie Marseille-Luminy, France), for providing us CD26-/- mice. Many thanks to professor Siniša Volarević, PhD, head of the department of Molecular Medicine and Biotechnology and professor Stipan Jonjić, PhD, head of the department of Histology and Embryology, School of Medicine, University of Rijeka, for allowing us to complete a part of experiments using the equipment at their departments.

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**6. Acknowledgements** 

**7. References** 


Role of Dipeptidyl Peptidase IV/CD26 in Inflammatory Bowel Disease 87

Munoz, E.; Blazquez, M.V.; Madueno, J.A.; Rubio, G. & Pena, J. (1992). CD26 induces T-cell

Murthy, S.N.; Cooper, H.S.; Shim, H.; Shah, R.S.; Ibrahim, S.A. & Sedergran, D.J. (1993).

Nagatsu, I.; Nagatsu, T. & Yamamoto, T. (1968). Hydrolysis of amino acid beta-

Nemoto, E.; Sugawara, S.; Takada, H.; Shoji, S. & Horiuch, H. (1999). Increase of

Ohkusa, T. (1985). (Production of experimental ulcerative colitis in hamsters by dextran

*Zasshi* (*The Japanese journal of gastro-enterology*) 82:1327-36. ISSN: 0446-6586 Ohman, L. & Simren, M. (2010). Pathogenesis of IBS: role of inflammation, immunity and neuroimmune interactions. *Nat Rev Gastroenterol Hepatol* 7:163-73. ISSN: 1759-5053 Ohtsuka, Y. & Sanderson, I.R. (2003). Dextran sulfate sodium-induced inflammation is

Okayasu, I.; Hatakeyama, S.; Yamada, M.; Ohkusa, T.; Inagaki, Y. & Nakaya, R. (1990). A

Puschel, G.; Mentlein, R. & Heymann, E. (1982). Isolation and characterization of dipeptidyl peptidase IV from human placenta. *Eur J Biochem* 126:359-65. ISSN: 0014-2956 Ravi, A.; Garg, P. & Sitaraman, S.V. (2007). Matrix metalloproteinases in inflammatory bowel disease: boon or a bane? *Inflamm Bowel Dis* 13:97-107. ISSN: 1536-4844 Reinhold , D.; Bank, U.; Buhling, F.; Tager, M.; Born, I.; Faust, J.; Neubert, K. & Ansorge, S.

Reinhold, D.; Kahne, T.; Tager, M.; Lendeckel, U.; Buhling, F.; Bank, U.; Wrenger, S.; Faust,

Rose, M.; Walter, O.B.; Fliege, H.; Hildebrandt, M.; Monnikes, H. & Klapp, B.F. (2003). DPP

Scheiffele, F. & Fuss, I.J. (2002). Induction of TNBS colitis in mice. *Curr Protoc Immunol*

colitis in mice. *Gastroenterology* 98:694-702. ISSN: 0016-5085

and thymocytes. *Immunol Lett* 58:29-35. ISSN: 0165-2478

*Nutr Metab Care* 11:518-21. ISSN: 1363-1950

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proliferation by tyrosine protein phosphorylation. *Immunology* 77:43-50. ISSN: 0019-2805

Treatment of dextran sulfate sodium-induced murine colitis by intracolonic

naphthylamides by aminopeptidases in human parotid salva and human serum.

CD26/dipeptidyl peptidase IV expression on human gingival fibroblasts upon stimulation with cytokines and bacterial components. *Infect Immun* 67:6225-33.

sulfate sodium and changes in intestinal microflora). *Nippon Shokakibyo Gakkai* 

enhanced by intestinal epithelial cell chemokine expression in mice. *Pediatr Res*

novel method in the induction of reliable experimental acute and chronic ulcerative

(1997a). Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-beta 1 (TGF-beta 1) in stimulated mouse splenocytes

J.; Neubert, K.; & Ansorge, S. (1997b). The effect of anti-CD26 antibodies on DNA synthesis and cytokine production (IL-2, IL-10 and IFN-gamma) depends on enzymatic activity of DP IV/CD26. *Adv Exp Med Biol* 421:149-55. ISSN: 0065-2598 Romijn, J.A.; Corssmit, E.P.; Havekes, L.M. & Pijl, H. (2008). Gut-brain axis. *Curr Opin Clin* 

IV and mental depression in Crohn's disease. *Adv Exp Med Biol* 524:321-31. ISSN:

Mentlein, R. (2004). Cell-surface peptidases. *Int Rev Cytol* 235:165-213. ISSN: 0074-7696 Mizoguchi, A. & Mizoguchi, E. (2010). Animal models of IBD: linkage to human disease.

*Curr Opin Pharmacol* 10:578-87. ISSN: 1471-4973

*Experientia* 24:347-8. ISSN: 0014-4754

ISSN: 0019-9567

53:143-7. ISSN: 0031-3998

cyclosporin. *Dig Dis Sci.* 38(9):1722-34. ISSN: 0163-2116


Hildebrandt, M.; Reutter, W.; Arck, P.; Rose, M. & Klapp, B.F. (2000). A guardian angel: the

nutrition and immune defence. *Clin Sci (Lond)* 99:93-104. ISSN: 0143-5221 Hildebrandt, M.; Rose, M.; Ruter, J.; Salama, A.; Monnikes, H. & Klapp, B.F. (2001).

Howarth, G.S.; Xian, C.J. & Read, L.C. (2000). Predisposition to colonic dysplasia is

Hyams, J.S.; Ferry, G.D.; Mandel, F.S.; Gryboski, J.D.; Kibort, P.M.; Kirschner, B.S.; Griffiths,

Ikushima, H.; Munakata, Y.; Ishii, T.; Iwata, S.; Terashima, M.; Tanaka, H.; Schlossman, S.F.

Ishii, T.; Ohnuma, K.; Murakami, A.; Takasawa, N.; Kobayashi, S.; Dang, N.H.; Schlossman,

Iwanaga, T.; Hoshi, O.; Han, H. & Fujita, T. (1994). Morphological analysis of acute ulcerative

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of the enzyme DPP IV. *Crit Rev Clin Lab Sci* 40:209-94. ISSN: 1040-8363 Mahler, M.; Bristol, I.J.; Leiter, E.H.; Workman, A.E.; Birkenmeier, E.H.; Elson, C.O. &

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**5** 

*University of Calgary* 

*Canada* 

**The Roles of Interleukin-17 and T Helper 17** 

Inflammatory bowel diseases (IBD) are caused by chronic inflammation of the gastrointestinal tract, affecting as many as 1.4 million persons in the United States, and 2.2 million persons in Europe (Loftus, 2004). Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of IBD, affect different regions of the intestinal tract and have distinct cytokine profiles. In CD, transmural inflammation can occur over the entire length of the gastrointestinal tract, whereas UC inflammation is restricted to the mucosa of the colon. The T helper (Th) paradigm was established by Mosmann et al (1986) who observed distinct cytokine patterns were produced by two types of fully differentiated effector T cells which they termed termed Th1 and Th2 cells. The initial cytokine profiles observed in IBD helped to classify CD as a Th1 disease, due to the increased production of the main Th1 effector cytokine, interferon-gamma (IFN-γ). UC was slightly more difficult to classify because levels of a central Th2 effector cytokine, IL-4, are not increased; however, other Th2 effector cytokines, such as IL-5 and IL-13 are produced at higher levels (Fuss et al, 1996). Therefore,

Conventional IBD therapies, including corticosteroids and anti-tumor necrosis factor-alpha (TNF-α) therapy, are aimed at reducing nonspecific inflammation. TNF-α is a central proinflammatory cytokine that contributes to the pathology of many autoimmune disorders. Anti-TNF-α was the first biological therapy introduced for patients with IBD in the late 1990s, and corticosteroid-refractory or fistulizing CD and refractory UC generally respond very well to anti-TNF-α treatment (Hoentjen & van Bodegraven, 2009; Rutgeerts et al., 2006). The initial identification of disease-specific inflammatory mediators in CD and UC, Th1 and Th2-associated cytokines respectively, lead to the development of more specific antiinflammatory treatment options, and the efficacies of these new biological agents have in turn helped evolve our understanding of IBD pathogenesis. Using mouse models of intestinal inflammation that resemble CD, and targeting the main cytokine that drives Th1 cellular development, IL-12, with an antibody to the IL-12p40 subunit either prevented the development of colitis, or completely cured established colitis (Liu et al., 2001; Neurath et al, 1995). These observations further supported the link between CD and Th1 responses, in addition to warranting the development of an anti-IL-12p40 antibody for human patients with CD. In clinical trials, anti-IL-12p40 therapy induced clinical responses and remissions in patients with active CD (Mannon et al., 2005; Sandborn et al., 2008), which lead to its

UC is not considered fully Th2, but rather a Th2-like disease (Fuss et al, 2004).

**1. Introduction** 

acceptance as a new therapy for CD.

**Cells in Intestinal Barrier Function** 

Elizabeth Trusevych, Leanne Mortimer and Kris Chadee


## **The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function**

Elizabeth Trusevych, Leanne Mortimer and Kris Chadee *University of Calgary Canada* 

### **1. Introduction**

88 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Schon, E.; Mansfeld, H.W.; Demuth, H.U.; Barth, A. & Ansorge, S. (1985). The dipeptidyl

Sedo, A. & Malik, R. (2001). Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? *Biochim Biophys Acta* 1550:107-16. ISSN: 0006-3002 Shah, S.A. & Feller, E.R. (2009). Inflammatory bowel disease. *Med Health R I* 92:72. ISSN:

Strober, W.; Fuss, I.J.; Ehrhardt, R.O.; Neurath, M.; Boirivant, M. & Ludviksson, B.R. (1998)

murine models of inflammation. *Scand J Immunol* 48:453-8. ISSN: 0300-9475 Thompson, M.A.; Ohnuma, K.; Abe, M.; Morimoto, C. & Dang, N.H. (2007).

Truelove, S.C. & Witts, L.J. (1955). Cortisone in ulcerative colitis; final report on a

Uhlig, H.H. & Powrie, F. (2009). Mouse models of intestinal inflammation as tools to

Vanderheyden, M.; Bartunek, J.; Goethals, M.; Verstreken, S.; Lambeir, A.M.; De Meester, I.

Varljen, J., Sincic, B.M.; Baticic, L.; Varljen, N.; Detel, D. & Lekic, A. (2005). Clinical relevance of

Varljen, J.; Detel, D.; Lupis, T. & Peršić, M. (2004). Serum Dipeptidyl Peptidase IV (DPP

Willheim, M.; Ebner, C.; Baier, K.; Kern, W.; Schrattbauer, K.; Thien, R.; Kraft, D.;

Yazbeck, R.; Howarth, G.S. & Abbott, C.A. (2009). Dipeptidyl peptidase inhibitors, an

Yazbeck, R.; Howarth, G.S.; Geier, M.S.; Demuth, H.U. & Abbott, C.A. (2008). Inhibiting

Yazbeck, R.; Sulda, M.L.; Howarth, G.S.; Bleich, A.; Raber, K.; von Horsten, S.; Holst, J.J. &

Yu, D.M.; Ajami, K.; Gall, M.G.; Park, J.; Lee, C.S.; Evans, K.A.; McLaughlin, E.A.; Pitman M.R.;

with T(H1) subsets. *J Allergy Clin Immunol* 100:348-55. ISSN: 0091-6749 Wirtz, S. & Neurath, M.F. (2007). Mouse models of inflammatory bowel disease. *Adv Drug* 

bench to bedside. *Clin Chem Lab Med* 47:248-52. ISSN: 1434-6621

*Biomed Biochim Acta* 44:K9-15. ISSN: 0232-766X

disorders. *Mini Rev Med Chem* 7:253-73. ISSN: 1389-5575

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peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes.

Mucosal immunoregulation and inflammatory bowel disease: new insights from

CD26/dipeptidyl peptidase IV as a novel therapeutic target for cancer and immune

understand the pathogenesis of inflammatory bowel disease. *Eur J Immunol*

& Scharpe, S. (2009). Dipeptidyl-peptidase IV and B-type natriuretic peptide. From

the serum dipeptidyl peptidase IV (DPP IV/CD26) activity in adult patients with Crohn's disease and ulcerative colitis. *Croatica Chemica Acta* 78:427-432. ISSN: 0011-1643

IV/CD26) Activity in Children with Inflammatory Bowel Disease. Pediatric Gastroenterology 2004 - Reports from the 2nd World Congress of Pediatric Gastroenterology, Hepatology and Nutrition. Medimont S.r.l., 559-563. ISBN 88-

Breiteneder, H.; Reinisch, W. & Scheiner, O. (1997). Cell surface characterization of T lymphocytes and allergen-specific T cell clones: correlation of CD26 expression

emerging drug class for inflammatory disease? *Trends Pharmacol Sci* 30:600-7. ISSN:

dipeptidyl peptidase activity partially ameliorates colitis in mice. *Front Biosci*

Abbott, C.A. (2010). Dipeptidyl peptidase expression during experimental colitis in

Abbott, C.A.; McCaughan, G.W. & Gorrell, M.D. (2009). The in vivo expression of dipeptidyl peptidases 8 and 9. *J Histochem Cytochem* 57:1025-40. ISSN: 1551-5044

Inflammatory bowel diseases (IBD) are caused by chronic inflammation of the gastrointestinal tract, affecting as many as 1.4 million persons in the United States, and 2.2 million persons in Europe (Loftus, 2004). Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of IBD, affect different regions of the intestinal tract and have distinct cytokine profiles. In CD, transmural inflammation can occur over the entire length of the gastrointestinal tract, whereas UC inflammation is restricted to the mucosa of the colon. The T helper (Th) paradigm was established by Mosmann et al (1986) who observed distinct cytokine patterns were produced by two types of fully differentiated effector T cells which they termed termed Th1 and Th2 cells. The initial cytokine profiles observed in IBD helped to classify CD as a Th1 disease, due to the increased production of the main Th1 effector cytokine, interferon-gamma (IFN-γ). UC was slightly more difficult to classify because levels of a central Th2 effector cytokine, IL-4, are not increased; however, other Th2 effector cytokines, such as IL-5 and IL-13 are produced at higher levels (Fuss et al, 1996). Therefore, UC is not considered fully Th2, but rather a Th2-like disease (Fuss et al, 2004).

Conventional IBD therapies, including corticosteroids and anti-tumor necrosis factor-alpha (TNF-α) therapy, are aimed at reducing nonspecific inflammation. TNF-α is a central proinflammatory cytokine that contributes to the pathology of many autoimmune disorders. Anti-TNF-α was the first biological therapy introduced for patients with IBD in the late 1990s, and corticosteroid-refractory or fistulizing CD and refractory UC generally respond very well to anti-TNF-α treatment (Hoentjen & van Bodegraven, 2009; Rutgeerts et al., 2006). The initial identification of disease-specific inflammatory mediators in CD and UC, Th1 and Th2-associated cytokines respectively, lead to the development of more specific antiinflammatory treatment options, and the efficacies of these new biological agents have in turn helped evolve our understanding of IBD pathogenesis. Using mouse models of intestinal inflammation that resemble CD, and targeting the main cytokine that drives Th1 cellular development, IL-12, with an antibody to the IL-12p40 subunit either prevented the development of colitis, or completely cured established colitis (Liu et al., 2001; Neurath et al, 1995). These observations further supported the link between CD and Th1 responses, in addition to warranting the development of an anti-IL-12p40 antibody for human patients with CD. In clinical trials, anti-IL-12p40 therapy induced clinical responses and remissions in patients with active CD (Mannon et al., 2005; Sandborn et al., 2008), which lead to its acceptance as a new therapy for CD.

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 91

(Fujino et al., 2003; Rovedatti et al., 2009). Furthermore, polymorphisms in the *IL-17A* and *IL-17F* genes have been linked to UC and animal models indicate that they are fundamentally involved in the etiology of IBD (Arisawa et al., 2008). However, their precise roles in pathogenesis are not entirely clear. This chapter will focus on the cytokines IL-17A and IL-17F, and review what is known about their contributions to mucosal barrier function

The gastrointestinal tract forms the largest surface in contact with the external environment. The intestinal mucosal barrier separates the internal intestinal tissues from an estimated 1014 organisms (Savage, 1977), and is composed of a physical barrier as well as specialized

The physical barrier is comprised of an outer mucus layer less than a millimeter thick, and a single layer of epithelial cells joined together by tight junctions (Figure 1). The main structural component of the outer mucus layer is the heavily O-glycosylated glycoprotein MUC2, which is produced by goblet cells and gives mucus its viscous properties. The outer mucus layer was recently discovered to contain within it two distinct layers: an outer loose mucus layer with high numbers of commensal bacteria, and a dense inner layer that is sterile, containing high concentrations of antimicrobial molecules including nonspecific antimicrobial peptides and specific antimicrobial immunoglobulins (IgA) (Johansson et al., 2008). Commensal bacteria contribute to the function of the mucosal barrier by inducing the production of IgA, recruiting intraepithelial lymphocytes, and providing a physical

The second component of the physical mucosal barrier is the single layer of epithelial cells supporting the outer mucus layer. The majority of epithelial cells are transporting enterocytes, but specialized epithelial cell types contribute to mucosal barrier integrity by producing the main constituents of the mucus layer, which minimizes microbial contact with the epithelium. Additionally, epithelial cells have a dense glycocalyx overlaying microvillar projections that prevent microbial attachment (Linden et al., 2008; L. Shen & Turner, 2006). The epithelial barrier needs to be selective to allow the absorption of essential nutrients while preventing the entry of potentially noxious compounds. As depicted in Figure 1, tight junctions that connect the epithelial cells allow the cellular barrier to respond to changes in the environment by regulating the tight junction protein composition, which leads to general or ion-selective changes in paracellular permeability

In both animal models of IBD and the clinical disease in humans, changes in the physical mucosal barrier have been observed. In patients suffering from UC, MUC2 protein levels are significantly decreased during active phases of the disease, resulting in a thinner protective mucus layer (Hinoda et al., 1998; Tytgat et al., 1996). In animal models of chronic intestinal inflammatory conditions that cycle between active and quiescent phases, paracellular permeability remains increased regardless of the inflammatory state, whereas transcellular permeability is only increased during active inflammation (Porras et al., 2006). Similar observations have been made in humans, where patients with quiescent CD have significantly increased intestinal permeability when compared to controls (Wyatt et al.,

in the gastrointestinal tract with special emphasis on IBD.

immune cells, primed to react if the physical barrier is breached.

blockade to prevent the colonization of pathogens (Umesaki et al., 1999).

**2.1 Anatomy and function of the physical barrier** 

**2. The intestinal mucosal barrier** 

(Arrieta et al., 2006).

Around the same time as anti-IL-12p40 therapy was being tested, discrepancies within the Th1/Th2 paradigm observed over the previous two decades were beginning to be resolved (Steinman, 2007). Two models in particular provided the first inconsistencies with the Th1/Th2 hypothesis: experimental autoimmune encephalomyelitis (EAE) and collageninduced arthritis (CIA). EAE is a mouse model of human multiple sclerosis, caused by cellmediated tissue damage that results in delayed-type hypersensitivity (DTH). DTH reactions are cell-mediated immune reactions to a challenge antigen, leading to swelling, induration, and redness appearing 24 to 72 hours after antigen exposure. Initially, DTH was believed to be mediated by a Th1 response (Cher & Mosmann, 1987). Therefore, it was hypothesized that EAE would worsen with the addition of the Th1 effector cytokine, IFN-γ. Interestingly, the results were just the opposite and IFN-γ administration ameliorated EAE damage (Billiau et al., 1988; Voorthuis et al., 1990). Similarly, CIA as a second model of autoimmune tissue destruction was also predicted to worsen with the administration of IFN-γ. Although the disease did worsen when IFN-γ was given before the administration of the adjuvant, it was ameliorated when IFN-γ was given after the adjuvant (Jacob et al., 1989; Nakajima et al., 1991). These puzzling inverse relationships between disease states thought to be controlled by Th1 responses and the presence of IFN-γ, eventually lead to the discovery of IL-23 and it's role as a master regulator of a new Th cell subset. IL-23, like IL-12, is a heterodimeric cytokine comprised of two subunits: a unique p19 subunit and a p40 subunit that is also shared by IL-12 (Oppmann et al., 2000). After it was discovered that IL-12 and IL-23 share a common subunit, divergent functions of these cytokines were unraveled, and the autoimmune inflammation in both EAE and CIA was found to result from the actions of IL-23, and not the Th1 associated cytokine IL-12 (Cua et al., 2003; Murphy et al., 2003). In the same regard, when models of innate and adaptive chronic intestinal inflammation were reevaluated, IL-23 was found to play a greater role than IL-12 in the induction of inflammation (Hue et al., 2006; Kullberg et al., 2006).

Around the time that IL-23 was found to be a central mediator of autoimmune inflammation, it was also discovered as a master regulator of an emerging Th cell subset, Th17 (Aggarwal et al., 2003). This was a significant event, as it shifted the long-standing Th1/Th2 paradigm of inflammation to include a novel subset of adaptive Th cells. Consequently, all inflammatory conditions involving the adaptive immune response have needed re-evaluation. Th17 cells have high expression of the transcription factors RORα and ROR-γt, produce the cytokines IL-17A, IL-17F and IL-22, have high surface expression of the IL-23R as well as the chemokine receptor CCR6, and can also secrete the CCR6 ligand, CCL20 (O'Connor et al., 2010). Importantly, the CCL20-CCR6 ligand-receptor pair plays an important chemoattractant role at mucosal surfaces (Schutyser et al., 2003). In addition to Th17 cells, CCR6 is also expressed on T regulatory (Treg) cells that function to maintain homeostatic conditions (Lim et al., 2008). By producing CCL20, Th17 cells are able to promote the migration of additional Th17 cells as well as Treg cells (Yamazaki et al., 2008), and both cell types are enriched at mucosal surfaces.

Since their characterization, Th17 cells have been shown to play an important protective role in infectious immunity where they promote the clearance of extracellular pathogens by enhancing neutrophil recruitment and promoting the expression of antimicrobial factors. Additionally, Th17 cells have been associated with many autoimmune diseases, such as rheumatoid arthritis, dermatitis, psoriasis, asthma, multiple sclerosis, as well as IBD (Hemdan et al., 2010). Studies of human IBD have shown that the Th17 effector cytokines IL-17A and IL-17F are both increased in the affected mucosa and sera of CD and UC patients (Fujino et al., 2003; Rovedatti et al., 2009). Furthermore, polymorphisms in the *IL-17A* and *IL-17F* genes have been linked to UC and animal models indicate that they are fundamentally involved in the etiology of IBD (Arisawa et al., 2008). However, their precise roles in pathogenesis are not entirely clear. This chapter will focus on the cytokines IL-17A and IL-17F, and review what is known about their contributions to mucosal barrier function in the gastrointestinal tract with special emphasis on IBD.

## **2. The intestinal mucosal barrier**

90 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Around the same time as anti-IL-12p40 therapy was being tested, discrepancies within the Th1/Th2 paradigm observed over the previous two decades were beginning to be resolved (Steinman, 2007). Two models in particular provided the first inconsistencies with the Th1/Th2 hypothesis: experimental autoimmune encephalomyelitis (EAE) and collageninduced arthritis (CIA). EAE is a mouse model of human multiple sclerosis, caused by cellmediated tissue damage that results in delayed-type hypersensitivity (DTH). DTH reactions are cell-mediated immune reactions to a challenge antigen, leading to swelling, induration, and redness appearing 24 to 72 hours after antigen exposure. Initially, DTH was believed to be mediated by a Th1 response (Cher & Mosmann, 1987). Therefore, it was hypothesized that EAE would worsen with the addition of the Th1 effector cytokine, IFN-γ. Interestingly, the results were just the opposite and IFN-γ administration ameliorated EAE damage (Billiau et al., 1988; Voorthuis et al., 1990). Similarly, CIA as a second model of autoimmune tissue destruction was also predicted to worsen with the administration of IFN-γ. Although the disease did worsen when IFN-γ was given before the administration of the adjuvant, it was ameliorated when IFN-γ was given after the adjuvant (Jacob et al., 1989; Nakajima et al., 1991). These puzzling inverse relationships between disease states thought to be controlled by Th1 responses and the presence of IFN-γ, eventually lead to the discovery of IL-23 and it's role as a master regulator of a new Th cell subset. IL-23, like IL-12, is a heterodimeric cytokine comprised of two subunits: a unique p19 subunit and a p40 subunit that is also shared by IL-12 (Oppmann et al., 2000). After it was discovered that IL-12 and IL-23 share a common subunit, divergent functions of these cytokines were unraveled, and the autoimmune inflammation in both EAE and CIA was found to result from the actions of IL-23, and not the Th1 associated cytokine IL-12 (Cua et al., 2003; Murphy et al., 2003). In the same regard, when models of innate and adaptive chronic intestinal inflammation were reevaluated, IL-23 was found to play a greater role than IL-12 in the induction of inflammation

Around the time that IL-23 was found to be a central mediator of autoimmune inflammation, it was also discovered as a master regulator of an emerging Th cell subset, Th17 (Aggarwal et al., 2003). This was a significant event, as it shifted the long-standing Th1/Th2 paradigm of inflammation to include a novel subset of adaptive Th cells. Consequently, all inflammatory conditions involving the adaptive immune response have needed re-evaluation. Th17 cells have high expression of the transcription factors RORα and ROR-γt, produce the cytokines IL-17A, IL-17F and IL-22, have high surface expression of the IL-23R as well as the chemokine receptor CCR6, and can also secrete the CCR6 ligand, CCL20 (O'Connor et al., 2010). Importantly, the CCL20-CCR6 ligand-receptor pair plays an important chemoattractant role at mucosal surfaces (Schutyser et al., 2003). In addition to Th17 cells, CCR6 is also expressed on T regulatory (Treg) cells that function to maintain homeostatic conditions (Lim et al., 2008). By producing CCL20, Th17 cells are able to promote the migration of additional Th17 cells as well as Treg cells (Yamazaki et al., 2008),

Since their characterization, Th17 cells have been shown to play an important protective role in infectious immunity where they promote the clearance of extracellular pathogens by enhancing neutrophil recruitment and promoting the expression of antimicrobial factors. Additionally, Th17 cells have been associated with many autoimmune diseases, such as rheumatoid arthritis, dermatitis, psoriasis, asthma, multiple sclerosis, as well as IBD (Hemdan et al., 2010). Studies of human IBD have shown that the Th17 effector cytokines IL-17A and IL-17F are both increased in the affected mucosa and sera of CD and UC patients

(Hue et al., 2006; Kullberg et al., 2006).

and both cell types are enriched at mucosal surfaces.

The gastrointestinal tract forms the largest surface in contact with the external environment. The intestinal mucosal barrier separates the internal intestinal tissues from an estimated 1014 organisms (Savage, 1977), and is composed of a physical barrier as well as specialized immune cells, primed to react if the physical barrier is breached.

#### **2.1 Anatomy and function of the physical barrier**

The physical barrier is comprised of an outer mucus layer less than a millimeter thick, and a single layer of epithelial cells joined together by tight junctions (Figure 1). The main structural component of the outer mucus layer is the heavily O-glycosylated glycoprotein MUC2, which is produced by goblet cells and gives mucus its viscous properties. The outer mucus layer was recently discovered to contain within it two distinct layers: an outer loose mucus layer with high numbers of commensal bacteria, and a dense inner layer that is sterile, containing high concentrations of antimicrobial molecules including nonspecific antimicrobial peptides and specific antimicrobial immunoglobulins (IgA) (Johansson et al., 2008). Commensal bacteria contribute to the function of the mucosal barrier by inducing the production of IgA, recruiting intraepithelial lymphocytes, and providing a physical blockade to prevent the colonization of pathogens (Umesaki et al., 1999).

The second component of the physical mucosal barrier is the single layer of epithelial cells supporting the outer mucus layer. The majority of epithelial cells are transporting enterocytes, but specialized epithelial cell types contribute to mucosal barrier integrity by producing the main constituents of the mucus layer, which minimizes microbial contact with the epithelium. Additionally, epithelial cells have a dense glycocalyx overlaying microvillar projections that prevent microbial attachment (Linden et al., 2008; L. Shen & Turner, 2006). The epithelial barrier needs to be selective to allow the absorption of essential nutrients while preventing the entry of potentially noxious compounds. As depicted in Figure 1, tight junctions that connect the epithelial cells allow the cellular barrier to respond to changes in the environment by regulating the tight junction protein composition, which leads to general or ion-selective changes in paracellular permeability (Arrieta et al., 2006).

In both animal models of IBD and the clinical disease in humans, changes in the physical mucosal barrier have been observed. In patients suffering from UC, MUC2 protein levels are significantly decreased during active phases of the disease, resulting in a thinner protective mucus layer (Hinoda et al., 1998; Tytgat et al., 1996). In animal models of chronic intestinal inflammatory conditions that cycle between active and quiescent phases, paracellular permeability remains increased regardless of the inflammatory state, whereas transcellular permeability is only increased during active inflammation (Porras et al., 2006). Similar observations have been made in humans, where patients with quiescent CD have significantly increased intestinal permeability when compared to controls (Wyatt et al.,

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 93

Adaptive immune responses are antigen specific and typically facilitate expeditious removal of pathogens. In the gut however, adaptive immune tolerance is crucial for maintaining quiescent relationships with the microbial flora and food antigens. In this regard, the intestine is a prime inductive site for large numbers of adaptive Treg cells that home back to the intestinal mucosa, where they help to maintain intestinal tolerance (Belkaid & Oldenhove, 2008; Coombes et al., 2007). Thus, innate and adaptive immune cells in the gut are primed for action so that they can maintain a tolerant immune environment, while still

IL-17 is a central pro-inflammatory cytokine at mucosal surfaces, with important functions in innate and adaptive immunity, as well as host defense against extracellular pathogens. Originally named cytotoxic T-lymphocyte antigen (CTLA)-8, IL-17 was first described in the mid 1990s (prior to the identification of Th17 cells) as a cytokine produced by activated CD4+ T cells that acts on stromal cells to up regulate inflammatory and hematopoietic processes (Fossiez et al., 1996; Rouvier et al., 1993; Yao et al., 1995a). IL-17 is now best known as the signature cytokine secreted from the recently characterized Th17 cells, however numerous innate cells can also produce IL-17, including innate-like γδ intraepithelial lymphocytes (IEL), natural killer (NK) T cells, lymphoid tissue inducer (LTi)-like cells, Paneth cells, and neutrophils, as well as other unidentified cell types (Buonocore et al., 2010; Cua & Tato, 2010; Doisne et al., 2011; L. Li et al., 2010; Maele et al., 2010; Michel et al., 2007; Shibata et al., 2007; Takahashi et al., 2006; Takatori et al., 2008). In the context of the intestinal mucosa, γδ IELs are currently the best-characterized innate sources of IL-17. γδ IELs reside at the intestinal mucosal surface between epithelial cells on the basolateral side of tight junctions. They play an essential role in the restitution of epithelial cells following mucosal injury through the production of growth factors, a distinct ability that does not occur in other mucosal T cell populations (Y. Chen et al, 2002). Additionally, γδ IELs play an essential role in controlling bacterial penetration across injured mucosal surfaces, and recruiting neutrophils following *Escherichia coli* infection by acting as the major source of early IL-17

Importantly, since the discovery of IL-17 additional IL-17 family members have been identified. The IL-17 cytokine family consists of six members in mammals: IL-17A (also called IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F (X. Zhang et al., 2011). IL-17F shares 50% sequence homology with IL-17A, is also produced by Th17 cells, binds the same receptor as IL-17A and in turn shares certain biological activities (Hymowitz et al., 2001). IL-17A and IL-17F are either produced as homodimeric cytokines or as heterodimers composed of IL-17A/F (Wright et al., 2007). When acting on fibroblasts, endothelial cells, or epithelial cells, both IL-17A and IL-17F induce the production of proinflammatory cytokines (notably IL-6 and IL-8), chemokines, antimicrobial peptides, and matrix metalloproteinases (Iwakura et al., 2011; Starners et al., 2001). Despite their similar pro-inflammatory actions, IL-17A and IL-17F appear to have distinct roles in mediating inflammatory processes and autoimmune diseases (*discussed later*). IL-17B, IL-17C, and IL-17D are the least well-characterized members of the IL-17 family. IL-17B and IL-17C have 27% homology with IL-17A, but are not produced by activated T cells and do not induce the same pro-inflammatory cytokines as IL-17A and IL-17F (H. Li et al., 2000). IL-17D, which is most similar to IL-17B with 27% sequence identity, is highly expressed in skeletal muscle,

being able to rapidly respond to invading pathogens.

(Ismail et al., 2009; Shibata et al., 2007).

**3. Interleukin-17** 

1993). It is believed that the sustained increase in paracellular permeability, indicative of epithelial cell layer dysfunction, contributes to the chronic nature of the disease.

Fig. 1. The physical intestinal mucosal barrier. A single layer of epithelial cells linked together at the apical junctional complex (AJC) and an overlying mucus layer form the physical mucosal barrier. AJCs are comprised of tight junctions and adherens junctions. The protein composition of the tight junction is dynamic, and different claudin-family proteins as well as varying levels of occludin and the junctional adhesion molecule (JAM) allow for specific alterations of paracellular permeability. The foundation of the adherens junction is formed by contacts between epithelial cadherin (E-cadherin)-catenin complexes, which functions to connect neighboring epithelial cells and maintain cell polarity.

#### **2.2 Immune cells of the mucosal barrier: Surveillance and tolerance**

In addition to the physical boundary, there are immune cells and gut associated lymphoid tissues (GALT) situated within and below the epithelium. In a healthy intestine these cells and tissues strike a balance between immunity and tolerance, and maintain barrier function. The intestinal tract harbors vast populations of leukocytes. Innate immune cells typically mediate the first line of host defense, and in the intestine these include dendritic cells, macrophages, natural killer (NK) cells, γδ T cells, NKT cells and polymorphonuclear cells (Meresse & Cerf-Bensussan, 2009). Innate immunity evolved to recognize molecular signatures within the products of microbes that are essential to microbial survival. The innate immune system is comprised of pathogen recognition receptors (PRRs), such as toll-like receptors (TLR) and nucleotide-binding and oligomerization domain-like receptors (NLR), which recognize pathogen-associated molecular patterns (PAMPs). Binding of PAMPs to their cognate PRR activates signaling pathways that in turn activate host defense mechanisms. Different cell types have distinct immune functions; they express different combinations and levels of PRRs, and the downstream targets of PRR signaling are cell specific (Wells et al., 2010). Consequently, PAMP-PRR signaling mediates cell specific responses that enable the surrounding tissue to adapt to the dynamic intestinal environment.

Additionally, intestinal tissues are unique in that they harbor large numbers of adaptive immune cells expressing effector or memory phenotypes (Mowat, 2003). These include IgA and IgG secreting plasma B cells and canonical αβ T cells located in the lamina propria. Adaptive immune responses are antigen specific and typically facilitate expeditious removal of pathogens. In the gut however, adaptive immune tolerance is crucial for maintaining quiescent relationships with the microbial flora and food antigens. In this regard, the intestine is a prime inductive site for large numbers of adaptive Treg cells that home back to the intestinal mucosa, where they help to maintain intestinal tolerance (Belkaid & Oldenhove, 2008; Coombes et al., 2007). Thus, innate and adaptive immune cells in the gut are primed for action so that they can maintain a tolerant immune environment, while still being able to rapidly respond to invading pathogens.

## **3. Interleukin-17**

92 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

1993). It is believed that the sustained increase in paracellular permeability, indicative of

epithelial cell layer dysfunction, contributes to the chronic nature of the disease.

Fig. 1. The physical intestinal mucosal barrier. A single layer of epithelial cells linked together at the apical junctional complex (AJC) and an overlying mucus layer form the physical mucosal barrier. AJCs are comprised of tight junctions and adherens junctions. The protein composition of the tight junction is dynamic, and different claudin-family proteins as well as varying levels of occludin and the junctional adhesion molecule (JAM) allow for specific alterations of paracellular permeability. The foundation of the adherens junction is formed by contacts between epithelial cadherin (E-cadherin)-catenin complexes,

which functions to connect neighboring epithelial cells and maintain cell polarity.

In addition to the physical boundary, there are immune cells and gut associated lymphoid tissues (GALT) situated within and below the epithelium. In a healthy intestine these cells and tissues strike a balance between immunity and tolerance, and maintain barrier function. The intestinal tract harbors vast populations of leukocytes. Innate immune cells typically mediate the first line of host defense, and in the intestine these include dendritic cells, macrophages, natural killer (NK) cells, γδ T cells, NKT cells and polymorphonuclear cells (Meresse & Cerf-Bensussan, 2009). Innate immunity evolved to recognize molecular signatures within the products of microbes that are essential to microbial survival. The innate immune system is comprised of pathogen recognition receptors (PRRs), such as toll-like receptors (TLR) and nucleotide-binding and oligomerization domain-like receptors (NLR), which recognize pathogen-associated molecular patterns (PAMPs). Binding of PAMPs to their cognate PRR activates signaling pathways that in turn activate host defense mechanisms. Different cell types have distinct immune functions; they express different combinations and levels of PRRs, and the downstream targets of PRR signaling are cell specific (Wells et al., 2010). Consequently, PAMP-PRR signaling mediates cell specific responses that enable the

Additionally, intestinal tissues are unique in that they harbor large numbers of adaptive immune cells expressing effector or memory phenotypes (Mowat, 2003). These include IgA and IgG secreting plasma B cells and canonical αβ T cells located in the lamina propria.

**2.2 Immune cells of the mucosal barrier: Surveillance and tolerance** 

surrounding tissue to adapt to the dynamic intestinal environment.

IL-17 is a central pro-inflammatory cytokine at mucosal surfaces, with important functions in innate and adaptive immunity, as well as host defense against extracellular pathogens. Originally named cytotoxic T-lymphocyte antigen (CTLA)-8, IL-17 was first described in the mid 1990s (prior to the identification of Th17 cells) as a cytokine produced by activated CD4+ T cells that acts on stromal cells to up regulate inflammatory and hematopoietic processes (Fossiez et al., 1996; Rouvier et al., 1993; Yao et al., 1995a). IL-17 is now best known as the signature cytokine secreted from the recently characterized Th17 cells, however numerous innate cells can also produce IL-17, including innate-like γδ intraepithelial lymphocytes (IEL), natural killer (NK) T cells, lymphoid tissue inducer (LTi)-like cells, Paneth cells, and neutrophils, as well as other unidentified cell types (Buonocore et al., 2010; Cua & Tato, 2010; Doisne et al., 2011; L. Li et al., 2010; Maele et al., 2010; Michel et al., 2007; Shibata et al., 2007; Takahashi et al., 2006; Takatori et al., 2008). In the context of the intestinal mucosa, γδ IELs are currently the best-characterized innate sources of IL-17. γδ IELs reside at the intestinal mucosal surface between epithelial cells on the basolateral side of tight junctions. They play an essential role in the restitution of epithelial cells following mucosal injury through the production of growth factors, a distinct ability that does not occur in other mucosal T cell populations (Y. Chen et al, 2002). Additionally, γδ IELs play an essential role in controlling bacterial penetration across injured mucosal surfaces, and recruiting neutrophils following *Escherichia coli* infection by acting as the major source of early IL-17 (Ismail et al., 2009; Shibata et al., 2007).

Importantly, since the discovery of IL-17 additional IL-17 family members have been identified. The IL-17 cytokine family consists of six members in mammals: IL-17A (also called IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F (X. Zhang et al., 2011). IL-17F shares 50% sequence homology with IL-17A, is also produced by Th17 cells, binds the same receptor as IL-17A and in turn shares certain biological activities (Hymowitz et al., 2001). IL-17A and IL-17F are either produced as homodimeric cytokines or as heterodimers composed of IL-17A/F (Wright et al., 2007). When acting on fibroblasts, endothelial cells, or epithelial cells, both IL-17A and IL-17F induce the production of proinflammatory cytokines (notably IL-6 and IL-8), chemokines, antimicrobial peptides, and matrix metalloproteinases (Iwakura et al., 2011; Starners et al., 2001). Despite their similar pro-inflammatory actions, IL-17A and IL-17F appear to have distinct roles in mediating inflammatory processes and autoimmune diseases (*discussed later*). IL-17B, IL-17C, and IL-17D are the least well-characterized members of the IL-17 family. IL-17B and IL-17C have 27% homology with IL-17A, but are not produced by activated T cells and do not induce the same pro-inflammatory cytokines as IL-17A and IL-17F (H. Li et al., 2000). IL-17D, which is most similar to IL-17B with 27% sequence identity, is highly expressed in skeletal muscle,

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 95

Although the majority of the defined roles played by IL-17 in mucosal barrier function are related to innate and adaptive immune functions, IL-17 has also been found to directly regulate components of the physical mucosal barrier. In colonic epithelial monolayers IL-17A enhances tight junction formation by increasing claudins 1 and 2 association with the membrane (Kinugasa et al., 2000). Direct application of IL-17A to T84 monolayers increased transepithelial resistance and decreased manitol flux through monolayers. Thus, IL-17A may have an important role in maintaining tight junctions and epithelial restitution during repair processes. In airway epithelial cells IL-17A induces mucin gene expression, and it may have similar inductive effects in the intestine on goblet cells (Chen et al., 2003). IL-17A also induces expression of β-defensins in the colon (Ishigame et al., 2009). Furthermore, in subepithelial myofibroblasts, which sit just below the epithelium, IL-17A reduced TNF-αinduced secretion of pro-inflammatory cytokines, demonstrating that IL-17 is not implicitly a pro-inflammatory cytokine. Additionally, IL-17 receptor-deficient mice show increased dissemination of *S. typhimurium* from the gut (Raffatellu et al., 2008). Taken together, it appears IL-17 can dynamically regulate components of the physical intestinal epithelial

There are numerous locations in the gut where adaptive immune responses are initiated. These include organized lymphoid tissue such as Peyer's patches and isolated lymphoid follicles (ILF) that are embedded directly in the epithelial wall, and mesenteric lymph nodes (MLN), which are connected to the intestinal mucosa by draining lymphatic vessels (Figure 2, Mowat, 2003). Furthermore, there is evidence that adaptive responses occur directly in the

Under homeostatic conditions, intestinal luminal contents are constitutively sampled and processed by professional antigen presenting cells (pAPC). pAPC present processed antigen to the naive T cell population, which has an infinite repertoire of antigen-specific receptors. Upon presentation of antigen to a T cell bearing a cognate receptor, the pAPC drives an antigen specific T cell response. Depending on the accompanying signals from the pAPC and surrounding environment, the T cell may become activated into an effector cell, anergic (unresponsive to antigen) or apoptotic. Classically there are three types of cells that act as pAPC: B cells, macrophages and dendritic cells. Arguably, antigen acquisition by dendritic cells is most critical for priming adaptive immune responses, as dendritic cells are the most

In Peyer's patches and ILF, antigen is transported from the lumen by microfold (M) cells to dendritic cells located in the follicle associated epithelium or the underlying subepithelial dome. From there, dendritic cells move into local T cell/follicular areas or drain to the MLN to initiate adaptive responses (Artis, 2008; Kelsall, 2008; Mowat, 2003). The other site for antigen entry is the non-follicular associated epithelium overlying the lamina propria. Under normal conditions antigen is moved across the non-follicular associated epithelium by receptor-mediated transport (Kelsall, 2008) and by dendritic cells located in the lamina propria, which project dendrites through the tight junctions into the lumen (Figure 2, Chieppa et al., 2006; Rescigno et al., 2001). When the epithelium is damaged, as occurs in

barrier, and the barrier is dysfunctional when IL-17 signaling is impaired.

lamina propria via dendritic cell and epithelial cell signaling (He et al., 2007).

**4. Adaptive immunity and Th17 cell development** 

IBD and pathogenic infections, antigens also enter directly.

**4.1 Induction of adaptive immunity** 

efficient class of pAPC.

**3.2 Interleukin-17 and the mucosal barrier** 

brain, adipose, heart, and lung tissue, but poorly expressed in activated T cells. However, similar to IL-17A and IL-17F, IL-17D can induce the expression of IL-6 and IL-8 from endothelial cells (Starnes et al. 2002). Lastly, IL-17E has the most divergent primary sequence compared to IL-17A with 16% homology, and plays a role in pro-allergic type 2 immune responses (Angkasekwinai et al., 2007; Lee et al., 2001; Pan et al., 2001).

Despite the varying degrees of sequence homology and varying functions, the C-terminal region of each IL-17 family member is quite conserved, containing 4 cysteine and 2 serine residues. Three IL-17 crystal structures have been resolved thus far: IL-17A with its neutralizing antibody, IL-17F, and IL-17F with its receptor IL-17RA. These structures have demonstrated the 6 conserved residues adopt a cysteine knot fold, which differs from the canonical cysteine knot found in TGF-β and neurotrophin proteins due to the absence of two cysteine residues (Ely et al., 2009; Gerhardt et al., 2009; Hymowitz et al, 2001).

## **3.1 IL-17 receptor and signaling**

Cytokine receptors are generally classified into six main categories: IL-1 receptors, class I cytokine receptors, class II cytokine receptors, TNF receptors, tyrosine kinase receptors and chemokine receptors (Wang et al., 2009). The IL-17 receptors do not belong to any of these categories based on their unique structure and cytokine interaction (X. Zhang et al., 2011).

The IL-17 receptor family contains 5 members: IL-17RA (or IL-17R), IL-17RB, IL-17RC, IL-17RD, and IL-17RE. IL-17B is known to signal through IL-17RB, IL-17C through IL-17RE, and IL-17E through IL-17RA/IL-17RB (Iwakura et al., 2011; Wright et al., 2008). The receptor for IL-17D remains unknown. IL-17RA and IL-17RC are normally required for IL-17A, IL-17F, and IL-17A/F signaling (Iwakura et al., 2011). However, the IL-17RA is highly expressed on mouse T cells, while IL-17RC is undetectable, and only IL-17A but not IL-17F can induce signaling (Ishigame et al., 2009). Thus, it appears in some cell types IL-17RC is dispensable for IL-17RA signaling. This has lead to the hypotheses that IL-17RA forms either a homodimeric signaling complex or that other subunits can pair with IL-17RA in some cell types that do not express IL-17RC (Gaffen, 2009). Clarification of the receptor complexes for IL-17A and IL-17F is important for understanding how a cell or tissue responds to IL-17A versus IL-17F and will undoubtedly reveal crucial aspects of tissue specific Th17 responses.

Signaling through IL-17 receptors triggers pathways that are usually associated with innate immune signaling (F. Shen et al., 2005; Park et al., 2005). Classical Th1 and Th2 cytokines activate JAK/STAT signaling, however IL-17A and IL-17F mediate signaling through nuclear factor (NF)-κB, NF-κB activator 1 (Act1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) (Chang et al., 2006; Schwandner et al., 2000; Yao et al., 1995b). This mode of signaling is similar to those used by TLRs and the IL-1 receptor family, which function in innate immunity. Furthermore, IL-17A and IL-17F generally induce events that are typical of early inflammation (Gaffen, 2009). Upon receptor binding, IL-17A and IL-17F induce expression of many pro-inflammatory genes including: the cytokines TNF, IL-1, IL-6, granulocye-colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF; the chemokines CXCL1, CXCL5, IL-8, CCL2, and CCL7; antimicrobial defensins, and S100 proteins; as well as matrix metalloproteinases (MMP)-1, -3, and -13 (Iwakura et al., 2011). In this regard, signaling by IL-17A and IL-17F through an IL-17 receptor complex is considered to mediate innate-like inflammatory events.

#### **3.2 Interleukin-17 and the mucosal barrier**

94 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

brain, adipose, heart, and lung tissue, but poorly expressed in activated T cells. However, similar to IL-17A and IL-17F, IL-17D can induce the expression of IL-6 and IL-8 from endothelial cells (Starnes et al. 2002). Lastly, IL-17E has the most divergent primary sequence compared to IL-17A with 16% homology, and plays a role in pro-allergic type 2

Despite the varying degrees of sequence homology and varying functions, the C-terminal region of each IL-17 family member is quite conserved, containing 4 cysteine and 2 serine residues. Three IL-17 crystal structures have been resolved thus far: IL-17A with its neutralizing antibody, IL-17F, and IL-17F with its receptor IL-17RA. These structures have demonstrated the 6 conserved residues adopt a cysteine knot fold, which differs from the canonical cysteine knot found in TGF-β and neurotrophin proteins due to the absence of two

Cytokine receptors are generally classified into six main categories: IL-1 receptors, class I cytokine receptors, class II cytokine receptors, TNF receptors, tyrosine kinase receptors and chemokine receptors (Wang et al., 2009). The IL-17 receptors do not belong to any of these categories based on their unique structure and cytokine interaction (X. Zhang et al., 2011). The IL-17 receptor family contains 5 members: IL-17RA (or IL-17R), IL-17RB, IL-17RC, IL-17RD, and IL-17RE. IL-17B is known to signal through IL-17RB, IL-17C through IL-17RE, and IL-17E through IL-17RA/IL-17RB (Iwakura et al., 2011; Wright et al., 2008). The receptor for IL-17D remains unknown. IL-17RA and IL-17RC are normally required for IL-17A, IL-17F, and IL-17A/F signaling (Iwakura et al., 2011). However, the IL-17RA is highly expressed on mouse T cells, while IL-17RC is undetectable, and only IL-17A but not IL-17F can induce signaling (Ishigame et al., 2009). Thus, it appears in some cell types IL-17RC is dispensable for IL-17RA signaling. This has lead to the hypotheses that IL-17RA forms either a homodimeric signaling complex or that other subunits can pair with IL-17RA in some cell types that do not express IL-17RC (Gaffen, 2009). Clarification of the receptor complexes for IL-17A and IL-17F is important for understanding how a cell or tissue responds to IL-17A versus IL-17F and will undoubtedly reveal crucial aspects of tissue

Signaling through IL-17 receptors triggers pathways that are usually associated with innate immune signaling (F. Shen et al., 2005; Park et al., 2005). Classical Th1 and Th2 cytokines activate JAK/STAT signaling, however IL-17A and IL-17F mediate signaling through nuclear factor (NF)-κB, NF-κB activator 1 (Act1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) (Chang et al., 2006; Schwandner et al., 2000; Yao et al., 1995b). This mode of signaling is similar to those used by TLRs and the IL-1 receptor family, which function in innate immunity. Furthermore, IL-17A and IL-17F generally induce events that are typical of early inflammation (Gaffen, 2009). Upon receptor binding, IL-17A and IL-17F induce expression of many pro-inflammatory genes including: the cytokines TNF, IL-1, IL-6, granulocye-colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF; the chemokines CXCL1, CXCL5, IL-8, CCL2, and CCL7; antimicrobial defensins, and S100 proteins; as well as matrix metalloproteinases (MMP)-1, -3, and -13 (Iwakura et al., 2011). In this regard, signaling by IL-17A and IL-17F through an IL-17 receptor complex is considered to mediate innate-like

immune responses (Angkasekwinai et al., 2007; Lee et al., 2001; Pan et al., 2001).

cysteine residues (Ely et al., 2009; Gerhardt et al., 2009; Hymowitz et al, 2001).

**3.1 IL-17 receptor and signaling**

specific Th17 responses.

inflammatory events.

Although the majority of the defined roles played by IL-17 in mucosal barrier function are related to innate and adaptive immune functions, IL-17 has also been found to directly regulate components of the physical mucosal barrier. In colonic epithelial monolayers IL-17A enhances tight junction formation by increasing claudins 1 and 2 association with the membrane (Kinugasa et al., 2000). Direct application of IL-17A to T84 monolayers increased transepithelial resistance and decreased manitol flux through monolayers. Thus, IL-17A may have an important role in maintaining tight junctions and epithelial restitution during repair processes. In airway epithelial cells IL-17A induces mucin gene expression, and it may have similar inductive effects in the intestine on goblet cells (Chen et al., 2003). IL-17A also induces expression of β-defensins in the colon (Ishigame et al., 2009). Furthermore, in subepithelial myofibroblasts, which sit just below the epithelium, IL-17A reduced TNF-αinduced secretion of pro-inflammatory cytokines, demonstrating that IL-17 is not implicitly a pro-inflammatory cytokine. Additionally, IL-17 receptor-deficient mice show increased dissemination of *S. typhimurium* from the gut (Raffatellu et al., 2008). Taken together, it appears IL-17 can dynamically regulate components of the physical intestinal epithelial barrier, and the barrier is dysfunctional when IL-17 signaling is impaired.

## **4. Adaptive immunity and Th17 cell development**

#### **4.1 Induction of adaptive immunity**

There are numerous locations in the gut where adaptive immune responses are initiated. These include organized lymphoid tissue such as Peyer's patches and isolated lymphoid follicles (ILF) that are embedded directly in the epithelial wall, and mesenteric lymph nodes (MLN), which are connected to the intestinal mucosa by draining lymphatic vessels (Figure 2, Mowat, 2003). Furthermore, there is evidence that adaptive responses occur directly in the lamina propria via dendritic cell and epithelial cell signaling (He et al., 2007).

Under homeostatic conditions, intestinal luminal contents are constitutively sampled and processed by professional antigen presenting cells (pAPC). pAPC present processed antigen to the naive T cell population, which has an infinite repertoire of antigen-specific receptors. Upon presentation of antigen to a T cell bearing a cognate receptor, the pAPC drives an antigen specific T cell response. Depending on the accompanying signals from the pAPC and surrounding environment, the T cell may become activated into an effector cell, anergic (unresponsive to antigen) or apoptotic. Classically there are three types of cells that act as pAPC: B cells, macrophages and dendritic cells. Arguably, antigen acquisition by dendritic cells is most critical for priming adaptive immune responses, as dendritic cells are the most efficient class of pAPC.

In Peyer's patches and ILF, antigen is transported from the lumen by microfold (M) cells to dendritic cells located in the follicle associated epithelium or the underlying subepithelial dome. From there, dendritic cells move into local T cell/follicular areas or drain to the MLN to initiate adaptive responses (Artis, 2008; Kelsall, 2008; Mowat, 2003). The other site for antigen entry is the non-follicular associated epithelium overlying the lamina propria. Under normal conditions antigen is moved across the non-follicular associated epithelium by receptor-mediated transport (Kelsall, 2008) and by dendritic cells located in the lamina propria, which project dendrites through the tight junctions into the lumen (Figure 2, Chieppa et al., 2006; Rescigno et al., 2001). When the epithelium is damaged, as occurs in IBD and pathogenic infections, antigens also enter directly.

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 97

theory any possible antigen can be recognized. Classical naive T cells express an αβ T cell receptor and a co-receptor, which comes in two flavors: CD4 or CD8. Accordingly, CD4 expressing T cells are called CD4+ T cells and CD8 expressing T cells are called CD8+ T cells. CD4+ T cells are also called T helper cells because following establishment of the adaptive phase by innate defenses, CD4+ T cells become the central coordinators of the adaptive immune response. The primary effector function of CD4+ T cells is to help and regulate other immune cells. Upon encountering their cognate antigen on mature pAPCs, CD4+ T cells

There are currently four well characterized lineages of Th cells: Th1, Th2, Treg, and Th17 (Figure 3). Naïve CD4+ T cells differentiate into Th1 cells in the presence of IFNγ and IL-12, which enhances the expression of the principal Th1 transcription factors, T-box family of transcription factors (T-bet) and the signal transducers and activators of transcription protein 4 (STAT4). Effector cytokines produced by Th1 cells include IFNγ, TNFα and IL-2, which help to clear intracellular pathogens. Th2 cells differentiate in the presence of IL-4, which activates STAT6 and leads to the expression of the transcription factor GATA binding protein 3 (GATA3). Th2-derived cytokines, including IL-4, IL-5 and IL-13, are important in

Fig. 3. T helper cell differentiation. After encountering an antigen-presenting cell (APC) within the periphery, naïve T helper (Th0) cells are able to differentiate into one of four Th subsets based on the cytokine milieu present. In the presence of interleukin (IL)-12, the activation of transcription factors STAT4 and T-bet lead to Th1 development, whereas IL-4 results in the activation of STAT6 and GATA3, leading to Th2 development. In the presence of TGF-β, Th0 cells will differentiate into inducible T regulatory (iTreg) cells following transcription of STAT5 and Foxp3, unless IL-6 (in mouse) or IL-21 (in human) is present in addition to TGF-β, in which case Th17 cells will develop following ROR-γt transcription.

proliferate and differentiate into antigen-specific effector cells.

mediating asthma and allergic responses (Zhu & Paul, 2010).

**4.2.1 T Helper cell differentiation** 

Fig. 2. Schematic of organized gastrointestinal lymphoid tissues. Antigens can be transported from the lumen to antigen presenting cells, such as dendritic cells (DC), by the specialized M cells of Peyer's patches where adaptive immune responses can be generated. Additionally, DCs are able to directly sample luminal antigens by projecting dendrites through the intestinal barrier. DCs can then migrate to local T cell areas, or drain to mesenteric lymph nodes (MLN) through lymphatic vessels. Other immune cells in the lamina propria include: mucosal macrophages, γδ T cells, αβ T cells and IgA-secreting B cells.

Dendritic cells are equipped to recognize microbial products with an array of PRR and in doing so, undergo a process of maturation in order to become proficient antigen presenters for naive T cells. In addition to down-regulating their phagocytic machinery and upregulating antigen processing pathways, dendritic cells secrete an array of immunomodulatory cytokines. At first, they express a mixed cytokine profile. However, a dominant cytokine profile emerges and this dictates the type of adaptive immunity that develops (Wilson et al., 2009). The specific constellation of PRRs that are engaged on a dendritic cell is what determines their cytokine profile.

#### **4.2 Adaptive immune cells**

The defining feature of an adaptive immune system is antigen-specific immunity. The first encounter with antigen leads to clonal expansion of a few antigen-specific lymphocytes, which target immune responses towards their cognate antigens. Some of these cells become longlived memory cells and they enable the immune system to remember antigen that has already been encountered, so that upon re-exposure a tailored immune response is quickly recalled. The cells of the adaptive immune system are T and B-lymphocytes. Each lymphocyte bears a surface receptor of a single specificity that binds antigen in a highly specific manner. T and B cell development generates an infinitely diverse repertoire of T and B cell receptors, so that in theory any possible antigen can be recognized. Classical naive T cells express an αβ T cell receptor and a co-receptor, which comes in two flavors: CD4 or CD8. Accordingly, CD4 expressing T cells are called CD4+ T cells and CD8 expressing T cells are called CD8+ T cells. CD4+ T cells are also called T helper cells because following establishment of the adaptive phase by innate defenses, CD4+ T cells become the central coordinators of the adaptive immune response. The primary effector function of CD4+ T cells is to help and regulate other immune cells. Upon encountering their cognate antigen on mature pAPCs, CD4+ T cells proliferate and differentiate into antigen-specific effector cells.

#### **4.2.1 T Helper cell differentiation**

96 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Fig. 2. Schematic of organized gastrointestinal lymphoid tissues. Antigens can be transported from the lumen to antigen presenting cells, such as dendritic cells (DC), by the specialized M cells of Peyer's patches where adaptive immune responses can be generated. Additionally, DCs are able to directly sample luminal antigens by projecting dendrites through the intestinal barrier. DCs can then migrate to local T cell areas, or drain to mesenteric lymph nodes (MLN) through lymphatic vessels. Other immune cells in the lamina propria include: mucosal

Dendritic cells are equipped to recognize microbial products with an array of PRR and in doing so, undergo a process of maturation in order to become proficient antigen presenters for naive T cells. In addition to down-regulating their phagocytic machinery and upregulating antigen processing pathways, dendritic cells secrete an array of immunomodulatory cytokines. At first, they express a mixed cytokine profile. However, a dominant cytokine profile emerges and this dictates the type of adaptive immunity that develops (Wilson et al., 2009). The specific constellation of PRRs that are engaged on a dendritic cell is

The defining feature of an adaptive immune system is antigen-specific immunity. The first encounter with antigen leads to clonal expansion of a few antigen-specific lymphocytes, which target immune responses towards their cognate antigens. Some of these cells become longlived memory cells and they enable the immune system to remember antigen that has already been encountered, so that upon re-exposure a tailored immune response is quickly recalled. The cells of the adaptive immune system are T and B-lymphocytes. Each lymphocyte bears a surface receptor of a single specificity that binds antigen in a highly specific manner. T and B cell development generates an infinitely diverse repertoire of T and B cell receptors, so that in

macrophages, γδ T cells, αβ T cells and IgA-secreting B cells.

what determines their cytokine profile.

**4.2 Adaptive immune cells** 

There are currently four well characterized lineages of Th cells: Th1, Th2, Treg, and Th17 (Figure 3). Naïve CD4+ T cells differentiate into Th1 cells in the presence of IFNγ and IL-12, which enhances the expression of the principal Th1 transcription factors, T-box family of transcription factors (T-bet) and the signal transducers and activators of transcription protein 4 (STAT4). Effector cytokines produced by Th1 cells include IFNγ, TNFα and IL-2, which help to clear intracellular pathogens. Th2 cells differentiate in the presence of IL-4, which activates STAT6 and leads to the expression of the transcription factor GATA binding protein 3 (GATA3). Th2-derived cytokines, including IL-4, IL-5 and IL-13, are important in mediating asthma and allergic responses (Zhu & Paul, 2010).

Fig. 3. T helper cell differentiation. After encountering an antigen-presenting cell (APC) within the periphery, naïve T helper (Th0) cells are able to differentiate into one of four Th subsets based on the cytokine milieu present. In the presence of interleukin (IL)-12, the activation of transcription factors STAT4 and T-bet lead to Th1 development, whereas IL-4 results in the activation of STAT6 and GATA3, leading to Th2 development. In the presence of TGF-β, Th0 cells will differentiate into inducible T regulatory (iTreg) cells following transcription of STAT5 and Foxp3, unless IL-6 (in mouse) or IL-21 (in human) is present in addition to TGF-β, in which case Th17 cells will develop following ROR-γt transcription.

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 99

Zheng et al., 2008). More recent evidence also suggests there is an early Th17-like response during *C. rodentium* infection that is dependent on the activation of the innate immune receptors Nod1 and Nod2 (Geddes et al., 2011). Whether or not this will directly relate to the *NOD2* coding variants identified as risk factors for IBD (Hugot & Cho, 2002) remains to

Since the discovery of IL-23 as a critical regulator of Th17 responses and that there are increased numbers of Th17 cells in IBD patients (Kleinschek et al., 2009), the importance of Th17 cells and their effector cytokines has been an active area of IBD research. To help elucidate the precise role of the Th17 subset, three principle animal models of intestinal inflammation resembling CD have been employed: T cell transfer models of colitis, trinitobenzene sulfonic acid (TNBS)-induced colitis, and dextran sulfate sodium (DSS) induced colitis. With the T cell-transfer model, the initiation of colitis via an adaptive immune response is modeled through the transfer of naïve CD4+ T cells (CD45RBhigh) to immune-deficient mice that lack T cells and B cells, such as recombination activating gene (RAG)-deficient mice, or severe combined immune-deficient (SCID) mice. The naïve cells introduced develop into pro-inflammatory effector T cells in the absence of a mature immune cell population (CD45RBlow) containing Treg cells, and spontaneous intestinal inflammation develops (Powrie et al., 1994a). TNBS-induced colitis is also dependent on the adaptive immune system, where mucosal inflammation following the administration of the haptenizing agent TNBS is mediated by Th1 and Th17 responses (Alex et al, 2009). In contrast to the latter two models, DSS-induced colitis does not require T cells to initiate inflammation. DSS is thought to disrupt the epithelial barrier, resulting in the activation of lamina propria cells by the normal microflora. In the acute DSS model both Th1 and Th17 cells accumulate; however, if the DSS is given in several cycles to establish chronic inflammation, the cytokine profile shifts towards Th2 (Alex et al., 2009). Therefore, acute DSS can be used as a model for CD whereas chronic DSS is more representative of UC.

IL-23 has been found to critically mediate intestinal inflammation through both adaptive and innate immune pathways. Interestingly, an uncommon coding variant of the *IL23R* gene, which encodes a subunit of the IL-23 receptor, was found to confer strong protection against both CD and UC (Duerr et al., 2006). T cell transfer models show that IL-23 is required for spontaneous development of colitis by activated CD4+ T cells (Elson et al., 2007; Hue et al., 2006). Similarly, RAG deficient mice that are also IL-23p19 or IL-12p40 deficient (do not produce IL-23) do not develop spontaneous intestinal inflammation, whereas RAG deficient mice that lack IL-12p35 (do not produce IL-12) still develop colitis (Hue et al., 2006). In these experimental systems IL-23 and not IL-12 drives intestinal inflammation. Interestingly, though IL-23p19 deficient mice fail to develop intestinal inflammation, they still develop a systemic inflammatory response (Hue et al., 2006). This demonstrates that IL-23 driven inflammation by CD4+ T cells is localized to the gut. A transfer model with bacteria-reactive CD4+ T cells showed that neutralization of IL-23p19 with a monoclonal antibody attenuates intestinal inflammation and that individually, bacteria-reactive Th17 cells induce more inflammation than bacteria-reactive Th1 cells (Elson et al., 2007). The latter study also highlights that Th1 and Th17 cells have an overlapping ability to promote

be explored.

**6. Role of IL-17 in IBD pathogenesis** 

**6.1 The IL-23/Th17 axis and IBD** 

Most Treg cells, termed natural Treg (nTreg) cells, are fully differentiated before leaving the thymus, upon TCR stimulation and encountering IL-2 or IL-15. This results in the activation of STAT5 and leads to forkhead box (Fox)p3 expression, the characteristic transcription factor of Treg cells (Burchill et al., 2008). Once these cells leave the thymus, they can home to mucosal surfaces, including the GI tract where the presence of TGF-β helps them to maintain their regulatory phenotype (Barnes & Powrie, 2009). TGF-β is also able to induce the expression of Foxp3 in naïve T cells within the periphery, resulting in inducible Treg (iTreg) cells. Primarily through the production of IL-10, nTreg and iTreg cells share the same suppressive phenotype and function to maintain peripheral tolerance and prevent autoimmunity (Maloy et al., 2003; Read et al., 2000; Zheng & Rudensky, 2007).

Th17 cellular differentiation also depends on TGF-β, however with the additional presence of IL-6 in mice (Veldhoen et al., 2006), or IL-21 in humans (L. Yang et al., 2008), Foxp3 expression is inhibited and STAT3 activation leads to expression of the transcriptional regulator retinoic acid receptor-related orphan receptor-γt (RORγt), which drives Th17 differentiation (Ivanov et al., 2006). Once differentiated, Th17 cells are highly responsive to IL-21 and IL-23, cytokines that function to maintain the Th17 phenotype. The principle effector cytokines produced by Th17 cells include IL-17A, IL-17F, IL-21, and IL-22.

## **5. Role of IL-17 in enteric infections**

Several murine models of infectious disease highlight the presence and importance of IL-17 in intestinal inflammation: *Helicobacter hepaticus, Salmonella enterica* serotype *typhimurium*, and *Citrobacter rodentium*. In *H. hepaticus*-induced typhlocolitis, a model of T-cell independent innate inflammation, local increases in IL-23 induced the secretion of IL-17 from non-T cell sources (Hue et al., 2006). A similar study using the same *H. hepaticus* model of bacteria-driven innate colitis confirmed the IL-23-dependent increases in IL-17, and went on to characterize the IL-17-producing cells. This led to the identification of a novel innate lymphoid cell population that accumulates in the inflamed colon, and is able to mediate acute and chronic innate colitis in response to IL-23 stimulation (Buonocore et al., 2010).

In the second infectious model with *S. typhimurium*, initial inflammatory responses are important to contain the infection as localized gastroenteritis, and prevent the systemic spread of bacteria. Macrophages and dendritic cells infected with *S. typhimurium* are a major source of IL-23, and five hours post- *S. typhimurium* infection, IL-17 expression is markedly up regulated (Raffetulla et al., 2008, 2009). The increased IL-17 production resulted in IL-17-dependent intestinal epithelial induction of antimicrobial peptides (Raffatellu et al. 2009). In IL-23p19-deficient mice, the increased expression of IL-17 during *S. typhimurim* infection was abrogated. Although αβ T cells were found to be the predominant cell type expressing the IL-23R, there was a marked increase in γδ T cells expressing the IL-23R during *S. typimurium* infection. γδ T cell-deficient mice demonstrated a blunted expression of IL-17, suggesting that γδ T cells are a significant source, but not the only source of IL-17 during an acute bacterial infection (Godinez et al., 2009).

Lastly, *C. rodentium* is a non-invasive bacterium that transiently colonizes the large intestine of mice. In addition to serving as a model for attaching/effacing bacteria, *C. rodentium* infection can be used a model of IBD, as the infection-associated pathology shares many features with IBD (Mundy et al., 2005). The first evidence of IL-17 involvement in *C. rodentium* infection implicated its importance during the peak and late stages of infection, demonstrating a role for adaptive Th17 cells in clearing the infection (Symonds et al., 2009; Zheng et al., 2008). More recent evidence also suggests there is an early Th17-like response during *C. rodentium* infection that is dependent on the activation of the innate immune receptors Nod1 and Nod2 (Geddes et al., 2011). Whether or not this will directly relate to the *NOD2* coding variants identified as risk factors for IBD (Hugot & Cho, 2002) remains to be explored.

## **6. Role of IL-17 in IBD pathogenesis**

98 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

Most Treg cells, termed natural Treg (nTreg) cells, are fully differentiated before leaving the thymus, upon TCR stimulation and encountering IL-2 or IL-15. This results in the activation of STAT5 and leads to forkhead box (Fox)p3 expression, the characteristic transcription factor of Treg cells (Burchill et al., 2008). Once these cells leave the thymus, they can home to mucosal surfaces, including the GI tract where the presence of TGF-β helps them to maintain their regulatory phenotype (Barnes & Powrie, 2009). TGF-β is also able to induce the expression of Foxp3 in naïve T cells within the periphery, resulting in inducible Treg (iTreg) cells. Primarily through the production of IL-10, nTreg and iTreg cells share the same suppressive phenotype and function to maintain peripheral tolerance and prevent

Th17 cellular differentiation also depends on TGF-β, however with the additional presence of IL-6 in mice (Veldhoen et al., 2006), or IL-21 in humans (L. Yang et al., 2008), Foxp3 expression is inhibited and STAT3 activation leads to expression of the transcriptional regulator retinoic acid receptor-related orphan receptor-γt (RORγt), which drives Th17 differentiation (Ivanov et al., 2006). Once differentiated, Th17 cells are highly responsive to IL-21 and IL-23, cytokines that function to maintain the Th17 phenotype. The principle

Several murine models of infectious disease highlight the presence and importance of IL-17 in intestinal inflammation: *Helicobacter hepaticus, Salmonella enterica* serotype *typhimurium*, and *Citrobacter rodentium*. In *H. hepaticus*-induced typhlocolitis, a model of T-cell independent innate inflammation, local increases in IL-23 induced the secretion of IL-17 from non-T cell sources (Hue et al., 2006). A similar study using the same *H. hepaticus* model of bacteria-driven innate colitis confirmed the IL-23-dependent increases in IL-17, and went on to characterize the IL-17-producing cells. This led to the identification of a novel innate lymphoid cell population that accumulates in the inflamed colon, and is able to mediate acute and chronic innate colitis in response to IL-23 stimulation (Buonocore et al., 2010). In the second infectious model with *S. typhimurium*, initial inflammatory responses are important to contain the infection as localized gastroenteritis, and prevent the systemic spread of bacteria. Macrophages and dendritic cells infected with *S. typhimurium* are a major source of IL-23, and five hours post- *S. typhimurium* infection, IL-17 expression is markedly up regulated (Raffetulla et al., 2008, 2009). The increased IL-17 production resulted in IL-17-dependent intestinal epithelial induction of antimicrobial peptides (Raffatellu et al. 2009). In IL-23p19-deficient mice, the increased expression of IL-17 during *S. typhimurim* infection was abrogated. Although αβ T cells were found to be the predominant cell type expressing the IL-23R, there was a marked increase in γδ T cells expressing the IL-23R during *S. typimurium* infection. γδ T cell-deficient mice demonstrated a blunted expression of IL-17, suggesting that γδ T cells are a significant source, but not the

autoimmunity (Maloy et al., 2003; Read et al., 2000; Zheng & Rudensky, 2007).

effector cytokines produced by Th17 cells include IL-17A, IL-17F, IL-21, and IL-22.

only source of IL-17 during an acute bacterial infection (Godinez et al., 2009).

Lastly, *C. rodentium* is a non-invasive bacterium that transiently colonizes the large intestine of mice. In addition to serving as a model for attaching/effacing bacteria, *C. rodentium* infection can be used a model of IBD, as the infection-associated pathology shares many features with IBD (Mundy et al., 2005). The first evidence of IL-17 involvement in *C. rodentium* infection implicated its importance during the peak and late stages of infection, demonstrating a role for adaptive Th17 cells in clearing the infection (Symonds et al., 2009;

**5. Role of IL-17 in enteric infections** 

Since the discovery of IL-23 as a critical regulator of Th17 responses and that there are increased numbers of Th17 cells in IBD patients (Kleinschek et al., 2009), the importance of Th17 cells and their effector cytokines has been an active area of IBD research. To help elucidate the precise role of the Th17 subset, three principle animal models of intestinal inflammation resembling CD have been employed: T cell transfer models of colitis, trinitobenzene sulfonic acid (TNBS)-induced colitis, and dextran sulfate sodium (DSS) induced colitis. With the T cell-transfer model, the initiation of colitis via an adaptive immune response is modeled through the transfer of naïve CD4+ T cells (CD45RBhigh) to immune-deficient mice that lack T cells and B cells, such as recombination activating gene (RAG)-deficient mice, or severe combined immune-deficient (SCID) mice. The naïve cells introduced develop into pro-inflammatory effector T cells in the absence of a mature immune cell population (CD45RBlow) containing Treg cells, and spontaneous intestinal inflammation develops (Powrie et al., 1994a). TNBS-induced colitis is also dependent on the adaptive immune system, where mucosal inflammation following the administration of the haptenizing agent TNBS is mediated by Th1 and Th17 responses (Alex et al, 2009). In contrast to the latter two models, DSS-induced colitis does not require T cells to initiate inflammation. DSS is thought to disrupt the epithelial barrier, resulting in the activation of lamina propria cells by the normal microflora. In the acute DSS model both Th1 and Th17 cells accumulate; however, if the DSS is given in several cycles to establish chronic inflammation, the cytokine profile shifts towards Th2 (Alex et al., 2009). Therefore, acute DSS can be used as a model for CD whereas chronic DSS is more representative of UC.

## **6.1 The IL-23/Th17 axis and IBD**

IL-23 has been found to critically mediate intestinal inflammation through both adaptive and innate immune pathways. Interestingly, an uncommon coding variant of the *IL23R* gene, which encodes a subunit of the IL-23 receptor, was found to confer strong protection against both CD and UC (Duerr et al., 2006). T cell transfer models show that IL-23 is required for spontaneous development of colitis by activated CD4+ T cells (Elson et al., 2007; Hue et al., 2006). Similarly, RAG deficient mice that are also IL-23p19 or IL-12p40 deficient (do not produce IL-23) do not develop spontaneous intestinal inflammation, whereas RAG deficient mice that lack IL-12p35 (do not produce IL-12) still develop colitis (Hue et al., 2006). In these experimental systems IL-23 and not IL-12 drives intestinal inflammation. Interestingly, though IL-23p19 deficient mice fail to develop intestinal inflammation, they still develop a systemic inflammatory response (Hue et al., 2006). This demonstrates that IL-23 driven inflammation by CD4+ T cells is localized to the gut. A transfer model with bacteria-reactive CD4+ T cells showed that neutralization of IL-23p19 with a monoclonal antibody attenuates intestinal inflammation and that individually, bacteria-reactive Th17 cells induce more inflammation than bacteria-reactive Th1 cells (Elson et al., 2007). The latter study also highlights that Th1 and Th17 cells have an overlapping ability to promote

The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 101

In addition to Th17 cells, dysregulated Th1 and Foxp3+ iTreg responses are also involved. Yet, the precise nature of the relationship between Th17 cells and Th1 *as well as* Th17 cells and Foxp3+ iTregs is unclear. Furthermore, in the gut there appears to be multiple cellular sources of IL-17A and IL-17F, in addition to heterogeneous expression of their receptors, IL-17RA and IL-17RC. Our understanding of how IL-17A and IL-17F mediate their cell specific effects and how this plays out during steady states, infectious disease and chronic inflammation in the intestinal tract is currently in progress. Beneficial results have been obtained using antibodies to neutralize IL-12p40 in Crohn's disease and genome wide association studies implicate the IL-23-Th17 axis in both Crohn's disease and ulcerative colitis. Together these data suggest therapies specifically targeting Th17 responses might provide better treatments. However, animal models have also shown IL-17A and IL-17F to critically mediate host protection and components of normal barrier function. Thus given these roles, targeted interventions of IL-17A and IL-17F will need careful consideration. Inflammatory bowel diseases are a complex set of diseases involving pre-disposing genetic factors and environmental triggers. The emerging IL-23-Th17 axis represents one significant component of these diseases among several. Though progress has been made, a substantial amount of work remains to identify pathways and mechanisms that connect Th17 cells, IL-17A and IL-17F to the etiology of inflammatory bowel diseases. In particular, genome wide association studies have established a key role for innate immunity in these diseases. Most well known are *NOD2* and autophagy genes *ATG16L* and *IRGM* involved in bacterial detection and processing. In this regard, much less is known about IL-23, IL-17A and IL-17F in aberrant innate immune responses. For now we can ascertain that both innate and adaptive immunity coordinate an imbalanced relationship between host and microflora that leads to chronic intestinal inflammation, and that Th17 cells and the IL-17A/F cytokine

network participate in both arms of the immune system that has gone awry.

Aggarwal, S., Ghilardi, N., Xie, M., de Sauvage, F. J. & Gurney, A. L. (2003). Interleukin-23

Interleukin-17. *Journal of Biological Chemistry*. Vol:278, No:3, pp. 1910-1914 Alex, P., Zachos, N. C., Nguyen, T., Gonzales, L., Chen, T. E., Conklin, L. S., Centola, M. &

Angkasekwinai, P., Park, H., Wang, Y.H., Wang, Y.H., Chang, S. H., Corry, D. B., Lui, Y.J.,

Arrieta, M. C., Bistritz, L. & Meddings, J. B. (2006) Alterations in Intestinal Permeability. *Gut*.

Promotes a Distinct CD4 T Cell Activation State Characterized by the Production of

Li, X. (2009). Distinct Cytokine Patterns Identified from Multiplex Profiles of Murine DSS and TNBS-Induced Colitis. *Inflammatory Bowel Disease*. Vol:15, No:3,

Zhu, Z. & Dong, C. (2007). Interleukin 25 Promotes the Initiation of Proallergic Type 2 Responses. *Journal of Experimental Medicine*. Vol:204, No:7, pp. 1509-1517 Arisawa, T., Tahara, T., Shibata, T., Nagasaka, M., Nakamura, M., Kamiya, Y., Fujita, H.,

Nakamura, M., Yoshioka, D., Arima, Y., Okubo, M., Hirata, I. & Nakano, H. (2008). The Influence of Polymorphisms of Interleukin-17A and Interleukin-17F Genes on the Susceptibility to Ulcerative Colitis. *Journal of Clinical Immunology*. Vol:28, No:1,

**8. References** 

pp. 341-352

pp. 44-49

Vol:55, pp. 1512-1520

pathologic responses. Although IL-12 as a Th1 inducing cytokine is dispensable for initiating colitis, Th1 responses should not be considered insignificant in inflammatory bowel disease. Previous studies have shown that neutralization of IFN-γ (signature Th1 cytokine) prevents intestinal inflammation and severe wasting, and transfer of IFN-γ deficient T cells into RAG deficient mice fails to induce colitis (Ito & Fathman, 1997; O'Connor et al., 2009; Powrie et al., 1994b). Taken together, these results suggest that although IFN-γ still appears to be the main effector cytokine driving the cell-transfer colitis model, IL-23 and Th17 responses are essential to support the development of chronic inflammation.

## **6.2 Contributions of IL-17A and IL-17F to IBD**

There are multiple lines of evidence to suggest that blocking IL-17A and IL-17F would prevent intestinal inflammation as both cytokines robustly induce neutrophil recruitment and pro-inflammatory cytokines, blocking IL-23 prevents development of pathogenic Th17 cells and colitis in animal models, and blocking IL-23 signaling is beneficial for treating CD. Along these same lines, IL-17R-deficient mice are significantly protected from TNBSinduced colitis, despite no change in the levels of IL-23 or IL-12 and IFN-γ (Z. Zhang et al., 2006). Thus, it was unexpected that neutralization of IL-17A exacerbated intestinal inflammation in the dextran sodium sulfate (DSS) colitis model (Ogawa et al., 2004). Animals treated with an IL-17A monoclonal antibody had enhanced inflammatory cell infiltrates into the mucosa and submucosa, more severe mucosal injury and drastically increased weight loss. Moreover, addition of IL-17A attenuated the response (Ogawa et al, 2004). These results were confirmed in IL-17A knockout mice, which also developed more severe DSS-induced colitis (X. Yang et al., 2008). Interestingly, this same study showed that IL-17F knockout mice, unlike IL-17A knockouts, were protected from DSS-induced colitis. Colons of IL-17F deficient mice showed little pathology and extremely low levels of proinflammatory cytokines (Yang et al, 2008). Using a T cell transfer model, IL-17A secretion by Th17 cells was also protective against the development of intestinal inflammation, as IL-17A deficient T cells transferred into RAG deficient mice caused more severe disease than transferred wildtype T cells (O'Connor et al, 2009). Additionally, IL-17A has been shown to directly inhibit Th1 cells and suppress Th1 mediated intestinal inflammation (Awasthi & Kuchroo, 2009). Taken together, these data suggest that IL-17A has protective roles in acute tissue inflammation and that IL-17F has pathogenic functions. However, there has also been some evidence that IL-17A is not protective. T cells deficient in ROR-γt, and therefore unable to differentiate into Th17 effector cells, were unable to induce colitis when transferred to RAG-deficient mice, but treatment with IL-17A caused colitis after the transfer of ROR-γtdeficient cells (Leppkes et al., 2009). Therefore, additional work on the mechanisms, function, and regulation of IL-17A/F in the context of intestinal inflammation is required before confident and definitive conclusions can be drawn.

## **7. Conclusion**

Knowledge of Th17 cells and their characteristic cytokines IL-17A and IL-17F has rapidly progressed. Likewise, significant progress has been made towards understanding their role in regulating the gut environment. However, there are numerous outstanding questions. The Th17 subset is unequivocally associated with chronic inflammatory bowel diseases, and the current belief is that they are instigated by a loss of tolerance to the intestinal microflora. In addition to Th17 cells, dysregulated Th1 and Foxp3+ iTreg responses are also involved. Yet, the precise nature of the relationship between Th17 cells and Th1 *as well as* Th17 cells and Foxp3+ iTregs is unclear. Furthermore, in the gut there appears to be multiple cellular sources of IL-17A and IL-17F, in addition to heterogeneous expression of their receptors, IL-17RA and IL-17RC. Our understanding of how IL-17A and IL-17F mediate their cell specific effects and how this plays out during steady states, infectious disease and chronic inflammation in the intestinal tract is currently in progress. Beneficial results have been obtained using antibodies to neutralize IL-12p40 in Crohn's disease and genome wide association studies implicate the IL-23-Th17 axis in both Crohn's disease and ulcerative colitis. Together these data suggest therapies specifically targeting Th17 responses might provide better treatments. However, animal models have also shown IL-17A and IL-17F to critically mediate host protection and components of normal barrier function. Thus given these roles, targeted interventions of IL-17A and IL-17F will need careful consideration.

Inflammatory bowel diseases are a complex set of diseases involving pre-disposing genetic factors and environmental triggers. The emerging IL-23-Th17 axis represents one significant component of these diseases among several. Though progress has been made, a substantial amount of work remains to identify pathways and mechanisms that connect Th17 cells, IL-17A and IL-17F to the etiology of inflammatory bowel diseases. In particular, genome wide association studies have established a key role for innate immunity in these diseases. Most well known are *NOD2* and autophagy genes *ATG16L* and *IRGM* involved in bacterial detection and processing. In this regard, much less is known about IL-23, IL-17A and IL-17F in aberrant innate immune responses. For now we can ascertain that both innate and adaptive immunity coordinate an imbalanced relationship between host and microflora that leads to chronic intestinal inflammation, and that Th17 cells and the IL-17A/F cytokine network participate in both arms of the immune system that has gone awry.

#### **8. References**

100 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

pathologic responses. Although IL-12 as a Th1 inducing cytokine is dispensable for initiating colitis, Th1 responses should not be considered insignificant in inflammatory bowel disease. Previous studies have shown that neutralization of IFN-γ (signature Th1 cytokine) prevents intestinal inflammation and severe wasting, and transfer of IFN-γ deficient T cells into RAG deficient mice fails to induce colitis (Ito & Fathman, 1997; O'Connor et al., 2009; Powrie et al., 1994b). Taken together, these results suggest that although IFN-γ still appears to be the main effector cytokine driving the cell-transfer colitis model, IL-23 and Th17 responses are essential to support the development of chronic

There are multiple lines of evidence to suggest that blocking IL-17A and IL-17F would prevent intestinal inflammation as both cytokines robustly induce neutrophil recruitment and pro-inflammatory cytokines, blocking IL-23 prevents development of pathogenic Th17 cells and colitis in animal models, and blocking IL-23 signaling is beneficial for treating CD. Along these same lines, IL-17R-deficient mice are significantly protected from TNBSinduced colitis, despite no change in the levels of IL-23 or IL-12 and IFN-γ (Z. Zhang et al., 2006). Thus, it was unexpected that neutralization of IL-17A exacerbated intestinal inflammation in the dextran sodium sulfate (DSS) colitis model (Ogawa et al., 2004). Animals treated with an IL-17A monoclonal antibody had enhanced inflammatory cell infiltrates into the mucosa and submucosa, more severe mucosal injury and drastically increased weight loss. Moreover, addition of IL-17A attenuated the response (Ogawa et al, 2004). These results were confirmed in IL-17A knockout mice, which also developed more severe DSS-induced colitis (X. Yang et al., 2008). Interestingly, this same study showed that IL-17F knockout mice, unlike IL-17A knockouts, were protected from DSS-induced colitis. Colons of IL-17F deficient mice showed little pathology and extremely low levels of proinflammatory cytokines (Yang et al, 2008). Using a T cell transfer model, IL-17A secretion by Th17 cells was also protective against the development of intestinal inflammation, as IL-17A deficient T cells transferred into RAG deficient mice caused more severe disease than transferred wildtype T cells (O'Connor et al, 2009). Additionally, IL-17A has been shown to directly inhibit Th1 cells and suppress Th1 mediated intestinal inflammation (Awasthi & Kuchroo, 2009). Taken together, these data suggest that IL-17A has protective roles in acute tissue inflammation and that IL-17F has pathogenic functions. However, there has also been some evidence that IL-17A is not protective. T cells deficient in ROR-γt, and therefore unable to differentiate into Th17 effector cells, were unable to induce colitis when transferred to RAG-deficient mice, but treatment with IL-17A caused colitis after the transfer of ROR-γtdeficient cells (Leppkes et al., 2009). Therefore, additional work on the mechanisms, function, and regulation of IL-17A/F in the context of intestinal inflammation is required

Knowledge of Th17 cells and their characteristic cytokines IL-17A and IL-17F has rapidly progressed. Likewise, significant progress has been made towards understanding their role in regulating the gut environment. However, there are numerous outstanding questions. The Th17 subset is unequivocally associated with chronic inflammatory bowel diseases, and the current belief is that they are instigated by a loss of tolerance to the intestinal microflora.

inflammation.

**7. Conclusion** 

**6.2 Contributions of IL-17A and IL-17F to IBD** 

before confident and definitive conclusions can be drawn.


The Roles of Interleukin-17 and T Helper 17 Cells in Intestinal Barrier Function 103

Cua, D. J. & Tato, C. M. (2010). Innate IL-17-Producting Cells: The Sentinels of the Immune

Doisne, J. M., Soulard, V., Bécourt, C., Amniai, L., Henrot, P., Havenar-Daughton, C.,

Duerr, R. H., Taylor, K. D., Brant, S. R., Rioux, J. D., Silverberg, M. S., Daly, M. J., Steinhart,

Elson, C. O., Cong, Y., Weaver, C. T., Schoeb, T. R., McClanahan, T. K., Fick, R. B. &

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**6** 

Yutao Yan *Emory University Georgia State University* 

*United States* 

**Pathogenesis of Inflammatory Bowel Diseases** 

Ulcerative colitis (UC) and Crohn's disease (CD), collectively called inflammatory bowel disease (IBD), are idiopathic**,** chronic, and relapsing intestinal inflammatory disorder, characterized by abdominal pain and diarrhea. UC differs dramatically from CD with the respects of disease distribution, morphology, and histopathology; for example, CD can affect any part of the gastrointestinal (GI) tract, usually discontinuously. UC is confined to the colon, it is characterized by continuous inflammation, invariably involving the rectum, and is classified according to its proximal limit (proctitis, distal, or extensive colitis). Further, unlike CD, inflammation in UC is restricted to the mucosal surface, perhaps giving weight to the emerging concept of a defective mucosal barrier in disease pathogenesis. Histologically active UC typically consists of a neutrophilic mucosal infiltrate, goblet cell depletion, ''cryptitis,'' and prominent crypt abscesses. Acute inflammatory process in UC is associated with mucosal (particularly epithelial) cell destruction. Meantime, UC and CD share a lot of inflammatory similarities, such as epithelial barrier dysfunction, genetic susceptibility etc. IBD may result in significant morbidity and mortality, with compromised quality of life and life expectancy. While there is no cure for IBD, the last two decades have seen tremendous advances in our understanding of the pathophysiology of this intestinal inflammation. Even though the precise etiology of IBD remains elusive, it is accepted (Figure 1) that IBD arises from abnormal host–microbe interactions, including qualitative and quantitative changes in the composition of the microbiota, host genetic susceptibility, barrier function, as well as innate and adaptive immunity. In more detail, some defects occur in luminal bacterial antigen sampling by the epithelium, possibly mediated by toll-like receptors (TLRs) or nucleotide binding oligomerisation domain family (NODs), controlled by genetic factors (including NOD2 for CD etc). An over-response to the antigens then stimulates activated dendritic cells to generate Th1-type /Th17 T cells or Th2-type /NK T cells, which then generate cytokines, initiating a cascade of immunologic events resulting in tissue damage. Thus, the factors participating in what manifests as inflammation in UC and CD are part of a dynamic process in which autoantibodies are generated against mucosal antigens in a susceptible host. The autoantibodies are not primarily responsible for disease pathogenesis; rather, they mark for disease-related autoantigens, which likely cross react with bacterial antigens from the normal intestinal flora. In a genetically susceptible host, the interaction results in an exaggerated inflammatory response in which either a lack of regulatory cells or enhanced numbers of effector cells initiates disease. With time, antigenic spreading to host antigens (the autoantigens) occurs; therefore, removal of these bacteria

would no longer affect disease activity (Vanderlugt et al., 1996).

**1. Introduction** 


## **Pathogenesis of Inflammatory Bowel Diseases**

## Yutao Yan

*Emory University Georgia State University United States* 

#### **1. Introduction**

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Ulcerative colitis (UC) and Crohn's disease (CD), collectively called inflammatory bowel disease (IBD), are idiopathic**,** chronic, and relapsing intestinal inflammatory disorder, characterized by abdominal pain and diarrhea. UC differs dramatically from CD with the respects of disease distribution, morphology, and histopathology; for example, CD can affect any part of the gastrointestinal (GI) tract, usually discontinuously. UC is confined to the colon, it is characterized by continuous inflammation, invariably involving the rectum, and is classified according to its proximal limit (proctitis, distal, or extensive colitis). Further, unlike CD, inflammation in UC is restricted to the mucosal surface, perhaps giving weight to the emerging concept of a defective mucosal barrier in disease pathogenesis. Histologically active UC typically consists of a neutrophilic mucosal infiltrate, goblet cell depletion, ''cryptitis,'' and prominent crypt abscesses. Acute inflammatory process in UC is associated with mucosal (particularly epithelial) cell destruction. Meantime, UC and CD share a lot of inflammatory similarities, such as epithelial barrier dysfunction, genetic susceptibility etc. IBD may result in significant morbidity and mortality, with compromised quality of life and life expectancy. While there is no cure for IBD, the last two decades have seen tremendous advances in our understanding of the pathophysiology of this intestinal inflammation. Even though the precise etiology of IBD remains elusive, it is accepted (Figure 1) that IBD arises from abnormal host–microbe interactions, including qualitative and quantitative changes in the composition of the microbiota, host genetic susceptibility, barrier function, as well as innate and adaptive immunity. In more detail, some defects occur in luminal bacterial antigen sampling by the epithelium, possibly mediated by toll-like receptors (TLRs) or nucleotide binding oligomerisation domain family (NODs), controlled by genetic factors (including NOD2 for CD etc). An over-response to the antigens then stimulates activated dendritic cells to generate Th1-type /Th17 T cells or Th2-type /NK T cells, which then generate cytokines, initiating a cascade of immunologic events resulting in tissue damage. Thus, the factors participating in what manifests as inflammation in UC and CD are part of a dynamic process in which autoantibodies are generated against mucosal antigens in a susceptible host. The autoantibodies are not primarily responsible for disease pathogenesis; rather, they mark for disease-related autoantigens, which likely cross react with bacterial antigens from the normal intestinal flora. In a genetically susceptible host, the interaction results in an exaggerated inflammatory response in which either a lack of regulatory cells or enhanced numbers of effector cells initiates disease. With time, antigenic spreading to host antigens (the autoantigens) occurs; therefore, removal of these bacteria would no longer affect disease activity (Vanderlugt et al., 1996).

Pathogenesis of Inflammatory Bowel Diseases 113

histocompatibility complex (MHC) class II region on chromosome 6 has been clearly

Gene *NOD2* is the breakthrough discovery whose mutations are associated with CD lying either within or near the C-terminal, leucine-rich repeat domain, which is required for microbial sensing. NOD2 is expressed by many leukocytes, including antigen presenting cells, macrophages, and lymphocytes, as well as ileal Paneth cells, fibroblasts, and epithelial cells. Activation of NOD2 by microbial ligands activates the transcription factor nuclear factor κB (NF-κB) and mitogen activated protein kinase (MAPK) signaling, and functions as a positive regulator of immune defense (Hugot et al., 2001; Ogura et al., 2001). The NOD2 ligand muramyl dipeptide (MDP) is ubiquitous, indicating that broad classes of bacteria are capable of activating NOD2. However, the N-glycolyated form of muramyl dipeptide found in mycobacteria and actinomycetes more potently activates NOD2 compared to the Nacetylated form, found more frequently in gram-positive and gram-negative bacteria (Coulombe et al., 2009). Autophagy 16-like 1 (*ATG16L1*) has been strongly associated with CD and encodes a protein component of the autophagy complex (Levine & Deretic, 2007). ATG16L1 is broadly expressed, including in small intestinal Paneth cells (Cadwell et al., 2008) where it mediates exocytosis of secretory granules that contain antimicrobial peptides. IL-10 gene SNPs were found to be involved in the UC by GWAS analysis (Franke et al., 2008), and IL-10-/- mouse is one of the oldest and most widely used animal models of UC, in which spontaneous colitis develops in specific pathogen-free conditions (Kuhn et al., 1993). IL10 is expressed by many different cells of the adaptive and innate immune system including Th1, 2, and 17 cells, subsets of regulatory T cells, dendritic cells, macrophages, mast cells, and natural killer cells (Mosser et al., 2008). It has pleiotropic effects on T and B cells, and importantly limits the release of proinflammatory cytokines like TNF-a and IL-12. In the IL-10-/- mouse model, defective counter regulatory anticytokine responses result in inflammation affecting intestinal mucosa which is characterized by enlarged and branched crypts, reduced number of goblet cells, degeneration of superficial epithelial cells, and increased expression of MHC class II molecules in mouse colon. But these IL-10-/- mice require gut microbia to develop inflammation, giving rise to an attractive theory that IL10 could be involved in restricting the mucosal immune response to enteric flora (Louis et al., 2009, Sellon et al., 1998). Interestingly, this IL-10-/- model of UC have, in another hand, elegantly shown a protective role of IL10: transfer of IL10 producing regulatory T cells to immunodeficient mice prevents or cures colitis (Uhlig et al., 2006). Further, IL10 has been shown to exert a protective effect on carcinogenesis in mice (Erdman et al., 2003). The antiinflammatory response of IL-10 is mediated through IL10 receptor (IL10R) and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL10R is a heterotetrameric molecule; while IL10R1 is specific to IL10R, IL10R2 is found as a subunit of receptors to other cytokines, notably IL22, IL26, and IFNγ. Extracellular matrix gene 1 (ECM1) (Festen et al., 2010), E-cadherin gene (CDH1), Hepatocyte nuclear factor 4 alpha gene (HNF4a), and laminin B1 (Barrett et al., 2009) are another four genes implicated in mucosal barrier function, conferring risk of UC; ECM1 interacts with the basement membrane, inhibits matrix metalloproteinase 9 (MMP9), and strongly activate NFκB (Chan et al., 2007; Matsuda et al., 2003). The Wnt/beta-catenin signal transduction pathway has been shown to influence ECM1 expression (Kenny et al., 2005). E-cadherin is the first genetic correlation between colorectal cancer and UC, Chimeric mice with impaired E-cadherin function due to expression of dominant–negative N-cadherin developed colitis despite possessing an intact immune system (Hermiston & Gordon 1995a, 1995b). Notably, all of

demonstrated to be associated with UC (Toyoda et al., 1993).

Fig. 1. Pathogenesis of IBD. Many different factors, such as genetic factors, environmental factors, and intestinal non-pathogenic or pathogenic bacteria can damage the mucus, epithelium, or the tight junction, to initiate the inappropriate regulation or deregulation of the immune response, leading to the secretion of pro-inammatory cytokines, decrease in epithelial barrier function and initiation of the inamma tion-related signaling pathways. IEC: Intestinal epithelial cell; APC: Antigen presenting cell; TJ: Tight junction. This model adapted from the model presented previously (Yan 2008)

In this chapter, we are going to focus on the involvement of diverse of factors in the pathogenesis of IBD, try to shed some light on the clues of intervention of IBD.

## **2. Genetic factor**

Population-based studies provided compelling evidence that genetic susceptibility plays an essential role in the pathogenesis of IBD, evidence including an 8- to 10-fold greater risk among relatives of UC and CD and greater rates of concordance between twins in UC patients (15.4% in monozygotic vs 3.9% in dizygotic twins) and CD patients (30.3% in monozygotic vs 3.6% in dizygotic twins) (Cho & Brant, 2011). Some of genes encoding protein kinases like ERK1 (Hugot et al., 1996) and p38α (Hampe et al., 1999) are located in major IBD susceptibility regions on chromosome 16 and 6. Recently, substantial advances have been achieved in defining the genetic architecture of IBD since the genome-wide association study (GWAS) analysis heralded a new era of complex disease gene discovery with notable success in CD initially and latterly also in UC. To date, near 99 published IBD susceptibility loci have been discovered and replicated, of which minimum 28 are associated with both UC and CD, although 47 are specific to UC and 24 to CD (Thompson & Lees, 2011). Generally, these genetic loci could be grouped into different categories. Importantly, most of the genes have been linked to defects in innate and adaptive immunity and epithelial barrier function. The first susceptible locus identified in IBD is the major

Fig. 1. Pathogenesis of IBD. Many different factors, such as genetic factors, environmental factors, and intestinal non-pathogenic or pathogenic bacteria can damage the mucus, epithelium, or the tight junction, to initiate the inappropriate regulation or deregulation of the immune response, leading to the secretion of pro-inammatory cytokines, decrease in epithelial barrier function and initiation of the inamma tion-related signaling pathways. IEC: Intestinal epithelial cell; APC: Antigen presenting cell; TJ: Tight junction. This model

In this chapter, we are going to focus on the involvement of diverse of factors in the

Population-based studies provided compelling evidence that genetic susceptibility plays an essential role in the pathogenesis of IBD, evidence including an 8- to 10-fold greater risk among relatives of UC and CD and greater rates of concordance between twins in UC patients (15.4% in monozygotic vs 3.9% in dizygotic twins) and CD patients (30.3% in monozygotic vs 3.6% in dizygotic twins) (Cho & Brant, 2011). Some of genes encoding protein kinases like ERK1 (Hugot et al., 1996) and p38α (Hampe et al., 1999) are located in major IBD susceptibility regions on chromosome 16 and 6. Recently, substantial advances have been achieved in defining the genetic architecture of IBD since the genome-wide association study (GWAS) analysis heralded a new era of complex disease gene discovery with notable success in CD initially and latterly also in UC. To date, near 99 published IBD susceptibility loci have been discovered and replicated, of which minimum 28 are associated with both UC and CD, although 47 are specific to UC and 24 to CD (Thompson & Lees, 2011). Generally, these genetic loci could be grouped into different categories. Importantly, most of the genes have been linked to defects in innate and adaptive immunity and epithelial barrier function. The first susceptible locus identified in IBD is the major

pathogenesis of IBD, try to shed some light on the clues of intervention of IBD.

adapted from the model presented previously (Yan 2008)

**2. Genetic factor** 

histocompatibility complex (MHC) class II region on chromosome 6 has been clearly demonstrated to be associated with UC (Toyoda et al., 1993).

Gene *NOD2* is the breakthrough discovery whose mutations are associated with CD lying either within or near the C-terminal, leucine-rich repeat domain, which is required for microbial sensing. NOD2 is expressed by many leukocytes, including antigen presenting cells, macrophages, and lymphocytes, as well as ileal Paneth cells, fibroblasts, and epithelial cells. Activation of NOD2 by microbial ligands activates the transcription factor nuclear factor κB (NF-κB) and mitogen activated protein kinase (MAPK) signaling, and functions as a positive regulator of immune defense (Hugot et al., 2001; Ogura et al., 2001). The NOD2 ligand muramyl dipeptide (MDP) is ubiquitous, indicating that broad classes of bacteria are capable of activating NOD2. However, the N-glycolyated form of muramyl dipeptide found in mycobacteria and actinomycetes more potently activates NOD2 compared to the Nacetylated form, found more frequently in gram-positive and gram-negative bacteria (Coulombe et al., 2009). Autophagy 16-like 1 (*ATG16L1*) has been strongly associated with CD and encodes a protein component of the autophagy complex (Levine & Deretic, 2007). ATG16L1 is broadly expressed, including in small intestinal Paneth cells (Cadwell et al., 2008) where it mediates exocytosis of secretory granules that contain antimicrobial peptides. IL-10 gene SNPs were found to be involved in the UC by GWAS analysis (Franke et al., 2008), and IL-10-/- mouse is one of the oldest and most widely used animal models of UC, in which spontaneous colitis develops in specific pathogen-free conditions (Kuhn et al., 1993). IL10 is expressed by many different cells of the adaptive and innate immune system including Th1, 2, and 17 cells, subsets of regulatory T cells, dendritic cells, macrophages, mast cells, and natural killer cells (Mosser et al., 2008). It has pleiotropic effects on T and B cells, and importantly limits the release of proinflammatory cytokines like TNF-a and IL-12. In the IL-10-/- mouse model, defective counter regulatory anticytokine responses result in inflammation affecting intestinal mucosa which is characterized by enlarged and branched crypts, reduced number of goblet cells, degeneration of superficial epithelial cells, and increased expression of MHC class II molecules in mouse colon. But these IL-10-/- mice require gut microbia to develop inflammation, giving rise to an attractive theory that IL10 could be involved in restricting the mucosal immune response to enteric flora (Louis et al., 2009, Sellon et al., 1998). Interestingly, this IL-10-/- model of UC have, in another hand, elegantly shown a protective role of IL10: transfer of IL10 producing regulatory T cells to immunodeficient mice prevents or cures colitis (Uhlig et al., 2006). Further, IL10 has been shown to exert a protective effect on carcinogenesis in mice (Erdman et al., 2003). The antiinflammatory response of IL-10 is mediated through IL10 receptor (IL10R) and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL10R is a heterotetrameric molecule; while IL10R1 is specific to IL10R, IL10R2 is found as a subunit of receptors to other cytokines, notably IL22, IL26, and IFNγ. Extracellular matrix gene 1 (ECM1) (Festen et al., 2010), E-cadherin gene (CDH1), Hepatocyte nuclear factor 4 alpha gene (HNF4a), and laminin B1 (Barrett et al., 2009) are another four genes implicated in mucosal barrier function, conferring risk of UC; ECM1 interacts with the basement membrane, inhibits matrix metalloproteinase 9 (MMP9), and strongly activate NFκB (Chan et al., 2007; Matsuda et al., 2003). The Wnt/beta-catenin signal transduction pathway has been shown to influence ECM1 expression (Kenny et al., 2005). E-cadherin is the first genetic correlation between colorectal cancer and UC, Chimeric mice with impaired E-cadherin function due to expression of dominant–negative N-cadherin developed colitis despite possessing an intact immune system (Hermiston & Gordon 1995a, 1995b). Notably, all of

Pathogenesis of Inflammatory Bowel Diseases 115

Decreased concentrations of bacteria that produce butyrate and other short-chain fatty acids (SCFA) may compromise epithelial barrier integrity. (B) Defective host containment of commensal bacteria. Increased mucosal permeability can result in overwhelming exposure of bacterial to TLR ligands and antigens that activate pathogenic innate and T cell immune responses. (C) Defective host immunoregulation. Inflammation might arise from lack of tolerance to antigens present in autologous microflora; cells derived from inflamed intestinal tissues of patients with IBD are activated by exposure to sonicated samples of autologous or heterologous GI microflora, whereas cells from normal individuals respond only to sonicates of heterologous microflora (Duchmann et al., 1997). Antigen-presenting cells and epithelial cells overproduce cytokines due to ineffective down regulation, which results in TH1 and TH17 differentiation and inflammation. Dysfunction of regulatory T cells (Treg) leads to decreased secretion of IL-10 and TGF-β, and loss of immunological tolerance to microbial

Fig. 2. Proposed mechanisms by which bacteria and fungi induce chronic immune-mediated inflammation and injury of the intestines. This model adapted from the model presented in

the work by Dr Sartor (Packey & Sartor 2008) (a) Pathogenic bacteria. (b) Abnormal microbial compostion. (c) Defective host containment of commensal bacteria. (d) Defective

antigens (an overly aggressive T cell response.).

host immunoregulation.

these 4 genes are regulated or related to protein kinases, for example, HNF4alpha-DNA binding activity is dependent on its phosphorylation by protein kinase A (PKA) (Viollet et al., 1997), while its transcription activity was dependent on AMP-activated protein kinase(AMPK) (Hong et al., 2003).

Similarly, by GWAS analysis, the strongest association in CD was found in interleukin-12 receptor (IL23R) (Duerr et al., 2006)—as well as the previously identified NOD2 gene. Knockout of or antibodies to IL23 prevent the development of intestinal inflammation in such models (McGovern & Powrie, 2007). It is now evident that much of the function previously ascribed to IL12 appears to relate to IL23, both of these cytokines sharing a p40 subunit in their heterodimeric structures. The IL23/IL12 pathway has become the subject of intensive study in the field of immunology as it plays a key role in determining differentiation of nave T cells into effector Th1 cells (driven by IL12) or Th17 cells (driven by IL23). Some specific bacterial components such as peptidoglycan can differentially induce antigen presenting cells to produce IL23 rather than IL12, leading to distinct patterns of inflammatory response (Begum et al., 2004). Th17 cells are particularly interesting for their role in organ-specific inflammation—raising the hope that therapeutic disruption of the IL23 pathway will control such inflammation without impairing systemic immunity.

## **3. Microbiota and immune responses**

The human GI tract contains as many as 1014 individual bacteria, comprising over 500 different species. These commensal bacteria serves as a primary barrier between the intestinal epithelial cells and the external environment, which is critical to the healthy host, as it modulates intestinal development, maintains a healthy intestinal pH, promotes immune homeostasis, and enhances metabolism of drugs, hormones and carcinogens. Evidence from immunologic, microbiologic, and genetic studies implicates abnormal host-microbial interactions in the pathogenesis of UC. But the mechanisms underlying the involvement of microbiota are elusive, and the effects of microbiota are due to their interaction with other factors, such as immunologic factors, genetic factor or epithelial junction proteins. The postulated mechanisms (Packey & Sartor, 2008) are as followed with little modification: (A) Pathogenic bacteria or abnormal microbial composition. A traditional pathogen or functional alterations in commensal bacteria, including enhanced epithelial adherence, invasion, and resistance to killing by phagocytes or acquisition of virulence factors, can result in increased stimulation of innate and adaptive immune responses. Luminal bacterial concentrations are increased in IBD, microbial diversity is diminished, particularly in patients with active disease. The involvement of pathogen mycobacterium avium subspecies paratuberculosis (MAP) in the pathogenesis of IBD is still controversial. Commensal bacteria that undergo functional alterations might contribute to the pathogenesis of IBD. Escherichia coli are commensal aerobic Gram-negative bacteria that play an important role in maintaining normal intestinal homeostasis. Modifications of luminal bacteria concentrations, including E. coli, have been observed in Crohn's disease patients (Frank et al., 2007). Reduced numbers of *Bacteroides fragilis* might also contribute to inflammation because this prominent human symbiont has protective effects: it protects mice from colitis induction by *Helicobacter hepaticus*, a murine commensal bacterium with pathogenic properties (Mazmanian et al., 2005). *Faecalibacterium prausnitzii* has antiinflammatory properties; its numbers are reduced in patients with CD and associated with risk of postresection recurrence of ileal CD (Sokol et al., 2008). There is a decreased ratio of protective commensal bacterial species compared to aggressive species in patients with IBD.

these 4 genes are regulated or related to protein kinases, for example, HNF4alpha-DNA binding activity is dependent on its phosphorylation by protein kinase A (PKA) (Viollet et al., 1997), while its transcription activity was dependent on AMP-activated protein

Similarly, by GWAS analysis, the strongest association in CD was found in interleukin-12 receptor (IL23R) (Duerr et al., 2006)—as well as the previously identified NOD2 gene. Knockout of or antibodies to IL23 prevent the development of intestinal inflammation in such models (McGovern & Powrie, 2007). It is now evident that much of the function previously ascribed to IL12 appears to relate to IL23, both of these cytokines sharing a p40 subunit in their heterodimeric structures. The IL23/IL12 pathway has become the subject of intensive study in the field of immunology as it plays a key role in determining differentiation of nave T cells into effector Th1 cells (driven by IL12) or Th17 cells (driven by IL23). Some specific bacterial components such as peptidoglycan can differentially induce antigen presenting cells to produce IL23 rather than IL12, leading to distinct patterns of inflammatory response (Begum et al., 2004). Th17 cells are particularly interesting for their role in organ-specific inflammation—raising the hope that therapeutic disruption of the IL23 pathway will control such inflammation without impairing systemic immunity.

The human GI tract contains as many as 1014 individual bacteria, comprising over 500 different species. These commensal bacteria serves as a primary barrier between the intestinal epithelial cells and the external environment, which is critical to the healthy host, as it modulates intestinal development, maintains a healthy intestinal pH, promotes immune homeostasis, and enhances metabolism of drugs, hormones and carcinogens. Evidence from immunologic, microbiologic, and genetic studies implicates abnormal host-microbial interactions in the pathogenesis of UC. But the mechanisms underlying the involvement of microbiota are elusive, and the effects of microbiota are due to their interaction with other factors, such as immunologic factors, genetic factor or epithelial junction proteins. The postulated mechanisms (Packey & Sartor, 2008) are as followed with little modification: (A) Pathogenic bacteria or abnormal microbial composition. A traditional pathogen or functional alterations in commensal bacteria, including enhanced epithelial adherence, invasion, and resistance to killing by phagocytes or acquisition of virulence factors, can result in increased stimulation of innate and adaptive immune responses. Luminal bacterial concentrations are increased in IBD, microbial diversity is diminished, particularly in patients with active disease. The involvement of pathogen mycobacterium avium subspecies paratuberculosis (MAP) in the pathogenesis of IBD is still controversial. Commensal bacteria that undergo functional alterations might contribute to the pathogenesis of IBD. Escherichia coli are commensal aerobic Gram-negative bacteria that play an important role in maintaining normal intestinal homeostasis. Modifications of luminal bacteria concentrations, including E. coli, have been observed in Crohn's disease patients (Frank et al., 2007). Reduced numbers of *Bacteroides fragilis* might also contribute to inflammation because this prominent human symbiont has protective effects: it protects mice from colitis induction by *Helicobacter hepaticus*, a murine commensal bacterium with pathogenic properties (Mazmanian et al., 2005). *Faecalibacterium prausnitzii* has antiinflammatory properties; its numbers are reduced in patients with CD and associated with risk of postresection recurrence of ileal CD (Sokol et al., 2008). There is a decreased ratio of protective commensal bacterial species compared to aggressive species in patients with IBD.

kinase(AMPK) (Hong et al., 2003).

**3. Microbiota and immune responses** 

Decreased concentrations of bacteria that produce butyrate and other short-chain fatty acids (SCFA) may compromise epithelial barrier integrity. (B) Defective host containment of commensal bacteria. Increased mucosal permeability can result in overwhelming exposure of bacterial to TLR ligands and antigens that activate pathogenic innate and T cell immune responses. (C) Defective host immunoregulation. Inflammation might arise from lack of tolerance to antigens present in autologous microflora; cells derived from inflamed intestinal tissues of patients with IBD are activated by exposure to sonicated samples of autologous or heterologous GI microflora, whereas cells from normal individuals respond only to sonicates of heterologous microflora (Duchmann et al., 1997). Antigen-presenting cells and epithelial cells overproduce cytokines due to ineffective down regulation, which results in TH1 and TH17 differentiation and inflammation. Dysfunction of regulatory T cells (Treg) leads to decreased secretion of IL-10 and TGF-β, and loss of immunological tolerance to microbial antigens (an overly aggressive T cell response.).

Fig. 2. Proposed mechanisms by which bacteria and fungi induce chronic immune-mediated inflammation and injury of the intestines. This model adapted from the model presented in the work by Dr Sartor (Packey & Sartor 2008) (a) Pathogenic bacteria. (b) Abnormal microbial compostion. (c) Defective host containment of commensal bacteria. (d) Defective host immunoregulation.

Pathogenesis of Inflammatory Bowel Diseases 117

gastrointestinal mucosa (Targan et al., 1995). pANCA-primed B cells have been demonstrated in the mesenteric nodes and were not found in detectable amounts in the periphery (Targan et al., 1995). These findings represent further confirmation that the pANCAreactive antigen(s) also originates in the mucosa. Despite the fact that pANCA can be found in the peripheral circulation, the mucosal origin is the evidence that the antigen(s) to which pANCA reacts is mucosa specific and thus is more closely related to mucosal immune responses and mucosal inflammation. This finding corroborates that disease results from a defect in the hosts reaction to bacteria. The antigens to pANCA have been localized

to the nucleus of neutrophils by the use of electron microscopy (Billing et al., 1995).

MLCK SPAK

Fig. 3. Binding of microbial adjuvants to extracellular and intracellular patter-recognition receptros and initiate their function by activating preotein kinases. Toll-like receptors on the cell membrane selectively bind to various bacterial, viral or fungal components. This ligation activates conserved signaling pathways that activate NF?B and mitogen-activated protein

proinflammatory and antiinflammatory genes. This model adapted and modified from the

The intestinal mucosa must rapidly recognize detrimental pathogenic threats to the lumen to initiate controlled immune responses but maintain hyporesponsiveness to omnipresent harmless commensals. Pattern recognition receptors (PRRs) may play an essential role in allowing innate immune cells to discriminate between ''self'' and microbial ''non-self'' based on the recognition of broadly conserved molecular patterns. Toll-like receptors (TLRs), a class of transmembrane PRRs, play a key role in microbial recognition, induction of antimicrobial genes, and the control of adaptive immune responses. Polymorphisms in TLRs have been linked to Crohn's disease (Franchimont et al., 2004; Torok et al., 2004), and immunofluorescence studies reveal that epithelial TLR expression is markedly upregulated in IBD (Cario et al., 2000). TLR4, for example, is induced by proinflammatory cytokines and is highly expressedin IECs, resident macrophages and dendritic cells in active IBD (Cario, 2000; Hausmann et al., 2002). The functional variant Asp299Gly of TLR4 is associated with IBD and increased susceptibility to Gram-negative infections (Franchimont et al., 2004). Disrupted

kinases. These transcription factors stimulate the expression of a number of

http://www.nature.com/nrgastro/v3/n7/full/ncpgasther0528.html

model presented previously.

The bowel is the largest immunological organ of the body, with continuous interaction between the mucosal immune system and the intestinal flora. IBD is commonly regarded as the consequences of an enhanced inflammatory response or the lack of a down regulatory response to bacteria abnormality (Sartor et al., 2008; Xavier et al., 2007). The dysregulated immune response involving the innate (for example, TLR, DC, etc) and the adaptive immune system (e.g. effector T-cells, regulatory T-cells, eosinophils, neutrophils, etc) may follow or precede the macroscopic lesions. Th-1 and Th17 immune responses play a role in the pathogenesis of Crohn's disease (Sartor, 2008; Strober et al., 2007). The Th1 cytokine profile, which includes IFN-γ and IL-12 p40, is dominant in patients with Crohn's disease. Traditional Th1 responses are mediated by IFN-γ, the production of which is stimulated by IL-12, produced by antigen-presenting cells (APCs). Most experimental colitis models also have a dominant Th1 response, although in several models Th1 responses can change into Th2 (type 2 T-helper lymphocyte) responses as the inflammatory process matures (Spencer et al., 2002; Bamias et al., 2005). How we think about Th1 responses has been influenced by the discovery of an additional Th17 pathway. IL-17 mediates Th17 responses (Kolls & Linden, 2004). The production of this cytokine is stimulated by the production of IL-6, TGFβ and IL-23 by innate immune cells and APCs, especially dendritic cells. Bacterial colonization stimulates IL-23 expression by ileal dendritic cells (Becker et al., 2003). The levels of both IL-23 and IL-17 are increased in Crohn's disease tissues and most forms of experimental colitis (Fujino et al., 2003; Schmidt et al., 2005; Yen et al., 2006). Of pathogenic importance, the IL-12–IFN-γ and IL-23–IL-17 pathways seem to be mutually exclusive, since IFN-γ suppresses IL-17, and vice versa (Kolls et al., 2004). The immunopathogenesis of UC has been a more difficult disease to ascertain, neither IFN-γ (a major Th1 cytokine) nor IL-4 (the major Th2 cytokines) was increased (Fuss et al., 2008). In fact, IL-4 production was found to be decreased in cells extracted from UC tissue and only the fact that an additional Th2 cytokine IL-5 secretion by these cells was somewhat increased hinted that the disease may have a Th2 character. A further, enhanced level of IL-13 was noticed in lamina propria from UC specimens, whereas those from Crohn's disease specimens were producing IFN-γ (Fuss et al., 1996). Fuss (Fuss et al., 2004) found that antigen-presenting cells bearing a CD1d construct (and thus expressing CD1d on its surface, which presents lipid rather than protein antigens to T cells.) could only induce lamina propria mononuclear cells from UC patients but not that of Crohn ' s disease to produce IL-13. Thereby, the cytokine secretion profile seen in UC was produced from a non-classical CD1 dependent NK T cell whereas the cytokines produced in Crohn's disease were from that of an activated classical Th1 CD4 + T cell. In addition, Lamina propria cells enriched for NK T cells from the patients could be shown to be cytotoxic for epithelial cells and such cytotoxicity was further enhanced by IL-13. Antigens in the mucosal microflora activate NK T cells because of barrier dysfunction that, in turn, cause cytolysis of epithelial cells and the characteristic ulcerations associated with the disease. As suggested, enhancement of cytolytic activity was observed *in vitro* in the presence of IL-13. Further, IL-13 was shown to have direct effects on activation of cytokine, transcription. These studies demonstrated that TGF-β transcription was dependent upon IL-13. In short, UC is associated with an atypical Th-2 response mediated by a distinct subset of NK T cells that produce IL- 13 and are cytotoxic for epithelial cells (Fuss et al., 2008). Further, UC is characterized by the presence of various types of autoantibodies which confirm a key role of defect of host/bacterial interface to this disease. Approximately 70% of patients were diagnosed in the traditional manner with ulcerative colitis express pANCA (Saxon et al., 1990). The site of production of pANCA has been localized to the

The bowel is the largest immunological organ of the body, with continuous interaction between the mucosal immune system and the intestinal flora. IBD is commonly regarded as the consequences of an enhanced inflammatory response or the lack of a down regulatory response to bacteria abnormality (Sartor et al., 2008; Xavier et al., 2007). The dysregulated immune response involving the innate (for example, TLR, DC, etc) and the adaptive immune system (e.g. effector T-cells, regulatory T-cells, eosinophils, neutrophils, etc) may follow or precede the macroscopic lesions. Th-1 and Th17 immune responses play a role in the pathogenesis of Crohn's disease (Sartor, 2008; Strober et al., 2007). The Th1 cytokine profile, which includes IFN-γ and IL-12 p40, is dominant in patients with Crohn's disease. Traditional Th1 responses are mediated by IFN-γ, the production of which is stimulated by IL-12, produced by antigen-presenting cells (APCs). Most experimental colitis models also have a dominant Th1 response, although in several models Th1 responses can change into Th2 (type 2 T-helper lymphocyte) responses as the inflammatory process matures (Spencer et al., 2002; Bamias et al., 2005). How we think about Th1 responses has been influenced by the discovery of an additional Th17 pathway. IL-17 mediates Th17 responses (Kolls & Linden, 2004). The production of this cytokine is stimulated by the production of IL-6, TGFβ and IL-23 by innate immune cells and APCs, especially dendritic cells. Bacterial colonization stimulates IL-23 expression by ileal dendritic cells (Becker et al., 2003). The levels of both IL-23 and IL-17 are increased in Crohn's disease tissues and most forms of experimental colitis (Fujino et al., 2003; Schmidt et al., 2005; Yen et al., 2006). Of pathogenic importance, the IL-12–IFN-γ and IL-23–IL-17 pathways seem to be mutually exclusive, since IFN-γ suppresses IL-17, and vice versa (Kolls et al., 2004). The immunopathogenesis of UC has been a more difficult disease to ascertain, neither IFN-γ (a major Th1 cytokine) nor IL-4 (the major Th2 cytokines) was increased (Fuss et al., 2008). In fact, IL-4 production was found to be decreased in cells extracted from UC tissue and only the fact that an additional Th2 cytokine IL-5 secretion by these cells was somewhat increased hinted that the disease may have a Th2 character. A further, enhanced level of IL-13 was noticed in lamina propria from UC specimens, whereas those from Crohn's disease specimens were producing IFN-γ (Fuss et al., 1996). Fuss (Fuss et al., 2004) found that antigen-presenting cells bearing a CD1d construct (and thus expressing CD1d on its surface, which presents lipid rather than protein antigens to T cells.) could only induce lamina propria mononuclear cells from UC patients but not that of Crohn ' s disease to produce IL-13. Thereby, the cytokine secretion profile seen in UC was produced from a non-classical CD1 dependent NK T cell whereas the cytokines produced in Crohn's disease were from that of an activated classical Th1 CD4 + T cell. In addition, Lamina propria cells enriched for NK T cells from the patients could be shown to be cytotoxic for epithelial cells and such cytotoxicity was further enhanced by IL-13. Antigens in the mucosal microflora activate NK T cells because of barrier dysfunction that, in turn, cause cytolysis of epithelial cells and the characteristic ulcerations associated with the disease. As suggested, enhancement of cytolytic activity was observed *in vitro* in the presence of IL-13. Further, IL-13 was shown to have direct effects on activation of cytokine, transcription. These studies demonstrated that TGF-β transcription was dependent upon IL-13. In short, UC is associated with an atypical Th-2 response mediated by a distinct subset of NK T cells that produce IL- 13 and are cytotoxic for epithelial cells (Fuss et al., 2008). Further, UC is characterized by the presence of various types of autoantibodies which confirm a key role of defect of host/bacterial interface to this disease. Approximately 70% of patients were diagnosed in the traditional manner with ulcerative colitis express pANCA (Saxon et al., 1990). The site of production of pANCA has been localized to the gastrointestinal mucosa (Targan et al., 1995). pANCA-primed B cells have been demonstrated in the mesenteric nodes and were not found in detectable amounts in the periphery (Targan et al., 1995). These findings represent further confirmation that the pANCAreactive antigen(s) also originates in the mucosa. Despite the fact that pANCA can be found in the peripheral circulation, the mucosal origin is the evidence that the antigen(s) to which pANCA reacts is mucosa specific and thus is more closely related to mucosal immune responses and mucosal inflammation. This finding corroborates that disease results from a defect in the hosts reaction to bacteria. The antigens to pANCA have been localized to the nucleus of neutrophils by the use of electron microscopy (Billing et al., 1995).

Fig. 3. Binding of microbial adjuvants to extracellular and intracellular patter-recognition receptros and initiate their function by activating preotein kinases. Toll-like receptors on the cell membrane selectively bind to various bacterial, viral or fungal components. This ligation activates conserved signaling pathways that activate NF?B and mitogen-activated protein kinases. These transcription factors stimulate the expression of a number of proinflammatory and antiinflammatory genes. This model adapted and modified from the model presented previously.

http://www.nature.com/nrgastro/v3/n7/full/ncpgasther0528.html

The intestinal mucosa must rapidly recognize detrimental pathogenic threats to the lumen to initiate controlled immune responses but maintain hyporesponsiveness to omnipresent harmless commensals. Pattern recognition receptors (PRRs) may play an essential role in allowing innate immune cells to discriminate between ''self'' and microbial ''non-self'' based on the recognition of broadly conserved molecular patterns. Toll-like receptors (TLRs), a class of transmembrane PRRs, play a key role in microbial recognition, induction of antimicrobial genes, and the control of adaptive immune responses. Polymorphisms in TLRs have been linked to Crohn's disease (Franchimont et al., 2004; Torok et al., 2004), and immunofluorescence studies reveal that epithelial TLR expression is markedly upregulated in IBD (Cario et al., 2000). TLR4, for example, is induced by proinflammatory cytokines and is highly expressedin IECs, resident macrophages and dendritic cells in active IBD (Cario, 2000; Hausmann et al., 2002). The functional variant Asp299Gly of TLR4 is associated with IBD and increased susceptibility to Gram-negative infections (Franchimont et al., 2004). Disrupted

Pathogenesis of Inflammatory Bowel Diseases 119

proinflammatory signalling through NFκB in response to distinct bacterial ligands. NOD2 is constitutively or inducibly expressed in all kinds of cells throughout the gasterintestinal tract. MDP has been identified as (so far) the sole ligand of NOD2 (Inohara et al., 2003; Girardin et al., 2003). NOD2 has been found to exert antibacterial activity in intestinal epithelial cells limiting survival of enteric bacteria after invasion. Bacterial clearance of Salmonella typhimurium is strongly accelerated in IEC expressing a functional NOD2 protein, whereas L1007fsinsC mutant expressing IEC are virtually unable to clear the pathogen *in vitro* (Hisamatsu et al., 2003). NOD2 (Chin et al., 2002) knockout mice, exhibit a profoundly decreased ability to clear intracellular Listeria monocytogenes, inducing persistent immune activation by combined loss of antibacterial activity, dysregulation of cytokine production, and imbalance of T cell activation. Emerging studies have started to reveal the molecular mechanisms by which NOD2 influences innate immune responses in the intestinal mucosa. It seems that different NOD2 mutations may span a spectrum of diverse phenotypes, ranging from complete ''loss of function'' to maximal ''gain of function''. NOD2 mutations within NBD lead to constitutive ligand independent NFκB activation, causing a chronic systemic inflammatory disorder known as ''Blau syndrome''(Inohara et al., 2003a). Conversely, it has been suggested that CD associated NOD2 mutants which are predominantly found in the microbial ligand dependent LRR domain rather reflect ''loss of function'' phenotypes. Several *in vitro* transfection studies showed that human CD associated NOD2 mutants significantly abolish NFκB activation in response to MDP (Inohara et al., 2003b; Girardin et al., 2003; Chamaillard, 2003). However, paradoxically, macrophages within the intestinal lamina propria of CD patients overproduce NFκB targets, including exaggerated production of proinflammatory cytokines, such as TNF-a and IL-1β (Podolsky, 2002). Accordingly, a recent *in vivo* study now demonstrates that MDP stimulated macrophages isolated from mice generated with a murine NOD22932iC variant, homologous to the human NOD23020insC (=L1007fsinsC) variant, exhibit enhanced NFκB activation, increased apoptosis, and elevated IL-1β secretion (Maeda et al., 2005), possibly implying an important mechanism of how dysfunctional NOD2 may trigger intestinal inflammation in some types of CD. Thus this murine NOD2 frame-shift mutation in the LRR region may imbalance functions of both terminal parts of the whole protein: bacterial dysrecognition through the impaired LRR domain, ligand independent NFκB activation, as well as uncontrolled apoptosis and subsequent induction of IL-1β processing and release through the hyperactive CARD domains. The NOD2 gene product is most abundant in ileal Paneth cells (Lala et al., 2003; Ogura et al., 2003) which express a diverse population of microbicidal defensins restricting colonization or invasion of small intestinal epithelium by bacteria (Ouellette et al., 1994).

Stimulation with MDP elicits cryptidin secretion from Paneth cells (Ayabe et al., 2000).

epithelium, similar to TLR4-deficient mice treated with DSS (Fukata et al., 2006).

**4. Barrier dysfunction** 

In addition, NF-κB is normally grouped into one of the pro-inflammatory mediators, a protective role for epithelial NF-κB signaling by either bacteria, IL-1, or TNF stimulation of TLRs, or cytokine receptors is demonstrated by conditional ablation of NEMO (IκB kinase) in intestinal epithelial cells causing spontaneous severe colitis (Nenci et al. 2007). Blockade of epithelial NF-κB signaling led to increased bacterial translocation across the injured

Generally, intestinal barrier function consists of different level of defense lines, the mucus layer, commensal microbiota, epithelial cells themselves, the junction between lateral

TLR4 signalling could engender an inappropriate innate and adaptive immune response necessary to eradicate pathogens, which would result in severe inflammation. Polymorphisms of TLRs 1, 2 and 6 are associated with more extensive disease localization in IBD (Pierik et al., 2006). UC patients have an association between a TLR7 variant and the prevalence of pANCA antibodies, which crossreact with enteric bacterial antigens (Vermeire et al., 2004; Seibold et al., 1998). Blockade of bacterial signalling through NFκB in IECs potentiates chemically induced colitis in TLR4 and TLR9-deficient mice (Fukata et al., 2006; Lee et al., 2006). Individual TLRs differentially activate distinct signaling events via diverse cofactors and adaptors. To date, at least five different adaptor proteins have been identified in humans: MyD88, Mal/TIRAP, TRIF/TICAM-1, TRAM/Tirp/TICAM-2, and SARM (O'Neill et al., 2003). The first identified so-called ''classical'' pathway (Cario, 2005) involves recruitment of the adaptor molecule MyD88, activation of the serine/threonine kinases of the interleukin 1 receptor associated kinase (IRAK) family, subsequently leading to degradation of inhibitor kB (IkB) and translocation of nuclear factor kB (NFkB) to the nucleus, then result in activation of specific transcription factors, including NFkB, AP-1, Elk-1, CREB, STATs, and the subsequent transcriptional activation of genes encoding pro- and anti-inflammatory cytokines and chemokines as well as induction of costimulatory molecules. All of these various downstream effects are critically involved in the control of pathogen elimination, commensal homeostasis, and linkage to the adaptive immunity. Signaling through different TLRs can result in considerable qualitative differences in TH dependent immune responses by differential modulation of MAPKs and the transcription factor c-FOS (Agrawal et al., 2003). So TLR signalling protects intestinal epithelial barrier and maintains tolerance, but aberrant TLR signalling may stimulate diverse inflammatory responses leading to UC. TLR comprise a family of (so far) 11 type-I transmembrane receptors. Different pathogen associated molecular patterns selectively activate different TLRs: (Lipoptroteins) TLR1, 2 and 6; (dsRNA) TLR3; (LPS) TLR4; (Flagellin) TLR5; (ssRNA) TLR7 and 8; (CpG DNA) TLR9. These signals all converge on a single pathway via myeloid differentiation primary response protein MyD88, which activates NFκB. the NFκB pathway was thought to have predominantly pro inflammatory activities and NFκB is activated in the tissues of UC patients and its inhibition can attenuate experimental colitis (Neurath et al., 1996). In intestine, tolerance is an essential mucosal defence mechanism maintaining hyporesponsiveness to harmless lumenal commensals and their products. Several molecular immune mechanisms that ensure tolerance via TLRs in intestinal epithelial cells (IEC) have recently been described, for example, low expression of TLRs at resting conditions in IEC can maintain hyporesponsiveness to microbiota; high expression levels of the downstream signaling suppressor Tollip which inhibits IRAK activation (Otte et al., 2004), ligand induced activation of peroxisome proliferator activated receptor c (PPARc) which uncouples NFkB dependent target genes in a negative feedback loop (Dubuquoy et al., 2003. Kelly et al., 2004), and external regulators which may suppress TLR mediated signalling pathways. Commensal bacteria may assist the host in maintaining mucosal homeostasis by suppressing inflammatory responses and inhibiting specific intracellular signal transduction pathways (Neish et al., 2000), uncoupling NFkB dependent target genes in a negative feedback loop (Dubuquoy et al., 2003) which may lead to attenuation of colonic inflammation (Kelly et al., 2004).

NODs comprise at present more than 20 different members with C terminal ligand recognition (LRR) domain, central nucleotide binding domain (NBD), and N terminal caspase recruitment domains (CARDs). Recent research has mostly focused on two cytosolic receptors of this family, NOD1 and NOD2, which both play a major role in intestinal regulation of

TLR4 signalling could engender an inappropriate innate and adaptive immune response necessary to eradicate pathogens, which would result in severe inflammation. Polymorphisms of TLRs 1, 2 and 6 are associated with more extensive disease localization in IBD (Pierik et al., 2006). UC patients have an association between a TLR7 variant and the prevalence of pANCA antibodies, which crossreact with enteric bacterial antigens (Vermeire et al., 2004; Seibold et al., 1998). Blockade of bacterial signalling through NFκB in IECs potentiates chemically induced colitis in TLR4 and TLR9-deficient mice (Fukata et al., 2006; Lee et al., 2006). Individual TLRs differentially activate distinct signaling events via diverse cofactors and adaptors. To date, at least five different adaptor proteins have been identified in humans: MyD88, Mal/TIRAP, TRIF/TICAM-1, TRAM/Tirp/TICAM-2, and SARM (O'Neill et al., 2003). The first identified so-called ''classical'' pathway (Cario, 2005) involves recruitment of the adaptor molecule MyD88, activation of the serine/threonine kinases of the interleukin 1 receptor associated kinase (IRAK) family, subsequently leading to degradation of inhibitor kB (IkB) and translocation of nuclear factor kB (NFkB) to the nucleus, then result in activation of specific transcription factors, including NFkB, AP-1, Elk-1, CREB, STATs, and the subsequent transcriptional activation of genes encoding pro- and anti-inflammatory cytokines and chemokines as well as induction of costimulatory molecules. All of these various downstream effects are critically involved in the control of pathogen elimination, commensal homeostasis, and linkage to the adaptive immunity. Signaling through different TLRs can result in considerable qualitative differences in TH dependent immune responses by differential modulation of MAPKs and the transcription factor c-FOS (Agrawal et al., 2003). So TLR signalling protects intestinal epithelial barrier and maintains tolerance, but aberrant TLR signalling may stimulate diverse inflammatory responses leading to UC. TLR comprise a family of (so far) 11 type-I transmembrane receptors. Different pathogen associated molecular patterns selectively activate different TLRs: (Lipoptroteins) TLR1, 2 and 6; (dsRNA) TLR3; (LPS) TLR4; (Flagellin) TLR5; (ssRNA) TLR7 and 8; (CpG DNA) TLR9. These signals all converge on a single pathway via myeloid differentiation primary response protein MyD88, which activates NFκB. the NFκB pathway was thought to have predominantly pro inflammatory activities and NFκB is activated in the tissues of UC patients and its inhibition can attenuate experimental colitis (Neurath et al., 1996). In intestine, tolerance is an essential mucosal defence mechanism maintaining hyporesponsiveness to harmless lumenal commensals and their products. Several molecular immune mechanisms that ensure tolerance via TLRs in intestinal epithelial cells (IEC) have recently been described, for example, low expression of TLRs at resting conditions in IEC can maintain hyporesponsiveness to microbiota; high expression levels of the downstream signaling suppressor Tollip which inhibits IRAK activation (Otte et al., 2004), ligand induced activation of peroxisome proliferator activated receptor c (PPARc) which uncouples NFkB dependent target genes in a negative feedback loop (Dubuquoy et al., 2003. Kelly et al., 2004), and external regulators which may suppress TLR mediated signalling pathways. Commensal bacteria may assist the host in maintaining mucosal homeostasis by suppressing inflammatory responses and inhibiting specific intracellular signal transduction pathways (Neish et al., 2000), uncoupling NFkB dependent target genes in a negative feedback loop (Dubuquoy et al., 2003) which may

lead to attenuation of colonic inflammation (Kelly et al., 2004).

NODs comprise at present more than 20 different members with C terminal ligand recognition (LRR) domain, central nucleotide binding domain (NBD), and N terminal caspase recruitment domains (CARDs). Recent research has mostly focused on two cytosolic receptors of this family, NOD1 and NOD2, which both play a major role in intestinal regulation of proinflammatory signalling through NFκB in response to distinct bacterial ligands. NOD2 is constitutively or inducibly expressed in all kinds of cells throughout the gasterintestinal tract. MDP has been identified as (so far) the sole ligand of NOD2 (Inohara et al., 2003; Girardin et al., 2003). NOD2 has been found to exert antibacterial activity in intestinal epithelial cells limiting survival of enteric bacteria after invasion. Bacterial clearance of Salmonella typhimurium is strongly accelerated in IEC expressing a functional NOD2 protein, whereas L1007fsinsC mutant expressing IEC are virtually unable to clear the pathogen *in vitro* (Hisamatsu et al., 2003). NOD2 (Chin et al., 2002) knockout mice, exhibit a profoundly decreased ability to clear intracellular Listeria monocytogenes, inducing persistent immune activation by combined loss of antibacterial activity, dysregulation of cytokine production, and imbalance of T cell activation. Emerging studies have started to reveal the molecular mechanisms by which NOD2 influences innate immune responses in the intestinal mucosa. It seems that different NOD2 mutations may span a spectrum of diverse phenotypes, ranging from complete ''loss of function'' to maximal ''gain of function''. NOD2 mutations within NBD lead to constitutive ligand independent NFκB activation, causing a chronic systemic inflammatory disorder known as ''Blau syndrome''(Inohara et al., 2003a). Conversely, it has been suggested that CD associated NOD2 mutants which are predominantly found in the microbial ligand dependent LRR domain rather reflect ''loss of function'' phenotypes. Several *in vitro* transfection studies showed that human CD associated NOD2 mutants significantly abolish NFκB activation in response to MDP (Inohara et al., 2003b; Girardin et al., 2003; Chamaillard, 2003). However, paradoxically, macrophages within the intestinal lamina propria of CD patients overproduce NFκB targets, including exaggerated production of proinflammatory cytokines, such as TNF-a and IL-1β (Podolsky, 2002). Accordingly, a recent *in vivo* study now demonstrates that MDP stimulated macrophages isolated from mice generated with a murine NOD22932iC variant, homologous to the human NOD23020insC (=L1007fsinsC) variant, exhibit enhanced NFκB activation, increased apoptosis, and elevated IL-1β secretion (Maeda et al., 2005), possibly implying an important mechanism of how dysfunctional NOD2 may trigger intestinal inflammation in some types of CD. Thus this murine NOD2 frame-shift mutation in the LRR region may imbalance functions of both terminal parts of the whole protein: bacterial dysrecognition through the impaired LRR domain, ligand independent NFκB activation, as well as uncontrolled apoptosis and subsequent induction of IL-1β processing and release through the hyperactive CARD domains. The NOD2 gene product is most abundant in ileal Paneth cells (Lala et al., 2003; Ogura et al., 2003) which express a diverse population of microbicidal defensins restricting colonization or invasion of small intestinal epithelium by bacteria (Ouellette et al., 1994). Stimulation with MDP elicits cryptidin secretion from Paneth cells (Ayabe et al., 2000).

In addition, NF-κB is normally grouped into one of the pro-inflammatory mediators, a protective role for epithelial NF-κB signaling by either bacteria, IL-1, or TNF stimulation of TLRs, or cytokine receptors is demonstrated by conditional ablation of NEMO (IκB kinase) in intestinal epithelial cells causing spontaneous severe colitis (Nenci et al. 2007). Blockade of epithelial NF-κB signaling led to increased bacterial translocation across the injured epithelium, similar to TLR4-deficient mice treated with DSS (Fukata et al., 2006).

#### **4. Barrier dysfunction**

Generally, intestinal barrier function consists of different level of defense lines, the mucus layer, commensal microbiota, epithelial cells themselves, the junction between lateral

Pathogenesis of Inflammatory Bowel Diseases 121

altered, as in CD and UC, inflammatory responses are initiated against the commensal bacteria. An important component, often neglected due to lack of understanding, is the mucus layer that overlies the entire intestinal epithelium as a protective gel-like layer (Johansson et al., 2008). This thick and hyperviscous mucus layer secreted by goblet cells overlies the entire intestinal epithelium as a protective gel-like layer that can extend up to as much as 150 µm thick in mouse colon (and 800 µm thick in rat colon). There exist two different kinds of mucus layer-out layer and inner layer. The majority of microorganisms in the lumen can be found in the outer mucus layer, there is an inner, protected, and unstirred layer that is directly adjacent to the epithelial surface and is relatively sterile. The sterility of this layer contributes to the retention of a high concentration of antimicrobial proteins (such as cathelicidiens, defensins, and cryptidens) produced by various intestinal epithelial lineages, including enterocytes and Paneth cells. The inner firmly attached mucus layer forms a specialized physical barrier that excludes the resident bacteria from a direct contact with the underlining epithelium. This organization of the colon mucus, as based on the properties of the Muc2 mucin, should be ideal for excluding bacteria from contacting the epithelial cells and thus also the immune system. Alterations or the absence of these protective layers, as in the Muc2-/- mouse colon, allow bacteria to have a direct contact with epithelial cells, to penetrate lower into the crypts and also translocate into epithelial cells. That such a close contact between bacteria and epithelia can trigger an inflammatory response (Johansson et al., 2008; Shen et al., 2009). The surface mucus layer also impacts mucosal permeability, as demonstrated by spontaneous colitis in Muc-2- deficient mice (Bergstrom et al., 2010), and increased dextran sulphate sodium-induced colitis in intestinal trefoil factordeficient mice (Mashimo et al., 1996) and in human UC, particularly in mucus composition and concentration in phospholipids (Braun et al., 2009). Aberrant mucin assembly causes endoplasmic reticulum stress and spontaneous inflammation that resembles UC in mice (Kaser et al., 2008, Heazlewood et al., 2008); defects in the mucus layer could also influence the pattern of microbial colonization and the maintenance of microbial community structure and function. The importance of the Muc2 mucin in organizing the colon mucus protection is further strengthen by the report that two mouse strains with diarrhea and colon inflammation were shown to have two separate spontaneous mutations in the Muc2 mucin (Heazlewood et al. 2008). Importantly, the production of mucin is regulated by protein kinases, for example, resistin and resistin-like molecule (RELM) beta upregulated mucin expression which dependent on the kinase activities of protein kinase C (PKC), tyrosine kinases, and extracellular-regulated protein kinase (Krimi et al., 2008); Cathelicidin stimulates colonic mucus synthesis by up-regulating MUC1 and MUC2

expression through a mitogen-activated protein kinase pathway (Tai et al., 2008).

The intestinal defect was first reported in studies showing that the intestinal mucosa of patients with CD had a decreased ability to exclude large molecules (Hollander et al., 1986). The cellular components of the intestinal barrier consist of the complete array of columnar epithelial cell types (enterocyte, paneth cells, enteroendorine cells, and goblet cells) present within the intestine. These cells are polarized with an apical membrane and a basolateral membrane, and apical membrane composition is distinct from the basolateral membrane, for example, the nutrient transporters are located on the apical membrane; they use Na+ ions cotransport to provide the energy and directionality of transport. In contrast, the Na+K+- ATPase, which establishes the Na+ electrochemical gradient, is present on basolateral, but

**4.2 Epithelial cell and its tight junction** 

epithelial cells, innate and adaptive immune systems and enteric nerve system. Any stresses which interfere with any level of this defense lines could potentially lead to intestinal barrier dysfunction and result in intestinal inflammation.

Fig. 4. Merged figure (A) of Muc2immunostaining (green, B) *and FISH analysis using the general bacterial probe EUB338-Alexa Fluor* 555 (red, C) of distal colon, it was shown muc2 postive goblet cells and the outer mucus layer (Arrow) and inner mucus layer (Star) on the epithelium. The inner layer (Star) is devoid of bacteria, which can only be detected in the outer mucus layer. The inner mucus generates a spatial separation between the cells and the microflora. (Scale bar: 20μm.). (D) FISH using the EUB338-Alexa Fluor 555 probe staining bacteria and DAPI DNA staining in colon show a clear separation of the bacterial DNA and epithelial surface in WT mice, but not in Muc2 -/- mice. This separation corresponds to the inner mucus layer (s). (Scale bar: 100μm.). These models adapted from the models presented previously (Johansson 2008).

Epithelial cells form a continuous, polarized monolayer that is linked together by a series of dynamic junctional complexes. Except function as a physical barrier, epithelial cells maintain a mucosal defense system through the expression of a wide range of PRRs, such as TLRs and NODs. These PRRs form the backbone of the innate immune system through the rapid response and recognition of the unique and conserved microbial components, (Medzhitov & Janeway. 2002; Akira et al., 2006). Tight junctions are composed of transmembrane proteins (claudins, occludins, and junctional adhesion molecule [JAM]), peripheral membrane or scaffolding proteins (zonula occludens [ZO]), and intracellular regulatory molecules that include kinases and actin. An anatomically and immunologically compromised intestinal epithelial barrier allows direct contact of the intestinal mucosa with the luminal bacteria and plays a crucial role in the development and maintenance of IBD by initiating chronic inflammatory responses, although it is unclear whether this is a primary pathogenic process or secondary to inflammation. Since the contribution of genetic factors, microbiota and immune responses to the pathogenesis to IBD, we high light the involvement of mucus layer, tight junction itself in the pathogenesis of IBD.

#### **4.1 Mucus layer**

As mentioned in previous part of this chapter, the digestive tract is home to 1014 bacteria and bacteria genome is as many 10 times as human genome, which has evolved to ensure homeostasis. How to manage this enormous bacterial load without overt immune responses from the adaptive and innate systems is not well understood. When the equilibrium is

epithelial cells, innate and adaptive immune systems and enteric nerve system. Any stresses which interfere with any level of this defense lines could potentially lead to intestinal barrier

WT Muc2 -/-

AB C D E

Epithelial cells form a continuous, polarized monolayer that is linked together by a series of dynamic junctional complexes. Except function as a physical barrier, epithelial cells maintain a mucosal defense system through the expression of a wide range of PRRs, such as TLRs and NODs. These PRRs form the backbone of the innate immune system through the rapid response and recognition of the unique and conserved microbial components, (Medzhitov & Janeway. 2002; Akira et al., 2006). Tight junctions are composed of transmembrane proteins (claudins, occludins, and junctional adhesion molecule [JAM]), peripheral membrane or scaffolding proteins (zonula occludens [ZO]), and intracellular regulatory molecules that include kinases and actin. An anatomically and immunologically compromised intestinal epithelial barrier allows direct contact of the intestinal mucosa with the luminal bacteria and plays a crucial role in the development and maintenance of IBD by initiating chronic inflammatory responses, although it is unclear whether this is a primary pathogenic process or secondary to inflammation. Since the contribution of genetic factors, microbiota and immune responses to the pathogenesis to IBD, we high light the

As mentioned in previous part of this chapter, the digestive tract is home to 1014 bacteria and bacteria genome is as many 10 times as human genome, which has evolved to ensure homeostasis. How to manage this enormous bacterial load without overt immune responses from the adaptive and innate systems is not well understood. When the equilibrium is

involvement of mucus layer, tight junction itself in the pathogenesis of IBD.

Fig. 4. Merged figure (A) of Muc2immunostaining (green, B) *and FISH analysis using the general bacterial probe EUB338-Alexa Fluor* 555 (red, C) of distal colon, it was shown muc2 postive goblet cells and the outer mucus layer (Arrow) and inner mucus layer (Star) on the epithelium. The inner layer (Star) is devoid of bacteria, which can only be detected in the outer mucus layer. The inner mucus generates a spatial separation between the cells and the microflora. (Scale bar: 20μm.). (D) FISH using the EUB338-Alexa Fluor 555 probe staining bacteria and DAPI DNA staining in colon show a clear separation of the bacterial DNA and epithelial surface in WT mice, but not in Muc2 -/- mice. This separation corresponds to the inner mucus layer (s). (Scale bar: 100μm.). These models adapted from the models presented

dysfunction and result in intestinal inflammation.

previously (Johansson 2008).

**4.1 Mucus layer** 

Merge Muc2 staining Bacterial fish

altered, as in CD and UC, inflammatory responses are initiated against the commensal bacteria. An important component, often neglected due to lack of understanding, is the mucus layer that overlies the entire intestinal epithelium as a protective gel-like layer (Johansson et al., 2008). This thick and hyperviscous mucus layer secreted by goblet cells overlies the entire intestinal epithelium as a protective gel-like layer that can extend up to as much as 150 µm thick in mouse colon (and 800 µm thick in rat colon). There exist two different kinds of mucus layer-out layer and inner layer. The majority of microorganisms in the lumen can be found in the outer mucus layer, there is an inner, protected, and unstirred layer that is directly adjacent to the epithelial surface and is relatively sterile. The sterility of this layer contributes to the retention of a high concentration of antimicrobial proteins (such as cathelicidiens, defensins, and cryptidens) produced by various intestinal epithelial lineages, including enterocytes and Paneth cells. The inner firmly attached mucus layer forms a specialized physical barrier that excludes the resident bacteria from a direct contact with the underlining epithelium. This organization of the colon mucus, as based on the properties of the Muc2 mucin, should be ideal for excluding bacteria from contacting the epithelial cells and thus also the immune system. Alterations or the absence of these protective layers, as in the Muc2-/- mouse colon, allow bacteria to have a direct contact with epithelial cells, to penetrate lower into the crypts and also translocate into epithelial cells. That such a close contact between bacteria and epithelia can trigger an inflammatory response (Johansson et al., 2008; Shen et al., 2009). The surface mucus layer also impacts mucosal permeability, as demonstrated by spontaneous colitis in Muc-2- deficient mice (Bergstrom et al., 2010), and increased dextran sulphate sodium-induced colitis in intestinal trefoil factordeficient mice (Mashimo et al., 1996) and in human UC, particularly in mucus composition and concentration in phospholipids (Braun et al., 2009). Aberrant mucin assembly causes endoplasmic reticulum stress and spontaneous inflammation that resembles UC in mice (Kaser et al., 2008, Heazlewood et al., 2008); defects in the mucus layer could also influence the pattern of microbial colonization and the maintenance of microbial community structure and function. The importance of the Muc2 mucin in organizing the colon mucus protection is further strengthen by the report that two mouse strains with diarrhea and colon inflammation were shown to have two separate spontaneous mutations in the Muc2 mucin (Heazlewood et al. 2008). Importantly, the production of mucin is regulated by protein kinases, for example, resistin and resistin-like molecule (RELM) beta upregulated mucin expression which dependent on the kinase activities of protein kinase C (PKC), tyrosine kinases, and extracellular-regulated protein kinase (Krimi et al., 2008); Cathelicidin stimulates colonic mucus synthesis by up-regulating MUC1 and MUC2 expression through a mitogen-activated protein kinase pathway (Tai et al., 2008).

#### **4.2 Epithelial cell and its tight junction**

The intestinal defect was first reported in studies showing that the intestinal mucosa of patients with CD had a decreased ability to exclude large molecules (Hollander et al., 1986). The cellular components of the intestinal barrier consist of the complete array of columnar epithelial cell types (enterocyte, paneth cells, enteroendorine cells, and goblet cells) present within the intestine. These cells are polarized with an apical membrane and a basolateral membrane, and apical membrane composition is distinct from the basolateral membrane, for example, the nutrient transporters are located on the apical membrane; they use Na+ ions cotransport to provide the energy and directionality of transport. In contrast, the Na+K+- ATPase, which establishes the Na+ electrochemical gradient, is present on basolateral, but

Pathogenesis of Inflammatory Bowel Diseases 123

defined by tight junction-associated pore-forming claudin proteins (Amasheh et al., 2002; Colegio et al., 2003; Simon et al., 1999). These pores have a radius that excludes molecules larger than 4 A (Van Itallie 2008; Watson et al., 2005). Thus, tight junctions show both size selectivity and charge selectivity, and these properties may be regulated individually or jointly by physiological or pathophysiological stimuli. It need to point out that barrier dysfunction may be caused by increased paracellular permeability, but mainly by epithelial damage, including erosion, and ulceration (Zeissig et al., 2004; Schulzke et al., 2006). In addition, in epithelial cells, the site of claudin protein polymerization to form strands depends on ZO family protein expression (Furuse & Tsukita, 2006), and cells lacking ZO-1

Generally, TJ proteins can be subdivided into ''tightening'' TJ proteins that strengthen epithelial barrier properties (such as occluding and claudin-1 and -4 etc) and ''leaky'' TJ proteins (like claudin-2) that selectively mediate paracellular permeability. Dysfunctional intestinal barrier is a feature of gut inflammation in humans and has been implicated as a pathogenic factor in IBD for the last 30 years. The factors responsible for barrier dysfunction in UC are similar to those in CD, including an increase in epithelial antigen transcytosis and a change in TJ structure with a reduction in TJ strand count and in the depth of the TJ main meshwork; although, in contrast to CD, strand breaks are not as frequent as in UC (Schmitz et al., 1999; Schurmann et al., 1999). Again, the downregulation of occludin and downregulation of several ''tightening'' TJ proteins like claudin-1 and -4, together with an upregulation of the pore-forming TJ protein claudin-2 contribute to the barrier defect observed in UC (Heller et al., 2005; Oshima et al., 2008). These disruptions of tight junction proteins could lead to a breakdown in the protective barrier and can be used as a portal of entry by the luminal bacteria. This breach in intestinal barrier can result in inflammatory infiltrate and enhanced production of cytokines and other mediators (such as neutrophil)

Mucosal permeability is influenced by several factors. The surface mucus layer also impacts mucosal permeability, as demonstrated by spontaneous colitis in Muc-2- deficient mice (Van der Sluis et al., 2006), and increased dextran sulphate sodium-induced colitis in intestinal trefoil factordeficient mice (Mashimo et al., 1996). Luminal microbiota can also compromise the intestinal barrier function (Packey & Sartor, 2008). The third is the integrity of the epithelial cell layer and the basement membrane. Molecularly this can be compromised by downregulating tight junction components Claudins 5 and 6, upregulating pore-forming Claudin 2 (Zessig et al., 2007 ), which can be accomplished by TNF and IL-13, or increasing epithelial apoptosis, which has been achieved in mice by blocking nuclear factor kappa-B (NFκB) signalling. Genetic factors are involved in the loss of intestinal barrier function (Cho & Brant, 2011). Dysregulated innate and adaptive immune system can lead to the enhanced epithelial permeability (Fuss, 2008). Finally, autonomic nerve system function affects epithelial permeability, as demonstrated by mice that develop fulminant jejunoileitis

The increased uptake of antigens and macromolecules from the intestinal lumen mediated through this epithelial barrier dysfunction can further exacerbate the inflammatory process, ending up in a vicious circle. In this manner, barrier dysfunction is a perpetuating principle during gastrointestinal inflammation. Since epithelial TJs are important in the maintenance of barrier function, regulatory changes in their function that are commonly found during intestinal inflammation can have severe consequences. For example, the resulting passive loss of solutes into the intestinal lumen and the subsequent osmotically driven water flow

and ZO-2 fail to form tight junctions at all.

that can further contribute to the altered barrier function.

following ablation of enteric glial cells (Bush et al., 1998).

not apical membranes. In addition, the lipid composition of the membrane differs; the apical membrane is enriched in sphingolipids and cholesterol relative to the basolateral membrane. One result of this cellular polarization is that the apical membranes of intestinal epithelial cells are generally impermeable to hydrophilic solutes in the absence of specific transporters. Thus, the presence of epithelial cells, particularly the apical membranes, contributes significantly to the mucosal barrier (Shen et al., 2009). Among the most important structures of the intestinal barrier are the epithelial tight junctions (TJs) that connect adjacent enterocytes together to determine paracellular permeability. The tight junction is composed of multiple proteins including transmembrane proteins such as occludin, tricellulin, claudins and junctional adhesion molecule (JAM). The intracellular portions of these transmembrane proteins interact with cytoplasmic peripheral membrane proteins, including zona occludens (ZO)-1,-2,-3 and cingulin (Mitic & Anderson. 1998). These tight junction and cytoplasmic proteins then interact with F-actin and myosin II, thereby anchoring the tight junction complex to the cytoskeleton. Once thought to be static, the association of these proteins with the tight junction is highly dynamic (Shen et al., 2009) and may play a role in epithelial barrier regulation. Occludin was the first tight junctionassociated integral membrane protein identified (Furuse et al., 1993). Although occludin knockout mice exhibit intact intestinal epithelial tight junctions and display no observable barrier defect (Schulzke et al., 2005, Saitou et al., 2000). But *in vitro* studies demonstrate crucial roles in tight junction assembly and maintenance (Yu et al., 2005; Suzuki et al., 2009; Elias et al., 2009). This suggests that further analysis of occludin knockout mice under stressed condition may reveal *in vivo* functions of occludin and provide new insight into mechanisms of tight regulation (Turner, 2006). Given the phylogenetic and structural similarities between occludin and tricellulin (Ikenouchi et al., 2005), it may be that the tricellulin accounts for normal intestinal barrier function in occludin knockout mice. This hypothesis could also be applied to inflammatory bowel disease, where intestinal epithelial occludin expression is reduced (Heller et al., 2005). The fact that occludin knockout mice exhibit intact intestinal epithelial barrier function led to the search for additional tight junctional components and ultimately to the discovery of the claudins (Furuse et al., 1998). The claudins are a large family of proteins that also interact with partners on neighboring cells to affect junctional adhesions via extracellular loops. At least 24 different claudin proteins are present in mammals (Van Itallie et al., 2003, 2004, 2006), and these proteins are the primary component of tight junction strands (Furuse et al., 2006). Claudins are expressed in a tissue-specific manner, studies on human intestine confirm the expression of claudins-1, -2, -3, -4, -5, -7, and -8 in the colon, expression of claudins-1, -2, -3, and -4 in the duodenum, and expression of claudins-2 and -4 in the jejunum (Burgel et al., 2002; Escaffit et al., 2005, Szakal et al., 2010; Wang et al., 2010; Zeissing et al., 2007).

The molecular anatomy of transport through tight junction is not yet clear, at least two routes allow transport across the tight junction, and the relative contributions of different paracellular transport are regulated independently (Fihn et al., 2000; Van Itallie, 2008; Watson et al., 2005). One route, the size-dependent pathway, allows paracellular transport of large solutes, including limited flux of proteins and bacterial lipopolysaccharides (Van Itallie 2008; Watson et al., 2005). Although at what size particles are excluded from the leak pathway has not been precisely defined, it is clear that materials as large as whole bacteria cannot pass. Flux across the leak pathway may be increased by cytokines and protein kinases, including IFNγ, TNF *(*Watson et al., 2005; Wang et al., 2005; Clayburgh et al., 2006), MAPKs, myosin II light chain kinase (MLCK) (Turner 2006) and SPAK (Yan et al., 2011). A second pathway is charge-dependent pathway, characterized by small pores that are

not apical membranes. In addition, the lipid composition of the membrane differs; the apical membrane is enriched in sphingolipids and cholesterol relative to the basolateral membrane. One result of this cellular polarization is that the apical membranes of intestinal epithelial cells are generally impermeable to hydrophilic solutes in the absence of specific transporters. Thus, the presence of epithelial cells, particularly the apical membranes, contributes significantly to the mucosal barrier (Shen et al., 2009). Among the most important structures of the intestinal barrier are the epithelial tight junctions (TJs) that connect adjacent enterocytes together to determine paracellular permeability. The tight junction is composed of multiple proteins including transmembrane proteins such as occludin, tricellulin, claudins and junctional adhesion molecule (JAM). The intracellular portions of these transmembrane proteins interact with cytoplasmic peripheral membrane proteins, including zona occludens (ZO)-1,-2,-3 and cingulin (Mitic & Anderson. 1998). These tight junction and cytoplasmic proteins then interact with F-actin and myosin II, thereby anchoring the tight junction complex to the cytoskeleton. Once thought to be static, the association of these proteins with the tight junction is highly dynamic (Shen et al., 2009) and may play a role in epithelial barrier regulation. Occludin was the first tight junctionassociated integral membrane protein identified (Furuse et al., 1993). Although occludin knockout mice exhibit intact intestinal epithelial tight junctions and display no observable barrier defect (Schulzke et al., 2005, Saitou et al., 2000). But *in vitro* studies demonstrate crucial roles in tight junction assembly and maintenance (Yu et al., 2005; Suzuki et al., 2009; Elias et al., 2009). This suggests that further analysis of occludin knockout mice under stressed condition may reveal *in vivo* functions of occludin and provide new insight into mechanisms of tight regulation (Turner, 2006). Given the phylogenetic and structural similarities between occludin and tricellulin (Ikenouchi et al., 2005), it may be that the tricellulin accounts for normal intestinal barrier function in occludin knockout mice. This hypothesis could also be applied to inflammatory bowel disease, where intestinal epithelial occludin expression is reduced (Heller et al., 2005). The fact that occludin knockout mice exhibit intact intestinal epithelial barrier function led to the search for additional tight junctional components and ultimately to the discovery of the claudins (Furuse et al., 1998). The claudins are a large family of proteins that also interact with partners on neighboring cells to affect junctional adhesions via extracellular loops. At least 24 different claudin proteins are present in mammals (Van Itallie et al., 2003, 2004, 2006), and these proteins are the primary component of tight junction strands (Furuse et al., 2006). Claudins are expressed in a tissue-specific manner, studies on human intestine confirm the expression of claudins-1, -2, -3, -4, -5, -7, and -8 in the colon, expression of claudins-1, -2, -3, and -4 in the duodenum, and expression of claudins-2 and -4 in the jejunum (Burgel et al., 2002; Escaffit et al., 2005,

Szakal et al., 2010; Wang et al., 2010; Zeissing et al., 2007).

The molecular anatomy of transport through tight junction is not yet clear, at least two routes allow transport across the tight junction, and the relative contributions of different paracellular transport are regulated independently (Fihn et al., 2000; Van Itallie, 2008; Watson et al., 2005). One route, the size-dependent pathway, allows paracellular transport of large solutes, including limited flux of proteins and bacterial lipopolysaccharides (Van Itallie 2008; Watson et al., 2005). Although at what size particles are excluded from the leak pathway has not been precisely defined, it is clear that materials as large as whole bacteria cannot pass. Flux across the leak pathway may be increased by cytokines and protein kinases, including IFNγ, TNF *(*Watson et al., 2005; Wang et al., 2005; Clayburgh et al., 2006), MAPKs, myosin II light chain kinase (MLCK) (Turner 2006) and SPAK (Yan et al., 2011). A second pathway is charge-dependent pathway, characterized by small pores that are defined by tight junction-associated pore-forming claudin proteins (Amasheh et al., 2002; Colegio et al., 2003; Simon et al., 1999). These pores have a radius that excludes molecules larger than 4 A (Van Itallie 2008; Watson et al., 2005). Thus, tight junctions show both size selectivity and charge selectivity, and these properties may be regulated individually or jointly by physiological or pathophysiological stimuli. It need to point out that barrier dysfunction may be caused by increased paracellular permeability, but mainly by epithelial damage, including erosion, and ulceration (Zeissig et al., 2004; Schulzke et al., 2006). In addition, in epithelial cells, the site of claudin protein polymerization to form strands depends on ZO family protein expression (Furuse & Tsukita, 2006), and cells lacking ZO-1 and ZO-2 fail to form tight junctions at all.

Generally, TJ proteins can be subdivided into ''tightening'' TJ proteins that strengthen epithelial barrier properties (such as occluding and claudin-1 and -4 etc) and ''leaky'' TJ proteins (like claudin-2) that selectively mediate paracellular permeability. Dysfunctional intestinal barrier is a feature of gut inflammation in humans and has been implicated as a pathogenic factor in IBD for the last 30 years. The factors responsible for barrier dysfunction in UC are similar to those in CD, including an increase in epithelial antigen transcytosis and a change in TJ structure with a reduction in TJ strand count and in the depth of the TJ main meshwork; although, in contrast to CD, strand breaks are not as frequent as in UC (Schmitz et al., 1999; Schurmann et al., 1999). Again, the downregulation of occludin and downregulation of several ''tightening'' TJ proteins like claudin-1 and -4, together with an upregulation of the pore-forming TJ protein claudin-2 contribute to the barrier defect observed in UC (Heller et al., 2005; Oshima et al., 2008). These disruptions of tight junction proteins could lead to a breakdown in the protective barrier and can be used as a portal of entry by the luminal bacteria. This breach in intestinal barrier can result in inflammatory infiltrate and enhanced production of cytokines and other mediators (such as neutrophil) that can further contribute to the altered barrier function.

Mucosal permeability is influenced by several factors. The surface mucus layer also impacts mucosal permeability, as demonstrated by spontaneous colitis in Muc-2- deficient mice (Van der Sluis et al., 2006), and increased dextran sulphate sodium-induced colitis in intestinal trefoil factordeficient mice (Mashimo et al., 1996). Luminal microbiota can also compromise the intestinal barrier function (Packey & Sartor, 2008). The third is the integrity of the epithelial cell layer and the basement membrane. Molecularly this can be compromised by downregulating tight junction components Claudins 5 and 6, upregulating pore-forming Claudin 2 (Zessig et al., 2007 ), which can be accomplished by TNF and IL-13, or increasing epithelial apoptosis, which has been achieved in mice by blocking nuclear factor kappa-B (NFκB) signalling. Genetic factors are involved in the loss of intestinal barrier function (Cho & Brant, 2011). Dysregulated innate and adaptive immune system can lead to the enhanced epithelial permeability (Fuss, 2008). Finally, autonomic nerve system function affects epithelial permeability, as demonstrated by mice that develop fulminant jejunoileitis following ablation of enteric glial cells (Bush et al., 1998).

The increased uptake of antigens and macromolecules from the intestinal lumen mediated through this epithelial barrier dysfunction can further exacerbate the inflammatory process, ending up in a vicious circle. In this manner, barrier dysfunction is a perpetuating principle during gastrointestinal inflammation. Since epithelial TJs are important in the maintenance of barrier function, regulatory changes in their function that are commonly found during intestinal inflammation can have severe consequences. For example, the resulting passive loss of solutes into the intestinal lumen and the subsequent osmotically driven water flow

Pathogenesis of Inflammatory Bowel Diseases 125

al., 2002; Dahan et al., 2008). Study also found that Erk activation is involved in claudin-4 protein expression and claudin-4 is involved in the maintenance of the intestinal epithelial cell barrier function (Pinton et al., 2010) as a "tightening" junction protein. Activation of p38/MAPK and Akt signal transduction pathways in the epithelial cells have also been implicated as key mediators of these protective effects (Resta-Lenert & Barrett. 2006). For example, *Lactobacillus GG* (LGG) prevents cytokine-induced apoptosis in both human and mouse intestinal epithelial cells through activating antiapoptotic Akt in a phosphatidylinositol-3κ-kinase (PI3K)-dependent manner and inhibiting proapoptotic p38/MAPK activation (Yan & Polk. 2002). The p38 family is composed of four members: α, β, γ and δ. Expression of the isoforms varies between tissues. Different ligands, via their respective receptors, are able to activate one or several of p38 targets TAK1, ASK1, MLK3, MEKK1-4 and TAO1-3 (Thalhamer et al., 2008). Several studies using the p38 inhibitor, SB203580, have indicated that p38 phosphorylation is increased significantly in IBD tissue (Waetzig et al., 2002; Dahan et al., 2008). This finding is substantiated further by an *in vitro* study, indicating that inhibition of p38 using the natural IL-1 receptor antagonist, in a colonocyte cell line, leads to reduced IL-6 and -8 production, and an *in vivo* study using a murine model of IBD, where inhibition of p38 reduced significantly cytokine mRNA and NFκB activation (Garat et al., 2003; Hollenbach et al., 2004). However, Heat-killed *L. brevis* SBC8803 induced Hsps, phosphorylated p38 MAPK, regulated the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-12, and improved the barrier function of intestinal epithelia under

Fig. 5. Molecular compostion of tight junctions. This model adapted from the model

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2413111/?tool=pubmed.

oxidant stress (Ueno et al., 2011).

presented previously:

results in ''leak flux diarrhea'', one of the main consequences of UC. The tight junction is, therefore, the rate-limiting step in transepithelial transport and the principal determinant of mucosal permeability. But it has to be pointed out that barrier dysfunction itself is not sufficient to cause intestinal diseases, such as in MLCK (Turner 2006) and SPAK (Yan et al., 2011) transgenic mouse models, these two different transgenic mice revealed increased transepithelial permeability, but neither of them demonstrated any UC characterization, for example, these mice develop normal, no significant weight loss, histologically normal crypts were found, no abscesses was noticed.

Recent molecular advances as well as studies of cellular physiology in model epithelia have instead revealed that both the permeability and selectivity of tight junctions can be modulated dynamically by a variety of signals (Mitic et al., 2000). Much of the progress in this field has rested on a significantly enhanced understanding of the proteins that make up the junction itself, as well as those components of the junction on its cytoplasmic face that link the junctional region both to the cellular cytoskeleton and to signal transduction modules (González-Mariscal et al., 2003).

## **5. Protein kinase and pathogenesis of IBD**

## **5.1 mitogen activated protein kinases (MAPK)**

Interestingly, protein kinases are associated with all different level of aspects, demonstrated promising potential as intervention targets against UC. Intracellular signaling cascades are the main route of communication between the plasma membrane and regulatory targets in various intracellular compartments. The evolutionarily conserved mitogen activated protein kinases (MAPK) signaling pathway plays an important role in transducing signals from diverse extra-cellular stimuli (including growth factors, cytokines and environmental stresses) to the nucleus in order to affect a wide range of cellular processes, such as proliferation, differentiation, development, stress responses and apoptosis. MAPK (Coskun et al., 2011) signaling cascades, which comprise up to seven levels of protein kinases, are sequentially activated by phosphorylation and also involved in intestinal inflammation. These families can be divided into two groups: the classical MAPKs, consisting of ERK1/2, p38, JNK and ERK5, and the atypical MAPKs, consisting of ERK3, ERK4, ERK7 and NLK (Coulombe & Meloche, 2007). The signalling pathways which the members of these families influence can be independent of each other or overlapping. The classical pathway leading to activation of ERK1/2 is through the upstream activation of the Raf MAPKKKs, which activate sequentially the MAPKKs, MEK1/2, which can specifically bind and phosphorylate ERK1/2. At this stage, and depending upon the signal being propagated, the ERK1/2 proteins commonly then phosphorylate the downstream MAPK activated proteins (MAPKAP) 1/2. However, other proinflammatory proteins such as cytosolic phospholipase A2 can be activated, as well as several transcription factors including Ets-1, Elk and c-myc. These transcription factors aid the inflammatory process by inducing other related cellular processes such as cell migration and proliferation. Interestingly, a role for ERK1/2, using an ERK1/2 inhibitor, was found in cells of the immune system and colonocytes in the development and progression of IBD, through its mediation in the signalling pathways induced by various cytokines, for example IL-21, and IL-1 (Caruso et al., 2007; Kwon et al., 2007). Indeed, several studies, cell line cultures and isolated crypts from human biopsies, have shown that it is not only over-expressed in IBD tissue (both colonocytes and cells in the underlying lamina propria), but that its phosphorylation state and therefore activation state is increased significantly during the active stages of IBD (Waetzig et

results in ''leak flux diarrhea'', one of the main consequences of UC. The tight junction is, therefore, the rate-limiting step in transepithelial transport and the principal determinant of mucosal permeability. But it has to be pointed out that barrier dysfunction itself is not sufficient to cause intestinal diseases, such as in MLCK (Turner 2006) and SPAK (Yan et al., 2011) transgenic mouse models, these two different transgenic mice revealed increased transepithelial permeability, but neither of them demonstrated any UC characterization, for example, these mice develop normal, no significant weight loss, histologically normal crypts

Recent molecular advances as well as studies of cellular physiology in model epithelia have instead revealed that both the permeability and selectivity of tight junctions can be modulated dynamically by a variety of signals (Mitic et al., 2000). Much of the progress in this field has rested on a significantly enhanced understanding of the proteins that make up the junction itself, as well as those components of the junction on its cytoplasmic face that link the junctional region both to the cellular cytoskeleton and to signal transduction

Interestingly, protein kinases are associated with all different level of aspects, demonstrated promising potential as intervention targets against UC. Intracellular signaling cascades are the main route of communication between the plasma membrane and regulatory targets in various intracellular compartments. The evolutionarily conserved mitogen activated protein kinases (MAPK) signaling pathway plays an important role in transducing signals from diverse extra-cellular stimuli (including growth factors, cytokines and environmental stresses) to the nucleus in order to affect a wide range of cellular processes, such as proliferation, differentiation, development, stress responses and apoptosis. MAPK (Coskun et al., 2011) signaling cascades, which comprise up to seven levels of protein kinases, are sequentially activated by phosphorylation and also involved in intestinal inflammation. These families can be divided into two groups: the classical MAPKs, consisting of ERK1/2, p38, JNK and ERK5, and the atypical MAPKs, consisting of ERK3, ERK4, ERK7 and NLK (Coulombe & Meloche, 2007). The signalling pathways which the members of these families influence can be independent of each other or overlapping. The classical pathway leading to activation of ERK1/2 is through the upstream activation of the Raf MAPKKKs, which activate sequentially the MAPKKs, MEK1/2, which can specifically bind and phosphorylate ERK1/2. At this stage, and depending upon the signal being propagated, the ERK1/2 proteins commonly then phosphorylate the downstream MAPK activated proteins (MAPKAP) 1/2. However, other proinflammatory proteins such as cytosolic phospholipase A2 can be activated, as well as several transcription factors including Ets-1, Elk and c-myc. These transcription factors aid the inflammatory process by inducing other related cellular processes such as cell migration and proliferation. Interestingly, a role for ERK1/2, using an ERK1/2 inhibitor, was found in cells of the immune system and colonocytes in the development and progression of IBD, through its mediation in the signalling pathways induced by various cytokines, for example IL-21, and IL-1 (Caruso et al., 2007; Kwon et al., 2007). Indeed, several studies, cell line cultures and isolated crypts from human biopsies, have shown that it is not only over-expressed in IBD tissue (both colonocytes and cells in the underlying lamina propria), but that its phosphorylation state and therefore activation state is increased significantly during the active stages of IBD (Waetzig et

were found, no abscesses was noticed.

modules (González-Mariscal et al., 2003).

**5. Protein kinase and pathogenesis of IBD 5.1 mitogen activated protein kinases (MAPK)** 

al., 2002; Dahan et al., 2008). Study also found that Erk activation is involved in claudin-4 protein expression and claudin-4 is involved in the maintenance of the intestinal epithelial cell barrier function (Pinton et al., 2010) as a "tightening" junction protein. Activation of p38/MAPK and Akt signal transduction pathways in the epithelial cells have also been implicated as key mediators of these protective effects (Resta-Lenert & Barrett. 2006). For example, *Lactobacillus GG* (LGG) prevents cytokine-induced apoptosis in both human and mouse intestinal epithelial cells through activating antiapoptotic Akt in a phosphatidylinositol-3κ-kinase (PI3K)-dependent manner and inhibiting proapoptotic p38/MAPK activation (Yan & Polk. 2002). The p38 family is composed of four members: α, β, γ and δ. Expression of the isoforms varies between tissues. Different ligands, via their respective receptors, are able to activate one or several of p38 targets TAK1, ASK1, MLK3, MEKK1-4 and TAO1-3 (Thalhamer et al., 2008). Several studies using the p38 inhibitor, SB203580, have indicated that p38 phosphorylation is increased significantly in IBD tissue (Waetzig et al., 2002; Dahan et al., 2008). This finding is substantiated further by an *in vitro* study, indicating that inhibition of p38 using the natural IL-1 receptor antagonist, in a colonocyte cell line, leads to reduced IL-6 and -8 production, and an *in vivo* study using a murine model of IBD, where inhibition of p38 reduced significantly cytokine mRNA and NFκB activation (Garat et al., 2003; Hollenbach et al., 2004). However, Heat-killed *L. brevis* SBC8803 induced Hsps, phosphorylated p38 MAPK, regulated the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β and IL-12, and improved the barrier function of intestinal epithelia under oxidant stress (Ueno et al., 2011).

Fig. 5. Molecular compostion of tight junctions. This model adapted from the model presented previously:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2413111/?tool=pubmed.

Pathogenesis of Inflammatory Bowel Diseases 127

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There are three JNK isoforms, JNK1, 2 and 3, of which there are 10 splice forms in total. Studies using a specific inhibitor against JNK1/2 in induced IBD in rodent models or with isolated colonic tissue found that proinflammatory cytokine production was reduced in conjunction with reduced inflammatory cell infiltration. Similarly, increased phosphorylation of JNK1/2 was seen in inflamed tissue from IBD patients (Dahan et al., 2008; Assi K et al., 2006; Mitsuyama et al., 2008). RDP58 (Loftberg et al., 2002) is a peptide consisting of 9 D-amino acids blocking p38 and JNK, further attenuate UC.

## **5.2 Serine and threonine kinase**

## **5.2.1 Ste20 related proline/alanine rich kinase (SPAK)**

SPAK is defined as a ste20-like proline-/alanine rich kinase that contains an N-terminal series of proline and alanine repeats (PAPA box) followed by a kinase domain, a nuclear localization signal, a consensus caspase cleavage motif, and a C-terminal regulatory region (Johnston et al., 2000). Colonic SPAK exists as a unique isoform that lacks the PAPA box and F-α helix loop in the N-terminus (Yan et al., 2007). The diversity of domains present in SPAK protein might be associated with a variety of biological roles. For example, SPAK has been shown to play roles in cell differentiation, cell transformation and proliferation, and regulation of chloride transport (Piechotta et al., 2002; Gagnon et al., 2006). More importantly, a linkage has been established between SPAK and inflammation, SPAK, as an upstream kinase to Na+-K+-2Cl− co-transporter 1 (NKCC1), can phosphorylate Thr203, Thr207, and Thr212 amino acids on NKCC1, which play an important role in inflammation (Topper et al., 1997). Furthermore, we have demonstrated that SPAK can activate p38 pathway (Yan et al., 2007) that is well known involving inflammation. SPAK caused an increase in intestinal permeability, and SPAK transgenic (TG) mice were more susceptible to experimental colitis. Additionally, increased cytokine production and bacterial translocation were associated with the increased colitis susceptibility (Yan Y et al., 2011).

#### **5.2.2 Myosin II light chain kinase (MLCK)**

MLCK is a specific Serine and threonine kinase which can phosphorylate MLC. It has been found that MLCK activity is required for TNF-induced acute diarrhea. Further, TNF treatment resulted in increased myosin light chain kinase expression (Wang et al., 2005), as a result of transcriptional activation (Graham et al. 2006) *in vitro* and *in vivo*. Constitutive MLCK activation accelerates onset and increases severity of experimental UC. MLCK inhibition, either pharmacologically or by genetic knockout, prevented both intestinal epithelial MLC phosphorylation and barrier dysfunction. More remarkably, MLCK inhibition also restored net water absorption, and therefore corrected the TNF-dependent diarrhea (Clayburgh et al., 2006).

## **6. Conclusions**

Different aspects of factors are implicated in the pathogenesis of a variety of human intestinal inflammatory disorders including IBD, continuing progress in the understanding of the involvement of these factors in intestinal barrier dysfunction, further in IBD pathogeneses offers hope for a new generation of therapeutic strategies targeted at the modulation of transcription factor activity.

#### **7. References**

126 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

There are three JNK isoforms, JNK1, 2 and 3, of which there are 10 splice forms in total. Studies using a specific inhibitor against JNK1/2 in induced IBD in rodent models or with isolated colonic tissue found that proinflammatory cytokine production was reduced in conjunction with reduced inflammatory cell infiltration. Similarly, increased phosphorylation of JNK1/2 was seen in inflamed tissue from IBD patients (Dahan et al., 2008; Assi K et al., 2006; Mitsuyama et al., 2008). RDP58 (Loftberg et al., 2002) is a peptide

SPAK is defined as a ste20-like proline-/alanine rich kinase that contains an N-terminal series of proline and alanine repeats (PAPA box) followed by a kinase domain, a nuclear localization signal, a consensus caspase cleavage motif, and a C-terminal regulatory region (Johnston et al., 2000). Colonic SPAK exists as a unique isoform that lacks the PAPA box and F-α helix loop in the N-terminus (Yan et al., 2007). The diversity of domains present in SPAK protein might be associated with a variety of biological roles. For example, SPAK has been shown to play roles in cell differentiation, cell transformation and proliferation, and regulation of chloride transport (Piechotta et al., 2002; Gagnon et al., 2006). More importantly, a linkage has been established between SPAK and inflammation, SPAK, as an upstream kinase to Na+-K+-2Cl− co-transporter 1 (NKCC1), can phosphorylate Thr203, Thr207, and Thr212 amino acids on NKCC1, which play an important role in inflammation (Topper et al., 1997). Furthermore, we have demonstrated that SPAK can activate p38 pathway (Yan et al., 2007) that is well known involving inflammation. SPAK caused an increase in intestinal permeability, and SPAK transgenic (TG) mice were more susceptible to experimental colitis. Additionally, increased cytokine production and bacterial translocation were associated with the

MLCK is a specific Serine and threonine kinase which can phosphorylate MLC. It has been found that MLCK activity is required for TNF-induced acute diarrhea. Further, TNF treatment resulted in increased myosin light chain kinase expression (Wang et al., 2005), as a result of transcriptional activation (Graham et al. 2006) *in vitro* and *in vivo*. Constitutive MLCK activation accelerates onset and increases severity of experimental UC. MLCK inhibition, either pharmacologically or by genetic knockout, prevented both intestinal epithelial MLC phosphorylation and barrier dysfunction. More remarkably, MLCK inhibition also restored net water absorption, and therefore corrected the TNF-dependent

Different aspects of factors are implicated in the pathogenesis of a variety of human intestinal inflammatory disorders including IBD, continuing progress in the understanding of the involvement of these factors in intestinal barrier dysfunction, further in IBD pathogeneses offers hope for a new generation of therapeutic strategies targeted at the

consisting of 9 D-amino acids blocking p38 and JNK, further attenuate UC.

**5.2.1 Ste20 related proline/alanine rich kinase (SPAK)** 

increased colitis susceptibility (Yan Y et al., 2011).

**5.2.2 Myosin II light chain kinase (MLCK)** 

diarrhea (Clayburgh et al., 2006).

modulation of transcription factor activity.

**6. Conclusions** 

**5.2 Serine and threonine kinase** 


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**Part 2** 

**Advances in Diagnosis of** 

**Inflammatory Bowel Disease** 

Vanderlugt, C.J., Miller, S.D. (1996).Epitope spreading. *Curr Opin Immunol.* 8:831–836.

