Dendritic cells and regulatory T cells in the gastrointestinal tract of healthy and diseased dogs with inflammatory bowel disease

ISBN 978-3-86345-156-1

Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH 35392 Gießen · Friedrichstraße 17 · Tel. 0641 / 24466 · Fax: 0641 / 25375

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in the gastrointestinal tract of healthy and diseased dogs with inflammatory bowel disease

Johannes Junginger

Department of Pathology

University of Veterinary Medicine Hannover 2013

Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.

1. Auflage 2013

© 2013 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH, Gießen

Printed in Germany

ISBN 978-3-86345-156-1

Verlag: DVG Service GmbH Friedrichstraße 17

35392 Gießen 0641/24466 info@dvg.de www.dvg.de

Dendritic cells and regulatory T cells in the gastrointestinal tract of healthy and diseased

dogs with inflammatory bowel disease

Thesis

Submitted in partial fulfilment of the requirements of the degree - Doctor of Veterinary Medicine -

Doctor medicinae veterinariae ( Dr. med. vet. )

by

Johannes Junginger Braunschweig

Hannover 2013

1. Referee: Prof. Dr. Marion Hewicker-Trautwein

2. Referee: PD Dr. Veronika M. Stein, PhD

Date of the oral examination: 7^th of May, 2013

Parts of the thesis have been published previously in Veterinary Research 2012, 43, 23 and Innate Immunity 2013, Epub ahead of print (DOI:

10.1177/1753425913485475).

Johannes Junginger received a scholarship from the German National Academic Foundation.

To my wife

1. Aims ...1

2. Introduction ...3

2.1. Gastrointestinal immunology... 3

2.2. Dendritic cells (DCs) ... 6

2.2.1. DCs as professional antigen presenting cells... 6

2.2.2. DC subsets... 9

2.2.3. Canine DCs... 9

2.2.4. Intestinal DCs... 11

2.3. Regulatory T cells (Tregs) ... 13

2.3.1. Tregs mediate cellular immunosuppression... 13

2.3.2. Tregs in domestic animals... 17

2.3.3. Tregs in the intestine... 18

2.4. Inflammatory bowel disease (IBD) ... 19

2.4.1. General remarks ... 19

2.4.2. Human IBD... 22

2.4.3. IBD in dogs... 22

2.4.4. Pathogenesis of IBD ... 25

3. Canine gut dendritic cells in the steady state and in inflammatory bowel disease...29

4. Immunohistochemical investigation of Foxp3 expression in the intestine in healthy and diseased dogs...31

5. Discussion...33

5.1. Canine gut DCs in the steady state ... 33

5.2. Tregs in the canine healthy gut... 35

5.3. DCs and Tregs in canine IBD ... 36

5.4. Potential therapeutic strategies for canine IBD... 39

5.5. Concluding remarks... 41

6. Summary...43

7. Zusammenfassung ...47

8. List of publications ...51

9. References...53

10. Acknowledgements ...83

List of abbrevations

AIEC adherent and invasive Escherichia coli ATG16L1 autophagy-related protein 16-1

B bacterium

CCR CC chemokine receptor

CD cluster of differentiation

CR complement receptor

CTLA cytotoxic T-lymphocyte antigen CX3CR1 CXC chemokine receptor for fractalkine

DC dendritic cell

E enterocyte

EGE eosinophilic gastroenteritis

F follicle

FAE follicle-associated epithelium

FcR receptor for crystallisable fragments of immunoglobulins

Foxp3 forkhead box P3

GALT gut-associated lymphoid tissue

GC germinal centre

hi high expression in flow cytometry

HUC histiocytic ulcerative colitis

IBD inflammatory bowel disease

iDC immature dendritic cell

IDO indoleamine 2,3-dioxygenase

IEL intraepithelial lymphocyte

IFR interfollicular region

Ig immunoglobulin

IL interleukin

ILF isolated lymph follicle

IL-23R interleukin 23 receptor

LPE lymphoplasmacytic enteritis

IPEX immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

iTreg induced regulatory T cell

LAG3 lymphocyte-activation gene 3

LI large intestine

lo low expression in flow cytometry

LP lamina propria

LPS lipopolysaccharide

M cell microfold cell

mDC mature dendritic cell

MHC major histocompatibility complex

MLN mesenteric lymph node

mRNA messenger ribonucleic acid

NCF neutrophil cytosolic factor NK cell natural killer cell

nTreg natural occurring regulatory T cell NOD2 nucleotide-binding oligomerization

domain-containing protein 2

PAMP pathogen-associated molecular pattern

PAS periodic acid-Schiff

pDC plasmacytoid dendritic cell

PP Peyer’s patch

Ref reference(s)

SED subepithelial dome

SI small intestine

S-IgA secretory immunoglobulin A

SNP single nucleotide polymorphism

spp. species

STAT3 signal transducer and activator of transcription 3 Tcon conventional non-regulatory T cell

TCR T cell receptor

TGF transforming growth factor

Th T helper cell

TJ tight junction

TLR toll-like receptor

TNF tumour necrosis factor

Treg regulatory T cell

Tr1 special subset of induced regulatory T cells

T0 naïve T cell

List of tables

Table 1. Phenotypes of intestinal DCs in animals and humans... 12

Table 2. Treg diversity. ... 15

Table 3. Differential diagnoses for IBD. ... 21

List of figures

Figure 2. Phenotypes and functions of DCs. ... 7

Figure 3. Pathogenesis of IBD... 25

Figure 4. Antigen uptake within the canine intestine... 34

Figure 5. Possible impact of DCs and Tregs on the development of canine IBD... 38

1. Aims

Alongside the gastrointestinal border, the mucosal immune system perpetually comes into contact with large amounts of both beneficial and potentially pathogenic antigens. Therefore, uptake followed by induction of either tolerance or protective immune responses towards luminal antigens are one of the principal goals of mucosal dendritic cells (DCs) (COOMBES et al. 2007). Particularly, induction of oral tolerance mainly depends on specialised DCs that mediate generation of regulatory T cells (Tregs) in mesenteric lymph nodes during the steady state followed by their homing to the intestinal lamina propria (HADIS et al. 2011). Moreover, breakdown of intestinal homoeostasis including loss of oral tolerance towards commensal microbiota is thought to be crucial for the pathogenesis of chronic idiopathic inflammatory intestinal disorders termed inflammatory bowel disease (IBD) in both men and dogs (MALOY and POWRIE 2011; CATCHPOLE and ALLENSPACH 2012).

As little is known about DCs and Tregs in the canine gut, the aim of this thesis was to investigate their distribution and phenotype alongside the gastrointestinal tract of dogs without alimentary disorders. Subsequently, they should further be analysed in dogs suffering from inflammatory gastrointestinal diseases – with special emphasize on IBD – to test the hypothesis that DCs and Tregs are involved in the pathogenesis of canine IBD. Therefore, DCs and Tregs were investigated in gastrointestinal tissue samples of dogs using immunohistochemistry and multicolour immunofluorescence after histopathological examination.

The results may have an impact on the knowledge about canine intestinal homoeostasis including the induction of oral tolerance in the steady state. Moreover, these investigations constitute the basis for further functional studies on DCs and Tregs in vitro. Additionally, the present study may give new insights into the pathogenesis of canine IBD.

2. Introduction

2.1. Gastrointestinal immunology

At body surfaces, mammalians share their life with a complex commensalic and symbiotic microflora (SUCHODOLSKI 2011). Therefore, it is important to adapt to this microbial load to protect the specific ecosystem while ensuring body integrity.

Alongside the gastrointestinal border, which holds the largest microbial population, many unspecific and innate defence mechanisms are established to prevent the penetration of the host (TIZARD 2012a). For instance, the intestinal mucosa is covered by a continuous epithelial layer closely connected by tight junctions. This cellular border establishes the anatomical separation of luminal organisms from the mucosa, expresses the pathogen recognition receptors, such as toll-like receptors (TLRs), and secretes several antimicrobial peptides (LOTZ et al. 2007). Additionally, specialised secreting goblet cells produce large amounts of mucin that form a mucous layer covering the epithelial surface and thus prevent microbial epithelial penetration (CORFIELD et al. 2000). Moreover, commensal bacteria themselves can block the proliferation of pathogens by influencing the local environment, such as pH and oxygen concentration (FUKUDA et al. 2011).

In addition to unspecific defence mechanisms, the gut contains a complex adaptive immunological network (Figure 1) that allows antigen specific interventions and ensures ‘toleration of the friend but elimination of the foe’ (WORBS et al. 2006;

RAMIRO-PUIG et al. 2008). This network includes mesenteric lymph nodes (MLNs) and the so-called gut-associated lymphoid tissue (GALT) comprising organised lymphoid structures – Peyer’s patches (PPs) and isolated lymphoid follicles (ILFs) – as well as diffuse parts including the lamina propria (LP) and surface epithelium (BRANDTZAEG et al. 2008).

M cell

IFR SED

GC F

Inductive sites Effector sites

PP

MLN Villus

YY YY

YY

Epitheliu m

DC

DC Bacteria

S-IgA

Plasma cell

B cell T cell

Figure 1. Organisation of the adaptive intestinal immune system.

The mucosal immune system comprises structured parts, such as PPs and MLNs, and diffuse elements including the LP and surface epithelium. Antigen uptake, processing and presentation to naïve T and B cells take place within PPs and MLN resulting in the induction of adaptive immune responses (inductive sites).

Subsequently, generated effector cells, including plasma cells and T lymphocytes, migrate to the intestinal mucosa, where they are randomly scattered throughout either the LP or the epithelium. Therefore, diffuse compartments host many effector functions, such as plasma cells contributing to the formation of S-IgA (effector sites).

However, antigen uptake may also occur within the LP.

DC = dendritic cell; F = follicle; GC = germinal centre; IFR = interfollicular region; LP

= lamina propria; M cell = microfold cell; MLN = mesenteric lymph node; PP = Peyer’s patch; SED = subepithelial dome; S-IgA = secretory immunoglobulin A.

PPs – firstly described in 1677 by the Swiss anatomist and physician Joseph Hans Conrad Peyer (MAKALA et al. 2002) – are prominent lymphoid structures of the small intestine that are believed to be the main source of immunoglobulin (Ig) A-producing plasma cells (FAGARASAN and HONJO 2003). Anatomically, PPs are covered by a

follicle-associated epithelium (FAE) containing distinct cells with a microfold apical cellular membrane (M cells) that are specialised in uptake of luminal antigens (VON ROSEN et al. 1981). Basally, M cells transfer the antigens to dendritic cells (DCs), which are located in subepithelial domes (SEDs). Subsequently, DCs present those antigens to naïve lymphocytes located in underlying follicles, which is followed by the formation of germinal centres and eventually results in the generation of specific effector lymphocytes (MAKALA et al. 2002). In addition to antigen presentation and generation of effector cells, which classically takes place in secondary lymphoid organs, the ileal PPs of sheep, cattle and pigs are important for primary B cell development and therefore act as primary lymphoid tissue (BARMAN et al. 1997;

YASUDA et al. 2006). In dogs, the small intestine comprises a total of 26-29 PPs that differ between anatomical parts (HOGENESCH et al. 1987). The jejunum and upper ileum contain small discrete PPs, while the PP that totally encircles the distal ileum measures 26-30 cm in length (HOGENESCH and FELSBURG 1992). Additionally, canine duodenal PPs normally show intrafollicular invaginations of the SED (HOGENESCH and FELSBURG 1990).

Although PPs were initially described in the small intestine, similar structures exist in the large bowel of several species, for instance as so-called lymphoglandular complexes in dogs (ATKINS and SCHOFIELD 1972). In addition to PPs and their large intestinal counterparts, the canine stomach contains many ILFs that are randomly scattered throughout the gastric mucosa without association to a FAE (KOLBJORNSEN et al. 1994).

Once they are generated in the so-called inductive sites, mature effector cells migrate into diffuse GALT parts where they exert their specific effector functions (BRANDTZAEG and JOHANSEN 2005; AGACE 2008). Therefore, the LP is a main reservoir of mature B cells, plasma cells and cluster of differentiation (CD) 4 positive T cells – making it difficult to decide whether the mucosa is inflamed or not by means of histopathological examination (DAY et al. 2008). LP plasma cells are proficient in the production of specialised mucosal dimeric immunoglobulins known as IgA (WOOF and KERR 2006). Once they are produced, IgA dimers bind to the basal surface of enterocytes followed by their transepithelial transport and secretion

(secretory IgA, S-IgA) into the intestinal lumen (JOHANSEN and KAETZEL 2011).

Luminal S-IgA is capable to agglutinate antigenic particles, to neutralise viruses and to prevent the adherence of invading microbes to the intestinal surface (WOOF and MESTECKY 2005). Similar to M cells of PPs, the intestinal LP also contains cells responsible for the detection and uptake of antigens followed by their migration to MLNs (BIMCZOK et al. 2005; SCHULZ et al. 2009).

In addition to the LP, the intestinal epithelium contains many intraepithelial lymphocytes (IELs) of which the majority are T cells predominantly expressing CD8 (HAYDAY et al. 2001). Functionally, IELs are suggested to exhibit both cytotoxic and regulatory properties (CORAZZA et al. 2000; LUCKSCHANDER et al. 2009).

Although they are not covered by the definition of organised GALT, the draining MLNs are important parts of the intestinal immune system as they perform antigen processing and generation of related effector cells similar to PPs (BRANDTZAEG et al. 2008). Furthermore, their role in intestinal homoeostasis seems to be independent of the presence of PPs, as mice lacking PPs but possessing MLNs can indeed develop oral tolerance (SPAHN et al. 2002). However, it remains unknown whether PPs and MLNs exhibit different functions or if they just comprise a double layer of defence.

2.2. Dendritic cells (DCs)

2.2.1. DCs as professional antigen presenting cells

In 1973, the Canadian immunologist Ralph M. Steinman described a novel cell type isolated from murine spleen, which was characterised by long tree-like arms, major histocompatibility complex (MHC) class II expression and the lack of T cell specific markers and macrophage characteristics (STEINMAN and COHN 1973). According to their morphology, they were termed as ‘dendritic cells’. Subsequently, detailed studies followed concerning their morphology and function (STEINMAN and COHN 1974; STEINMAN et al. 1974; STEINMAN et al. 1975; STEINMAN et al. 1979).

Today, more than three decades later, DCs are accepted as the linkage between

innate and acquired immunity and therefore Ralph M. Steinman received the Nobel Prize for his fundamental discovery in 2011 (CALLAWAY 2011).

Periphery

iDC

CD1 CR3/4

FcR TLR

Lymph node

mDC

CD40 C 80 D C 86 D MHC II

Treg T

TCR

T

IL MHC II

CD80/

86 CD

CD154

B CR

TLR

Figure 2. Phenotypes and functions of DCs.

At the body periphery, immature DCs (iDCs) express molecules involved in uptake of antigens, such as TLRs, CD1, FcR, CR3 (CD11b/CD18) and CR4 (CD11c/CD18).

Phagocytosed antigens are processed and linked to MHC II followed by migration of DCs to secondary lymphoid organs, where their maturation and stimulation of naïve T cells (T0) takes place (mature DCs, mDCs). This includes MHC II mediated antigen presentation, ligation of costimulatory molecules, such as CD40, CD80 and CD86, and the production of costimulatory ILs. Subsequently, T cells differentiate into several Th subsets responsible for distinct types of immune responses.

B = bacterium; CD = cluster of differentiation; CR = complement receptor; FcR = receptor for crystallisable fragments of immunoglobulins; IL = interleukin; MHC = major histocompatibility complex; TCR = T cell receptor; Th = T helper cell; TLR = toll-like receptor; Treg = regulatory T cell.

DCs are omnipresent cells widely distributed along the body periphery where they are specialised for antigen recognition and uptake (BANCHEREAU and STEINMAN 1998). Phenotypically, these so-called immature DCs (iDCs; Figure 2) display high expression levels of receptors related to antigen recognition, such as TLRs, glycolipid receptors CD1, receptors for crystallisable fragments of immunoglobulins (FcRs) and the complement receptor (CR) 3 (CD11b/CD18) and 4 (CD11c/CD18) (BANCHEREAU et al. 2000). Additionally, iDCs show high intracytoplasmic expression of MHC II but lack costimulatory molecules, such as CD40, CD80 and CD86. Once the antigen is recognised, iDCs perform phagocytosis and intracytoplasmic processing followed by the association of small antigen fragments, so-called epitopes, to MHC II molecules (INABA et al. 2000). Subsequently, iDCs start maturation by changing their phenotype and migrating to secondary lymphoid organs, where they travel to T cell areas, such as the paracortex of lymph nodes (STEINMAN et al. 1997). Accordingly, mature DCs (mDCs; Figure 2) present their epitopes associated with surface MHC II molecules to T cell antigen receptors (TCRs) of naïve T cells (GERMAIN 1994; TURLEY et al. 2000). Additionally, mDCs express high levels of CD40, CD80 and CD86 binding to CD154 or CD28 on T cells, which give them a costimulatory impulse (YOUNG et al. 1992; INABA et al. 1994).

Furthermore, mDCs produce several costimulatory interleukins (ILs) including IL-12 or IL-10 (BANCHEREAU et al. 2000). This stimulatory triad leads to the activation of naïve T cells resulting in their differentiation into contrasting T helper cell (Th) subsets, which are specific for different aspects of the broad spectrum of immune responses (ZHOU et al. 2009). As stimulation of naïve T cells can only be performed by DCs but not by macrophages or B cells, DCs constitute the most potent professional antigen presenting cells that orchestrate the mammalian immune system (BANCHEREAU et al. 2000). In addition to the induction of protective immunity against pathogens, specialised DCs can mediate tolerance and therefore are important for immune regulation (STEINMAN et al. 2003). Moreover, DCs have gained increasing notice as promising candidates for the development of novel therapeutic approaches (SHLOMCHIK et al. 1999; BLANCO et al. 2001; KALERGIS and RAVETCH 2002).

2.2.2. DC subsets

Due to their complex diversity, DCs consist of several subpopulations concerning their origin, phenotype and function. For example, so-called conventional DCs, derived from blood monocytes, have been arisen from bone marrow myeloid precursor cells (LIU and NUSSENZWEIG 2010). They differentiate into either nonlymphoid tissue DCs, which potentially can migrate to the sites of antigen presentation, or those who are resident in lymphoid tissue (LIU and NUSSENZWEIG 2010). In contrast to conventional DCs, plasmacytoid DCs, arisen from lymphoid precursors, produce large amounts of interferon in response to viral infections (CELLA et al. 1999; COLONNA et al. 2004).

Phenotypically, DCs can be classified according to the expression of different surface molecules. Based on their large heterogeneity and plasticity, various subsets of DCs exist and thus there is a wide phenotypic range instead of a single marker for these cells (SHORTMAN and LIU 2002; FRIES and GRIEBEL 2011).

Furthermore, DCs display general functional differences depending on their immunological environment. In the steady state, DCs conditioned by local factors, such as vitamin A metabolites and transforming growth factor (TGF) β, are suggested to mediate an antiinflammatory milieu responsible for the development of regulatory T cells (Tregs) (COOMBES et al. 2007). Therefore, these so-called steady state DCs are crucial for the induction and maintenance of homoeostasis and tolerance towards the commensal microflora. In contrast, certain pathogen-associated molecular patterns (PAMPs) can induce the production of proinflammatory molecules that result in the recruitment of inflammatory DCs being potent mediators of protective immune responses (COOMBES and POWRIE 2008).

2.2.3. Canine DCs

In dogs, DCs have been extensively described in vivo in the context of related neoplasia including the complex of cutaneous histiocytoma, reactive histiocytosis and histiocytic sarcoma (MOORE 2010). Neoplastic canine interstitial DCs are known to express CD1, MHC I and MHC II that makes them competent in antigen

presentation. They further express the β2-integrin CD11c, which is not only involved in cell adhesion but also, as a part of CR4 (CD11c/CD18), crucial for antigen recognition (VERBOVETSKI et al. 2002). Interestingly, macrophages predominantly express CD11b or – especially in the splenic red pulp and bone marrow – CD11d (DANILENKO et al. 1995; MOORE 2010). Moreover, canine mature DCs reveal costimulatory molecules of the B7 family including CD80 and CD86, which are essential for the interaction with T cells (MOORE 2010). Furthermore, canine DCs are confirmed to express CD1, CD11c, CD80 and MHC II in the context of several conditions and diseases, respectively, including canine marrow graft recipients (DEEG et al. 1988), arthropathies (HEWICKER-TRAUTWEIN et al. 1999; LEMBURG et al. 2004), skin disorders (RICKLIN et al. 2010) and systemic lupus erythematosus (HENRIQUES et al. 2012).

In addition to in vivo-investigations, canine blood- and bone marrow-derived DCs generated in vitro further express CD11b, CD14, CD40 and CD206 (YOSHIDA et al.

2003; BUND et al. 2010; RICKLIN GUTZWILLER et al. 2010). In contrast to mice and humans, these molecules are not exclusively restricted to canine DCs as they are also found on canine macrophages in vitro (BUENO et al. 2005; RICKLIN GUTZWILLER et al. 2010; GOTO-KOSHINO et al. 2011).

Despite the expression of surface molecules, cytoplasmic projections comprise the most specific morphological feature of DCs allowing to distinguish them from macrophages (QESKA et al. 2013). Additionally, ultrastructural findings – these are large formation of Golgi apparatus, endoplasmatic reticulum and presence of periodic microstructures parallel to the absence of lysosomal organelles or phagocytic vacuoles – constitute certain properties of canine DCs discriminating them from macrophages (IBISCH et al. 2005).

Taken together, these results suggest that an application of a panel of the aforementioned different surface markers in combination with morphological characteristics seems to be the most appropriate method to identify canine DCs in vivo.

2.2.4. Intestinal DCs

Alongside the gastrointestinal border, DCs get into contact with large amounts of antigens and have to decide whether they represent ‘friend or foe’ followed by induction of either tolerance or protective immunity (COOMBES and POWRIE 2008).

Thus, the maintenance of intestinal homoeostasis is closely related to the balance between steady state and inflammatory DCs, and consequently a failure of DC homoeostasis may promote exhaustive inflammation in the gut (NIESS 2008).

Within the gastrointestinal tract, they are widely distributed in both diffuse and organised GALT and approximately 10-15% of leucocytes in the murine intestinal LP are CD11c+ MHC II+ DCs (SUN et al. 2007). Due to their pivotal importance for mucosal immunology, intestinal DCs have been characterised in animals and humans (Table 1). In general, expression of MHC II and CD11c is widely conserved among different species. Additionally, several subsets are described based on the expression of further molecules. For example, murine intestinal DCs can be distinguished according to their expression of either the integrin CD103 (αEβ7) or the CXC chemokine receptor for fractalkine (CX3CR1) (SCHULZ et al. 2009). In the steady state, CD103+ DCs can migrate to MLNs and induce gut homing of primed T cells (JOHANSSON-LINDBOM et al. 2005). In the context of local TGF-β and retinoic acid, CD103+ DCs are further potent inducers of Tregs, which are essential for oral tolerance (COOMBES et al. 2007). In contrast, murine CX3CR1+ DCs constitute non-migratory gut-resident cells that seem to be involved in direct sampling of luminal antigens (NIESS et al. 2005; SCHULZ et al. 2009). Furthermore, intestinal DCs in mice can be subdivided based on their expression of CD8α, CD11b and the chemokine receptors (CCRs) 6 and 7 (IWASAKI and KELSALL 1999, 2000). In pigs, four phenotypically distinct DC subsets are proposed based on the expression of CD11b and CD172a (BIMCZOK et al. 2005). Interestingly, only CD11b+ DCs were detected in the draining lymph suggesting that porcine LP DCs but not those from PPs migrate to MLNs (BIMCZOK et al. 2005).

Despite their importance in mucosal immunology, only few reports focused on canine intestinal DCs (GERMAN et al. 1998; KATHRANI et al. 2011b). In these studies, intestinal mononuclear cells with macrophage and/or DC morphology were labelled

with single markers including CD11c or MHC II. However, these molecules are not exclusively expressed by canine DCs.

Table 1. Phenotypes of intestinal DCs in animals and humans.

Species,

Tissue Phenotype Ref

Mouse

LPSI CD103⁺ CX3CR1^- CD11b^hi/lo CD11c^hi 1 LPSI CD103^- CX3CR1⁺ CD11b^hi CD11c^hi/lo 2,3

LPLI CD40^lo CD80⁺ CD86⁺ MHC II^lo 4

PPSED CD8α^- CD11b⁺ CD11c⁺ MHC II⁺ CCR6⁺ CCR7⁺ 5, 6 PPIFR CD8α⁺ CD11b^- CD11c⁺ MHC II⁺ CCR6^- CCR7⁺ 5, 6 PP_{SED, IFR} CD8α^- CD11b^- CD11c⁺ MHC II⁺ 5, 6 PP pDC⁺ CD207⁺ CD208⁺ CD209⁺ CD303⁺ TLR8⁺ 7

MLN CD103⁺ CX3CR1^- CD11c^hi 8

Rat

LPSI, LI MHC II⁺ 9

Pig

LPSI CD1^- CD14^- CD16⁺ CD80⁺ MHC II⁺ 10

LPSI CD11b⁺ CD172a⁺ MHC II⁺ 11

PPSED CD11b^- CD172a⁺ MHC II⁺ 11

PPIFR CD11b^- CD172a^- MHC II⁺ 11

lymph CD11b⁺ CD172a^- MHC II⁺ 11

Sheep

LPSI CD11b⁺ CD11c⁺ CD68⁺ CD205⁺ CD209⁺ MHC II⁺ 12 PPSED CD11b⁺ CD11c⁺ CD68⁺ CD205⁺ CD209^- MHC II⁺ 12 PPF CD11b^- CD11c^- CD68⁺ CD205⁺ CD209^- MHC II⁺ 12 PPIFR CD11b⁺ CD11c⁺ CD68⁺ CD205⁺ CD209⁺ MHC II⁺ 12 Primate (rhesus macaque)

PPSED, IFR CD209⁺ 13

Dog*

LPSI MHC II⁺ 14

LPSI CD11c⁺ 15

Human

LP_LI CD25^- CD80^- CD83⁺ CD86⁺ 16

PPSED CD11c⁺ CD83⁺ CD209⁺ CCR6⁺ CCR7⁺ TLR4⁺ 17

PPSED CD11c^+/- CD209^+/- MHC II⁺ 13

PPGC CD11c⁺ CD209^- MHC II⁺ 13

PPIFR CD11c⁺ CD209^+/- MHC II⁺ 13

*In dogs, two studies described intestinal mononuclear cells with macrophage and/or DC morphology labelled with single markers, which are not exclusively found on canine DCs.

CCR = CC chemokine receptor; CD = cluster of differentiation; CX3CR1 = CXC chemokine receptor for fractalkine; F = follicle; GC = germinal centre; hi = high expression in flow cytometry; IFR = interfollicular region; LI = large intestine; lo = low expression in flow cytometry; LP = lamina propria; MHC = major histocompatibility complex; MLN = mesenteric lymh node; pDC = plasmacytoid dendritic cell; PP = Peyer’s patch; Ref = reference(s); SED = subepithelial dome; SI = small intestine;

TLR = toll-like receptor.

References: 1 (JOHANSSON-LINDBOM et al. 2005), 2 (NIESS et al. 2005) 3 (ATARASHI et al. 2008), 4 (CRUICKSHANK et al. 2005), 5 (IWASAKI and KELSALL 1999), 6 (IWASAKI and KELSALL 2000), 7 (ROCHEREAU et al. 2011), 8 (SCHULZ et al. 2009), 9 (MARIC et al. 1996), 10 (HAVERSON et al. 2000), 11 (BIMCZOK et al.

2005), 12 (AKESSON et al. 2008), 13 (JAMESON et al. 2002), 14 (GERMAN et al.

1998), 15 (KATHRANI et al. 2011b), 16 (TE VELDE et al. 2003), 17 (SALIM et al.

2009).

2.3. Regulatory T cells (Tregs)

2.3.1. Tregs mediate cellular immunosuppression

To avoid excessive and prolonged immune responses and to prevent destruction of beneficial and commensal antigens, the mammalian immune system comprises several regulatory mechanisms including anti-inflammatory cytokines and cells with regulatory capacity (TIZARD 2012b).

The glorious story about the prominent Th subset with regulatory properties, Tregs, was founded in 1969 by Nishizuka and Sakakura giving the first evidence that a subset of thymus-derived cells is involved in the prevention of autoimmune diseases (NISHIZUKA and SAKAKURA 1969). The direct regulatory intervention of thymic lymphocytes was hypothesized one year later (GERSHON and KONDO 1970) and therefore special suppressor T cells were suggested to be crucial for the control of immune homoeostasis (GERSHON et al. 1972). Although the concept of suppressor T cells lost favour and was critically discussed during the 1980s (MOLLER 1988), some immunologists perpetually believed in the existence of these cells (SAKAGUCHI et al. 1985; JANEWAY 1988; TADA 1988). In 1995, the idea of T cell mediated regulation was renewed by Shimon Sakaguchi and colleagues discovering

a special T cell subset, which expresses CD4 and the IL-2 receptor α-chain (CD25) molecule (SAKAGUCHI et al. 1995). These cells revealed regulatory capacity in vitro and in vivo and its depletion, for instance via application of monoclonal antibodies against CD25, resulted in development of autoimmune diseases – such as thyroiditis, gastritis, insulitis, sialoadenitis, adrenalitis, oophoritis, glomerulonephritis and polyarthritis – that were prevented by the transfer of CD4+ CD25+ cells (SAKAGUCHI et al. 1995; TAKAHASHI et al. 1998; THORNTON and SHEVACH 1998). Subsequently, the highly conserved transcription factor forkhead box P3 (Foxp3¹) – it’s mutation is responsible for the human ‘immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome’ (IPEX) – was shown to be mainly involved in the development and function of CD4+ CD25+ Tregs (BENNETT et al. 2001; FONTENOT et al. 2003; HORI et al. 2003). Accordingly, Foxp3 has been established as the most important marker for Tregs and the availability of specific antibodies allows their investigation in several animals and humans (BILLER et al.

2007; BANHAM et al. 2009; GARDEN et al. 2011). Today, approximately 10 years later, Tregs are accepted as the key mediators of cellular immune regulation and tolerance (LAN et al. 2007; SAKAGUCHI et al. 2008) and their discovery constitutes a key step not only in modern immunology but also for the development of new therapeutic strategies (LESLIE 2011).

1 designated in upper case letters (FOXP3) in humans;

Table 2. Treg diversity.

Name Phenotype Location, function Ref

nTregs CD4⁺ CD25⁺ Foxp3⁺ Helios⁺ Naturally occurring Tregs

derive in the thymus 1, 2

iTregs CD4⁺ CD25⁺ Foxp3⁺ Helios^-

Adaptive/induced Tregs arise in the periphery due to conversion of non-regulatory Tcons

1, 2

Th3 CD4⁺ Foxp3⁺ TGF-β⁺ IL-4⁺ IL-10⁺ Mucosal surfaces, induction and

maintenance of oral tolerance 3, 4

CD103⁺ CD4⁺ CD25^+/- Foxp3⁺ CD103⁺ CD103 mediates homing

to the lung, skin and gut 5, 6

CCR4⁺ CCR4⁺ Foxp3⁺ CCR4 mediates

homing to the skin 6

Tr1 CD4⁺ CD25^lo Foxp3^+/- TGF-β⁺, IL-4^-, IL-10⁺

Generated in vitro and in vivo (by iDCs) via IL-10, prevent murine colitis

7, 8

CCR = CC chemokine receptor; CD = cluster of differentiation; Foxp3 = forkhead box P3, iDC = immature dendritic cell; IL = interleukin; iTreg = induced regulatory T cell;

lo = low expression in flow cytometry; nTreg = natural occurring regulatory T cell; Ref

= reference(s); Tcon = conventional non-regulatory T cell; TGF = transforming growth factor; Th3, Tr1 = subsets of iTregs.

References: 1 (SUGIMOTO et al. 2006), 2 (THORNTON et al. 2010), 3 (CHEN et al.

1994), 4 (SAURER and MUELLER 2009), 5 (LEHMANN et al. 2002), 6 (SATHER et al. 2007), 7 (GROUX et al. 1997), 8 (RONCAROLO et al. 2006).

Generally, regulatory T cells can naturally occur in the thymus along a regulatory lineage (naturally occurring Tregs, nTregs) or may arise in the periphery – induced (iTregs) or ‘adaptive’ subsets – via the conversion of conventional non-regulatory T cells (Tcons) (FEUERER et al. 2009; GARDEN et al. 2011). This transformation can be mediated by either the influence of regulatory cytokines, such as IL-10 and TGF- β, or the interaction of Tcons with nTregs in a process termed ‘infectious tolerance’

(JONULEIT et al. 2002). In contrast to their thymus-derived counterparts, iTregs constitute a more heterogeneous group that comprises several subsets, summarised in Table 2. Despite the initial differentiation of naïve T cells into a regulatory Th

subtype, Tregs conserve potential plasticity that allows them to convert into contrasting subsets, such as Th1, Th2 and Th17 cells, in the context of a suitable pro-inflammatory microenvironment (LOCKSLEY 2009; MURAI et al. 2010).

Functionally, Tregs use various mechanisms to mediate their suppressive activity including contact-dependent and -independent pathways (VIGNALI et al. 2008). For example, Treg-derived inhibitory cytokines, such as IL-10, IL-35 and TGF-β, do not only drive peripheral generation of iTregs but also mediate direct modulation of Tcons (ASSEMAN et al. 1999; NAKAMURA et al. 2001; COLLISON et al. 2007; LI et al. 2007). Additionally, the secretion of granzymes – classically done by natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (LIEBERMAN 2003) – can also be performed by Tregs resulting in the lysis of addressed effector cells. Interestingly, murine Tregs deficient in granzyme-B show reduced regulatory activity in vitro (GONDEK et al. 2005). Besides, Tregs are reported to inhibit NK cells and cytotoxic T cells and therefore may indirectly avoid their granzyme B-mediated clearance of tumour cells (CAO et al. 2007). Moreover, Tregs are supposed to induce metabolic disruption, such as absorption of IL-2 via CD25, which leads to cytokine deprivation- mediated apoptosis of effector cells (PANDIYAN et al. 2007) or direct transfer of cyclic adenosine monophosphate (cAMP) – a potent inhibitory second messenger – into effector T cells through gap junctions (BODOR et al. 2012). Furthermore, Tregs can interact with DCs and therefore indirectly modulate the activation of effector T cells (TADOKORO et al. 2006). For example, the interaction of cytotoxic T- lymphocyte antigen (CTLA) 4, expressed on Tregs, with the costimulatory molecules CD80 and CD86 persuades DCs to produce the suppressive molecule indoleamine 2,3-dioxygenase (IDO) (READ et al. 2000; FALLARINO et al. 2003). Tregs additionally induce downregulation of CD80 and CD86 on DCs in vitro (CEDERBOM et al. 2000). Moreover, lymphocyte-activation gene 3 (LAG3 – also called CD223), expressed on Tregs, can bind DCs’ MHC II molecules with very high affinity and thereby inhibits their maturation (LIANG et al. 2008).

2.3.2. Tregs in domestic animals

Since Treg-specific antibodies, such as those against CD25 and Foxp3, are available, the literature concerning regulatory T cells in domestic animals has significantly increased (GARDEN et al. 2011). In cats, the existence of ‘suppressor cells’ was firstly hypothesised in 1982 (LANGWEILER and COCKERELL 1982) and until today, CD4+ CD25+ Foxp3+ Tregs have been characterised in cats with feline immunodeficiency virus infections (JOSHI et al. 2004; VAHLENKAMP et al. 2004;

LANKFORD et al. 2008; MEXAS et al. 2008). Tregs have also been investigated in pigs (KASER et al. 2008a, b; BOLZER et al. 2009) and they were further characterised in studies concerning foetal tolerance (GEORGIEVA 1984), renal and cardiac allotransplantation (MEZRICH et al. 2003; WU et al. 2003; WU et al. 2004) and infection with porcine reproductive and respiratory syndrome virus (SILVA- CAMPA et al. 2009; WONGYANIN et al. 2010). Furthermore, upregulation of Treg- related messenger ribonucleic acid (mRNA) is described in the liver of pigs infected with Ascaris suum (DAWSON et al. 2009). Tregs are also suggested to exist in cattle and most of Foxp3+ bovine T cells express both CD4 and CD25 (SEO et al. 2009).

Additionally, minor populations of CD8β+ and γδ+ Foxp3+ lymphocytes are described (GERNER et al. 2010). However, the regulatory properties of CD4+ CD25+ Foxp3+

bovine lymphocytes are still under discussion, as γδ+ T cells and CD14+ monocytes but not CD4+ CD25+ Foxp3+ cells displayed regulatory functions in one study (HOEK et al. 2009). Although ‘suppressor cells’ have already been reported in sheep in the 1980s (ELLIS and DEMARTINI 1985), increase of ovine Foxp3 has recently been shown within the skin of Psoroptes ovis-infested animals (MCNEILLY et al.

2010). Moreover, Foxp3 expression is described in other species including horses (HAMZA et al. 2012), baboons (PORTER et al. 2007; SINGH et al. 2009), macaques (ALLERS et al. 2010), chimpanzees (MANIGOLD et al. 2006), harbour seals and walrus (SEIBEL et al. 2010), as well as zebrafish (MITRA et al. 2010).

Interestingly, the concept of canine peripheral tolerance was firstly suggested in 1976 as a specific population of T cells seemed to avoid graft-versus-host reaction in dogs (WEIDEN et al. 1976). Approximately three decades later, a subset of canine CD4+

cells was stained with anti-mouse/rat Foxp3 monoclonal antibodies (clone FJK-16s)

(BILLER et al. 2007) and their crossreactivity with canine Foxp3 was subsequently confirmed (MIZUNO et al. 2009). In addition, specific antibodies against canine CD25 allowed the investigation of CD4+ CD25+ Foxp3+ T cells in the canine peripheral blood (MIZUNO et al. 2009; ABRAMS et al. 2010; RISSETTO et al. 2010; PINHEIRO et al. 2011). The regulatory function of these cells has been described in vitro (ABRAMS et al. 2010; PINHEIRO et al. 2011). Interestingly, canine Tregs, assessed by flow cytometry, reveal different intensities in Foxp3 expression and authors discussed Foxp3^high cells to be activated Tregs while Foxp3intermediate cells may represent a more heterogeneous subset of activated Tcons (PINHEIRO et al. 2011).

Thus, there is strong evidence for heterogeneity and plasticity of Tregs in dogs. Due to their crucial role in immune homoeostasis, Tregs have been studied in several canine disorders including neoplasia, atopic dermatitis and adverse food reactions (BILLER et al. 2007; KEPPEL et al. 2008; HORIUCHI et al. 2009; O'NEILL et al.

2009; HORIUCHI et al. 2010; TOMINAGA et al. 2010; VEENHOF et al. 2010;

VEENHOF et al. 2011).

2.3.3. Tregs in the intestine

The gastrointestinal mucosa harbours both nTregs and iTregs including CD103+, Tr1 and Th3 cells (Table 3) (CHEN et al. 1994; GROUX et al. 1997; LEHMANN et al.

2002).

In humans, mutations of FOXP3 are reported to cause IPEX, which mainly comprises inflammation of the intestine, highlighting the importance of Tregs for maintenance of intestinal homoeostasis (BENNETT et al. 2001). Underlining this hypothesis, scurfy mice suffer from a fatal lymphoproliferative disorder similar to IPEX, which is also associated with Foxp3 mutation (BRUNKOW et al. 2001). Interestingly, these animals remain healthy after receiving CD4+ CD25+ Tregs from wild type mice (FONTENOT et al. 2003) and the absence of intact Foxp3 is suggested to cause the scurfy phenotype (LAHL et al. 2007). Moreover, depletion of Foxp3 in mice without scurfy background also results in intestinal inflammatory and autoimmune diseases (VELTKAMP et al. 2006; MOES et al. 2010). Accordingly, active transfer of CD4+

CD25+ Tregs can cure murine colitis (LIU et al. 2003; MOTTET et al. 2003).

Furthermore, Tregs are significantly reduced in the colonic mucosa of Long-Evans Cinnamon rats – deficient in thymocyte development – that spontaneously develop colonic inflammation (ISHIMARU et al. 2008). These studies implicate an essential role of Tregs for intestinal immune regulation.

Physiologically, induction and expansion of Tregs in MLNs followed by their homing to the intestinal LP is thought to be crucial for the maintenance of oral tolerance (HADIS et al. 2011; VAN ESCH et al. 2011). Concerning this, commensal bacteria seem to play an important role in Treg induction in the gut (GEUKING et al. 2011;

LATHROP et al. 2011). Here, the presence of intestinal Tregs mainly depends on interventions of specialised intestinal DCs – primed by local factors including TGF-β and vitamin A metabolites under steady state conditions – that are potent inducers of Foxp3 (COOMBES et al. 2007; SUN et al. 2007).

2.4. Inflammatory bowel disease (IBD)

2.4.1. General remarks

Inflammatory bowel disease (IBD) constitutes a group of chronic emerging inflammatory disorders of the gastrointestinal tract that are described in humans and several domestic animals including dogs, cats and horses (SCHUMACHER et al.

2000; XAVIER and PODOLSKY 2007; HALL and GERMAN 2008). Chronic relapsing or progressing gastrointestinal signs, such as vomiting, diarrhoea, weight loss and abdominal pain, comprise the main clinical features of IBD (JERGENS et al. 1992;

BAUMGART and SANDBORN 2007). Multiple factors are suggested to support disease manifestation (see 2.4.4.) and exclusion of possible differential diagnoses (Table 3) constitutes the pitfalls during diagnostic procedures (BAUMGART and SANDBORN 2007; JERGENS and SIMPSON 2012). Besides clinical investigation – this involves case history, physical examination, haematology, serum biochemistry, ultrasonography, endoscopy, radiography, virology, microbiology and dietary interventions – histopathological examination of gastrointestinal biopsy specimens is

necessary for final diagnosis, as IBD is always associated with mucosal inflammation (BAUMGART and SANDBORN 2007; WASHABAU et al. 2010). Once IBD is diagnosed, therapeutic strategies include alteration of the lifestyle, pharmaceutical management – such as the application of anti-inflammatory drugs – and (in humans) surgical interventions (ABRAHAM and CHO 2009; SIMPSON and JERGENS 2011).

Additionally, alteration of the local cytokine milieu via treatment with anti-tumour necrosis factor (TNF) monoclonal antibodies or the application of IL-10-producing T cells or bacteria comprise novel therapeutic approaches in human patients (BAUMGART and SANDBORN 2007).

Besides naturally occurring IBD in humans and domestic animals, chronic intestinal inflammation – particularly colitis – can be experimentally induced by several mechanisms in laboratory animals, thus allowing investigation of possible underlying pathways of IBD. For example, colitis can be induced by application of chemicals, such as dextrane sulfonic acid or acetic acid, specific antigens, exposure to radiation or genetic modification of mice and rats (HOFFMANN et al. 2002; SOLLID and JOHANSEN 2008).

Table 3. Differential diagnoses for IBD.

IBD = inflammatory bowel disease; spp. = species.

References: BAUMGART et al. 2007, JERGENS et al. 2012.

HU M A N S Infection

Parasitic: Cryptospora spp., Entamoeba histolytica, Isospora spp., Trichuris trichura

Bacterial: Campylobacter spp., Chlamydia trachomatis,

Clostridium difficile, Escherichia coli, Mycobacterium tuberculosis, atypical mycobacteria, Salmonella spp., Shigella spp.,

Yersinia spp.

Viral: Cytomegalovirus, Herpes simplex virus, Human immunodeficiency virus

Fungal: Aspergillus spp., Candida spp.

Non-infectious inflammation Diverticulitis, graft versus host disease, radiation related, sarcoidosis

Intoxication

Antineoplastic chemotherapy, laxatives, non-steroidal anti- inflammatory drugs, postoperative diversion colitis Neoplasia

Adenocarcinoma, lymphoma, neuroendocrine tumours Vascular

Ischaemic colitis, vasculitis DO G S

Infection

Parasitic: Cryptosporidium spp., Giardia spp., Histoplasma spp., Prototheca spp., Toxoplasma spp., Tritrichomonas foetus Bacterial: Campylobacter jejuni, Escherichia coli, Mycobacteria spp., Salmonella spp.

Food allergy

Antibiotic-responsive enteropathy Intestinal lymphangiectasia Neoplasia

Adenocarcinoma, Lymphoma Insufficiency of exocrine pancreas

2.4.2. Human IBD

In humans, idiopathic IBD is reported with variable but increasing prevalence and its onset typically occurs in the second or third decade of life (MOLODECKY et al.

2012). Based on clinical and histopathological features, IBD classically occurs in two major contrasting forms (PODOLSKY 2002). Crohn’s disease – named after the US physician Burril B. Crohn – is commonly located within the terminal jejunum, ileum and colon (CROHN et al. 1984). Histopathologically, it is characterised by initial transmural inflammatory foci dominated by neutrophilic granulocytes followed by granuloma formation (CORNAGGIA et al. 2011). During progression, this form is often complicated by anal fistulae, strictures, obstructions or intestinal perforations (GALANDIUK et al. 2005). In contrast, ulcerative colitis – firstly described by the British physician Sir Samuel Wilks in 1859 - is mainly restricted to the large bowel and characterised by neutrophilic inflammation, ulceration and crypt abscesses (WILKS 1859). In addition to intestinal lesions, extraintestinal disease manifestations – those include arthritis, renal disease, pyrexia, mucocutaneous lesions, hepatobiliary complications and osteopenia – occur in up to 40% of patients with IBD (WILLIAMS et al. 2008). As Crohn’s disease and ulcerative colitis constitute two extremes of a wide spectrum of intestinal inflammatory diseases, both types can be subdivided – for example according to the phenotypic Montreal classification – based on the anatomical location, disease behaviour (concerning intestinal complications), age at diagnosis and severity (SATSANGI et al. 2006). Recently, serological and genetic data were added to develop a more specific reclassification of human IBD (VERMEIRE et al. 2012).

2.4.3. IBD in dogs

In dogs, idiopathic IBD mainly occurs in middle-aged animals but is uncommon in dogs younger than 6 months of age (CRAVEN et al. 2004). Although canine IBD may affect any breed, a predisposition of German shepherd dogs, Basenjis, Soft-coated Wheaten terriers and Shar Pei (GERMAN et al. 2003) as well as Weimaraners, Rottweilers, Border collies and Boxers (KATHRANI et al. 2011c) has been reported.

Histopathologically, mucosal inflammation comprises the main feature of canine IBD that can be further decorated by epithelial damage, increased numbers of IELs, villus stunting, lacteal dilation and loss of mucosal architecture (HALL and GERMAN 2008). As different types are classically distinguished based on their dominating cellular infiltrates, lymphoplasmacytic enteritis (LPE) constitutes the most common form of canine IBD (CRAVEN et al. 2004). Additionally, eosinophilic gastroenteritis (EGE), characterised by a mixed infiltration of inflammatory cells with dominance of eosinophilic granulocytes, is the second most frequently diagnosed type (CRAVEN et al. 2004). Moreover, several variants of LPE and EGE may occur depending on the anatomical site of inflammation – stomach, small intestine or colon.

Furthermore, histiocytic ulcerative colitis (HUC) is a rare granulomatous colitis of Boxers younger than 4 years of age that was firstly described in 1965 (VAN KRUININGEN et al. 1965; CRAVEN et al. 2011). Histopathology of colonic biopsies reveals numerous periodic acid-Schiff (PAS)-positive macrophages and therefore HUC shares some similarities with Whipple’s disease in humans, which is caused by Tropheryma whipplei (AFSHAR et al. 2010). As clinical improvement was obtained by antibiotic treatment, an infectious aetiology was suggested (HOSTUTLER et al.

2004). Subsequently, the presence of Escherichia coli was demonstrated in intestinal biopsies of diseased dogs using immunohistochemistry and fluorescence in situ hybridisation (VAN KRUININGEN et al. 2005). Interestingly, these organisms differ from those causing diarrhoea and they reveal a pathogen-like behaviour in vitro, such as adherence and invasion of epithelial cells (adherent and invasive E. coli, AIEC) and persistence within macrophages (SIMPSON et al. 2006). Furthermore, an aetiological role of Escherichia coli is supported by the fact that clinical remission is associated with antibiotic-related eradication of invasive Escherichia coli (MANSFIELD et al. 2009). In humans, AIEC have been isolated from the ileal and colonic mucosa of patients with Crohn’s disease and these organisms were able to induce similar granulomatous lesions in vitro (DARFEUILLE-MICHAUD et al. 2004;

BAUMGART et al. 2007; MARTINEZ-MEDINA et al. 2009). As HUC is almost exclusively reported in Boxers and rarely occurs in French bulldogs, a genome-wide association scan has been started to identify possible breed-specific genetic defects.

Subsequently, single nucleotide polymorphisms (SNPs) in the gene encoding for the neutrophil cytosolic factor (NCF) 2 were shown to be associated with HUC (CRAVEN et al. 2010; CRAVEN et al. 2011). This may indicate a possible defect in innate immunity, which possibly leads to an altered interaction with intestinal bacteria.

However, it is still under discussion if HUC represents a bacterial colitis rather than a special form of idiopathic IBD.

In contrast to HUC, idiopathic granulomatous inflammation of the intestine characterised by macrophage infiltration and formation of granulomas similar to Crohn’s disease is rarely reported in dogs (BRIGHT et al. 1994; LEWIS 1995).

Furthermore, systemic manifestations are uncommon in canine IBD and only thrombocytopenia is discussed as an extraintestinal alteration in affected dogs (RIDGWAY et al. 2001).

Despite some differences concerning anatomy, histopathology and frequency of complication, canine IBD shares many similarities with its human counterpart including clinical signs, diagnostic procedures, therapeutic strategies and the multi- factorial pathogenesis (see 2.4.4.) (JERGENS and SIMPSON 2012). Hence, canine IBD represents a useful spontaneous animal model to study the pathogenesis of Crohn’s disease and ulcerative colitis in humans.

2.4.4. Pathogenesis of IBD

Although the exact aetiology remains unknown, cumulative effects of multiple factors that lead to intestinal inflammation are suggested to induce IBD in susceptible individuals (Figure 3).

IBD

? ?

E

Intestinal inflammation

Factors Mucosa

Known factors

Unknown factors

Lymphocytes LP

Figure 3. Pathogenesis of IBD.

Several mechanisms may trigger intestinal inflammation (characterised by increased mucosal infiltration and changes of the epithelial border) that is limited by both intrinsic regulatory and inflammatory activity under physiological conditions.

Simultaneous accumulation of multiple factors may lead to an overload and breakdown of regulatory capacities that can enhance exacerbated inflammation progressing to IBD. Suggested factors include but may not be limited to genetic mutations, barrier dysfunction, environmental influences, intestinal dysbiosis, infections, immunologic hyperactivity or defects in immunoregulation and loss of oral tolerance.

E = enterocyte; IBD = inflammatory bowel disease; LP = lamina propria.

In humans, variants of several genes including ‘nucleotide-binding oligomerization domain-containing protein 2’ (NOD2), ‘autophagy-related protein 16-1’ (ATG16L1),

‘interleukin 23 receptor’ (IL-23R) and ‘signal transducer and activator of transcription 3’ (STAT3), which are important for both innate and adaptive immune functions, have been shown to be highly associated with IBD (CHO 2008). Similar to this, genetic susceptibility is also suggested for the canine counterpart as there are predispositions of several breeds, as mentioned above (GERMAN et al. 2003;

KATHRANI et al. 2011c). Recently, molecular analyses have shown polymorphisms of genes encoding for TLR4 and TLR5 to be associated with canine IBD (KATHRANI et al. 2010). Physiologically, TLRs – for example expressed by epithelial cells – recognise specific microbial structures also known as PAMPs (LOTZ et al. 2007).

TLRs are further suggested to differentiate between pathogenic and commensal bacteria – although both may share common PAMPs, such as lipopolysaccharide (LPS) – and TLR activation by the physiological microflora seems to be crucial for intestinal homoeostasis (RAKOFF-NAHOUM et al. 2004; KAMDAR et al. 2013). In German shepherd dogs, a specific haplotype of the TLR5 SNP ‘G22A’ is associated with increased IBD risk, whereas the TLR5 SNPs ‘C100T’ and ‘T1844C’ seem to be protective for IBD in various breeds (KATHRANI et al. 2010; KATHRANI et al.

2011a). In addition, the risk-associated TLR5 haplotype reveals hyper- responsiveness to flagellin in vitro, which is the major component of bacterial flagella (KATHRANI et al. 2012). Despite of genetic defects, the mRNA levels of TLR2, TLR4 and TLR9 are significantly increased in the mucosa of dogs suffering from IBD, while TLR5 mRNA is downregulated in German shepherd dogs compared with Greyhound controls (BURGENER et al. 2008; ALLENSPACH et al. 2010).

Before they get into contact with surface receptors, such as TLRs, large amounts of luminal antigens are excluded from the host’s immune system by mucosal S-IgA (PABST 2012). The role for intestinal homoeostasis is strongly supported by the alteration of intestinal microbiota in IgA-deficient mice and the concentrations of mucosal IgA are significantly decreased in human patients with IBD (ROGNUM et al.

1982; MACDERMOTT et al. 1989; SUZUKI et al. 2004). As already known, German shepherd dogs are deficient in IgA production (GERMAN et al. 2000; LITTLER et al.

2006). Additionally, a recent study demonstrated IgA to be reduced in the intestinal lumen, mucosa and peripheral blood of dogs with IBD (MAEDA et al. 2013).

Moreover, this study showed decreased mRNA levels of TGF-β in duodenal samples suggesting a deficiency in intestinal immune regulation (MAEDA et al. 2013). This is further supported by another study showing reduced mRNA of TGF-β and IL-10 in canine IBD (JERGENS et al. 2009). Similarly, depletion of Tregs results in chronic colitis in immunodeficient mice (VELTKAMP et al. 2006). In spite of the reduction of regulatory cytokines, several studies failed to detect a specific Th profile in canine IBD on the mRNA level (JERGENS et al. 2009; SCHMITZ et al. 2012). Although human IBD is dominated by a Th1/Th17 (Crohn’s disease) or Th2 pattern (ulcerative colitis), diseased dogs reveal a mixed Th profile composed of both Th1 and Th2 related cytokines and lack clear evidence for Th17 involvement (JERGENS et al.

2009; STROBER and FUSS 2011; SCHMITZ et al. 2012).

Despite adequate host-related defence mechanisms, commensal microbiota play a pivotal role in conditioning the mucosal immune system and maintaining intestinal homoeostasis (LATHROP et al. 2011). In humans, disturbances of the intestinal microflora are associated with IBD. Affected individuals reveal lower bacterial diversity as well as quantitative changes of bacterial phyla, such as decreased numbers of Fimbricutes and Bacteroides but higher amounts of Proteobacteria (REHMAN et al. 2010; KAUR et al. 2011). Similarly, investigations of the intestinal bacterial contents of dogs suffering from IBD showed dysbiotic changes in diseased animals. These include lower species diversity as well as decreased amounts of Fusobacteria, Bacteroidaceae, Prevotellaceae and Clostridiales, while there is higher abundance of Proteobacteria including Diaphorobacter and Acinetobacter (XENOULIS et al. 2008; SUCHODOLSKI et al. 2010; SUCHODOLSKI et al. 2012).

Additionally, AIECs seem to be associated with the development of HUC in Boxers, as mentioned above (2.4.3.). Although disturbances of the intestinal microflora are highly suggested to be involved in the pathogenesis of both human and canine IBD, it remains to be elucidated whether these changes are the cause or a consequence of the disease.

In summary, breakdown of intestinal homoeostasis including a loss of oral tolerance, aberrant immune activity and changes in commensal bacteria seem to be crucial for the development and progression of both human and canine IBD in susceptible individuals (MALOY and POWRIE 2011; JERGENS and SIMPSON 2012).

3. Canine gut dendritic cells in the steady state and in inflammatory bowel disease

JUNGINGER, J., LEMENSIECK, F., MOORE, P. F., SCHWITTLICK, U., NOLTE, I., HEWICKER-TRAUTWEIN, M.

Abstract

Alongside the intestinal border, dendritic cells (DCs) sample large amounts of endogenous and potentially pathogenic antigens followed by initiation of protective immune responses or induction of tolerance. Breakdown of oral tolerance towards commensal bacteria is suggested to be crucial for the development of both human and canine inflammatory bowel disease (IBD). The aim of this study was to investigate canine intestinal DCs in the steady state and in dogs with IBD using multicolour immunofluorescence. In the healthy gut, DC-like cells expressed MHC II, CD1a8.2, CD11c and in lower amounts CD11b within lamina propria, Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs), whereas those expressing CD80 and CD86 were only present in PPs and MLNs. Occasionally, DC-like cells were in contact with the intestinal lumen through transepithelial projections. In canine IBD, CD1a8.2+, CD11b+ and CD11c+ DC-like cells were decreased within the stomach, duodenum and colon, whereas the colonic mucosa revealed elevation of CD86+ DC-like cells. The complex network of DC-like cells in the gut indicates their important role in canine mucosal immunity including active sampling of luminal antigens. Furthermore, their shift in diseased dogs suggests a pathogenetic significance for canine IBD.

Innate Immunity 2013, Epub ahead of print.

DOI: 10.1177/1753425913485475