Immunomodulation of porcine leukocytes and dendritic cells ...and as most important one, dectin-1 (Battle et al., 1998; Brown and Gordon, 2001). Dectin-1 is a microbial sensor or pattern

Vakgroep Virologie, Parasitologie en Immunologie Laboratorium voor Immunologie

Immunomodulation of porcine leukocytes and dendritic cells by

-(1,3) glucans

Eva Sonck

Promotoren Prof. Dr. E. Cox

Prof. Dr. B.M. Goddeeris

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Diergeneeskundige Wetenschappen – Universiteit Gent, 2011

Table of contents

3

Table of contents

List of abbreviations ........................................................................................................................... 6

Introduction ....................................................................................................................................... 8

Part I Review of the literature ..................................................................................................... 12

Chapter 1 : -glucan receptors ...................................................................................................... 13

1.1. Dectin-1 ................................................................................................................................... 13

1.2. Other -glucan receptors ........................................................................................................ 21

1.3. Collaboration between dectin-1 and other innate immune receptors ................................... 23

Chapter 2 : -glucans .................................................................................................................... 25

2.1. Modulation of the immune system by -glucans.................................................................... 26

2.2. Defining factors of the immunomodulatory character of -glucans ...................................... 29

2.3. Possible mechanisms of -glucan uptake after oral administration ....................................... 36

Chapter 3 : Use of -glucans for oral immunization ....................................................................... 39

3.1. Challenges of effective oral immunization .............................................................................. 39

3.2. Targeting to pattern recognition receptors (PRRs) ................................................................. 40

References ................................................................................................................................... 46

Part II Aims of the study ............................................................................................................... 62

Aims of the study .......................................................................................................................... 63

Part III Experimental studies ......................................................................................................... 64

Chapter 4 : Identification of the Porcine C-Type Lectin dectin-1 ...................................................... 65

4.1. Abstract ................................................................................................................................... 66

4.2. Technical report....................................................................................................................... 66

4.3. Acknowledgements ................................................................................................................. 72

4.4. References ............................................................................................................................... 72

Chapter 5 : The effect of β-glucans on porcine leukocytes .............................................................. 74

5.1. Abstract ................................................................................................................................... 75

5.2. Introduction ............................................................................................................................. 75

5.3. Materials and Methods ........................................................................................................... 76

5.4. Results ..................................................................................................................................... 81

5.5. Discussion ................................................................................................................................ 88

5.6. Acknowledgements ................................................................................................................. 91

5.7. References ............................................................................................................................... 91

Table of contents

4

Chapter 6 : The specific dectin-1 inhibitor laminarin doesn’t block the -glucan induced effects in porcine leukocytes ........................................................................................................................ 94

6.1. Abstract ................................................................................................................................... 95

6.2. Introduction ............................................................................................................................. 95

6.3. Materials and Methods ........................................................................................................... 96

6.4. Results and discussion ............................................................................................................. 98

6.5. Acknowledgements ............................................................................................................... 104

6.6. References ............................................................................................................................. 105

Chapter 7 : Varying effect of different β-glucans on the maturation of porcine monocyte-derived dendritic cells ............................................................................................................................. 107

7.1. Abstract ................................................................................................................................. 108

7.2. Introduction ........................................................................................................................... 108

7.3. Materials and Methods ......................................................................................................... 110

7.4. Results ................................................................................................................................... 112

7.5. Discussion .............................................................................................................................. 118


7.7. References ............................................................................................................................. 121

Chapter 8 : β-glucan rich Yeast shells as potential carriers for antigen targeting to dendritic cells 125

8.1. Abstract ................................................................................................................................. 126

8.2. Introduction ........................................................................................................................... 126

8.3. Materials and Methods ......................................................................................................... 128

8.4. Results ................................................................................................................................... 131

8.5. Discussion .............................................................................................................................. 134


8.7. References ............................................................................................................................. 136

Chapter 9 : General discussion and future perspectives ............................................................... 140

9.1. Introduction ........................................................................................................................... 141

9.2. The expression pattern of porcine dectin-1 ......................................................................... 141

9.3. different immunostimulating effects on leukocytes ............................................................. 142

9.4. Does dectin-1 play a role in the -glucan induced activity of leukocytes? ........................... 145

9.5. The effect of different -glucan preparations on DC maturation ......................................... 147

9.6. Are -glucans potential candidates for DC targeting? .......................................................... 151

9.7. Main conclusions and future perspectives ............................................................................ 152

9.8. References ............................................................................................................................. 154

Table of contents

5

Summary ....................................................................................................................................... 159

Samenvatting ................................................................................................................................. 163

Curriculum Vitae ............................................................................................................................ 168

Publications ................................................................................................................................... 169

Abstracts and posters ..................................................................................................................... 170

Dankwoord .................................................................................................................................... 171

List of abbreviations

6


APC antigen presenting cell BMDC bone marrow-derived dendritic cells CARD caspase recruit domain CD cluster of differentiation CLR C-type lectin receptor CR3 complement receptor 3 CRD carbohydrate recognition domain CTL cytotoxic T-lymphocyte CTLD C-type lectin-like domains DAP DNA activating protein DC dendritic cell ERK extracellular signal regulated kinase ETEC enterotoxigenic Escherichia coli FOS fructooligosaccharides GALT gut-associated lymphoid tissue GM-CSF granulocyte macrophage colony-stimulating factor GOS galactooligosaccharides GTPase guanosine triphosphatase HPAEC high performance anionic exchange chromatography ICAM intercellular adhesion molecule-1 IFN interferon

IKK inhibitor of B kinase IL interleukin ImDC immature dendritic cell ITAM immunoreceptor tyrosine-based activation motif LacCer lactosylceramide LPS lipopolysaccharide mAb monoclonal antibody MAC macrophage antigen MAMP microbe-associated molecular pattern MAP mitogen-activated protein M-cells microfold cells MHC major histocompatibility complex MIP macrophage-inflammatory protein MLN mesenteric lymph nodes MLR mixed lymphocyte reaction MoDC monocyte- derived dendritic cell Mw molecular weight NFAT nuclear factor of activated T-cells

NF-B nuclear factor kappa B NK cell natural killer cell PAMP pathogen-associated molecular pattern PBMC pheripheral blood monomorphonuclear cell


7

PEI polyethylenimine PKC protein kinase C

PLC2 phospholipase-C2 PRR pattern recognition receptor PP peyer’s patches ppm parts per million Ran-BP Ran- binding protein ROS reactive oxygen species SCFA short-chain fatty acids SH2 Src-homology 2 siRNA small interfering RNA SPR surface plasmon resonance Syk spleen tyrosine kinase Th T helper cell TGF transforming growth factor TNF tumor necrosis factor TLR Toll-like receptor Treg regulatory T-cell tRNA transfer RNA

Introduction

8

Introduction

At weaning, the young pig is often faced with major challenges including separation from their mother

and littermates, a new environment and the change from highly-digestible liquid food (milk) to a more

complex and less-digestible solid diet (Lalles et al., 2007). Feed transition and stress can cause

gastrointestinal disturbances making newly weaned pigs highly susceptible to enteric diseases (Pluske et

al., 2002). Therefore, weaning is often associated with reduced feed intake, body weight loss and

diarrhea, and causes large economical losses in the pig industry. One of the most abundant cause of

postweaning diarrhea in young pigs are enterotoxigenic Escherichia coli (ETEC) infections. Porcine ETEC

strains isolated from diarrheic pigs express 5 different fimbriae of which F4 and/or F18 fimbriae

producing strains are causing postweaning diarrhea. F4 fimbriae are the most prevalent and best

characterized (Fairbrother et al., 2005). F4 ETEC bacteria adheres via F4 fimbriae to specific receptors

expressed by the intestinal epithelium, enabling colonization of the intestine and the production of

enterotoxins leading to the induction of severe diarrhea (Van den Broeck et al., 1999b). Subtherapeutic

use of antibiotics has widely been applied to nursery pigs to solve post-weaning problems (Barton,

2000). However, their long term and extensive use has resulted in the development of antibiotic

resistance. Therefore, all commonly-used antimicrobial growth promoters have been banned in the EU

member states (Khaksefidi and Rahimi, 2005; Montagne et al., 2003). A wide variety of acceptable

alternatives to antibiotics have been suggested including substances which enhance the natural defense

mechanisms (Verstegen and Williams, 2002). Such immunomodulators act completely different from

antibiotics. For some antibiotics, it has been suggested that they improve the efficiency of animal growth

through the inhibition of certain bacteria of the intestinal microflora and of enteropathogens, leading to

a reduction in the energy used by the gastro-intestinal immune system (Gaskins et al., 2002). The aim of

immunomodulating substances is to help the individual to develop ‘appropriate’ responses of both

innate and acquired immunity (Gallois et al., 2009). Substances well known for their immunomodulating

effect are non-digestible carbohydrates. The effects of prebiotic carbohydrates are well studied, focusing

mainly on the effect of fructo- (FOS) and galactooligosaccharides (GOS) (Vos et al., 2007). Prebiotic

agents affect the prevalence and/or activity of one or a limited number of microbiota in the colon, and

the indirect immunomodulatory effects of these carbohydrates are often studied under the assumption

that the prebiotic effect is causative for the observed immunomodulation (Gibson and Roberfroid, 1995).

In addition, prebiotics might also affect the host and/or the microbiota on the basis of their

carbohydrate structure by activating or blocking cellular receptors (Vos et al., 2007).

Introduction

9

In contrast with prebiotics, -glucans are widely studied non-digestible carbohydrates that induce direct

receptor-mediated, immunomodulatory effects . In human and mice, multiple -glucan binding receptors

have been described, including complement receptor 3 (CR3), lactosylceramide, scavenger receptors,

and as most important one, dectin-1 (Battle et al., 1998; Brown and Gordon, 2001). Dectin-1 is a

microbial sensor or pattern recognition receptor (PRR) and is expressed on different immune cells

throughout the gastrointestinal tract. -glucan structures, which are found in the cell wall of different

microorganisms, are referred to as pathogen-associated molecular patterns (PAMPs), although they are

not unique to microbes that can cause disease. -glucans are recognized by -glucan receptors on innate

immune cells including neutrophils, monocytes, macrophages and dendritic cells (DCs) (Goodridge et al.,

2009b). These cells play an important role in oxidative burst and phagocytosis, and through cytokine

production and antigen presentation, they in turn activate the adaptive immune system. Cytokines are

used for inter-cell communication and the produced cocktail of cytokines promotes the differentiation of

T helper (Th) cells and ultimately the outcome of an immune response. An appropriate response is

crucial for the well functioning of the intestine. In the newly weaned pig, an ideal immunomodulating

agent needs to help to induce an active immune response to pathogens, as well as give the ability to

generate tolerance to food antigens or commensal bacteria (Bailey et al., 2001). In this respect,

understanding the nature of adaptive immune responses induced by -glucans is necessary in order to

use their powerful modulating properties in newly weaned pigs. Moreover, as recently weaned pigs are

highly susceptible to enteric pathogens, it would be interesting to use their immunomodulating potential

in oral immunization against a specific pathogen or disease. In addition, through the expression of many

PRRs including dectin-1, together with their capacity to link innate and adaptive immunity, DCs play a

pivotal role in immunomodulation, making them an attractive target for oral immunization (Caminschi et

al., 2009). However, oral vaccine delivery has been challenging and progress in this area has been quite

slow because of the numerous potential obstacles it is associated with, including proteolytic degradation

in the gastrointestinal system, poor absorption of large molecules and oral tolerance (Mishra et al., 2010;

Poonam, 2007). Many approaches for mucosal delivery have been proposed and biodegradable

polymeric carriers are one of the most promising (Mishra et al., 2010). By encapsulating the antigen in

polymeric matrices, biodegradable carrier systems can enhance the mucosal immune system in several

ways. Polymer-based approaches are designed to protect the antigen in the gut, to target the antigen to

the gut-associated lymphoid tissue or to prolong the residence time of the antigen in the intestine

through bioadhesion. Besides the advantage of delivering the antigens to a specific target site, where

they are released from the carrier, some polymers can also act as adjuvant by itself (Mishra et al., 2010;

Introduction

10

Payne et al., 1995). As particularly recently weaned pigs are highly susceptible to enteric pathogens, it

would be useful to investigate if -glucans are powerful candidates as antigen delivery carrier for oral

immunization.

Chapter one and two will provide background information on -glucan receptors and the main

properties of -glucans, while the third chapter is focused on the potential role of -glucans in oral

immunization. Finally, the aims and the experimental approach are explained.

11

Part I : Review of the literature

12

Part I Review of the literature

Chapter 1 : β-glucan receptors

13

Chapter 1 : -glucan receptors

Different -glucan receptors have been described in human and mouse. In the following overview, each

-glucan receptor will be discussed, focusing particularly on dectin-1, since this receptor has been

reported in recent years as the major -glucan receptor.

1.1. Dectin-1 Dectin-1 was initially described as a dendritic cell (DC)-specific glycoprotein receptor of about 43 kDa by

employing the subtractive cDNA cloning strategy on the XS52 DC line (Ariizumi et al., 2000). With respect

to its function, dectin-1 was identified as an endogenous, costimulatory ligand of T-cells involved in

stimulation of T-cell proliferation (Ariizumi et al., 2000). Later it emerged as a -glucan receptor by

screening a murine macrophage cDNA expression library with the -glucan-rich particle zymosan (Brown

and Gordon, 2001).

1.1.1. Structure Dectin-1 belongs to the family of the C-type lectin receptors (CLRs). The CLRs are a large superfamily of

proteins that possess one or more structurally related C-type lectin-like domains (CTLDs). Based on the

phylogenetic relationships between CTLDs, dectin-1 is classified into group V or the common group of

the ‘type II NK cell receptors’ (Drickamer, 1999; Zelensky and Gready, 2005). The NK cell receptor-like C-

type lectins are traditionally associated with the control of cellular cytotoxicity, through recognition of

MHC class I molecules and other protein ligands. They consist of several families of related molecules,

which are mostly encoded by a single genomic region, known as the NK complex (Yokoyama et al., 1991).

The extracellular part of this type of receptors is characterized by one single C-terminal carbohydrate

recognition domain (CRD) and a variable length-neck region (Figure 1.1). The N-terminal cytoplasmic tail

also varies in length and commonly contains internalization motifs or binding sites for cytosolic

proteins. Dectin-1 has a similar structure to the other members of the NK cell receptor-like C-type lectin

family, with some exceptions. First, the receptor lacks cysteine residues in its stalk region (Ariizumi et al.,

2000), which are required for dimerization, explaining why the receptor appears to be expressed as a

monomer (Brown, 2006). Second, dectin-1 contains an immunoreceptor tyrosine-based activation motif

(ITAM)-like motif (Figure 1.1) and actually works as an activating receptor in the absence of any adaptor

molecule. By contrast, all other known activating NK-cell-receptor like C-type lectins associate with


14

signaling molecules to carry out cellular activation. The structure of its ITAM is also unique. Compared

with the consensus sequence containing two repeats of YxxL/I (tyrosine, 2 amino acid residues, leucine

or isoleucine) (van Vliet et al., 2005), three amino acid residues are inserted between the first tyrosine

and the following leucine or isoleucine (YxxxL/I) of the tandem repeat. Furthermore, dectin-1 is unusual

in that the CRD of this molecule lacks the highly conserved Glu-Pro-Asn (EPN) and Gln-Pro-Asp (QPD)

sequences involved in calcium coordination and essential for recognizing mannose- and galactose-

containing ligands in classic Ca2+-dependent C-type lectins (Brown and Gordon, 2001; Drickamer, 1992).

Nevertheless, although other NK-cell-receptor-like C-type lectins normally recognize proteinaceous

ligands, dectin-1 is able to recognize carbohydrates in a Ca2+ independent manner (Ariizumi et al., 2000).

Although the precise mechanism of binding is still unclear, mutagenic analysis has indicated that in the

CRD of dectin-1, at least two residues, Trp221 and His223, are crucial in the formation of the dectin-1 -

glucan binding site (Adachi et al., 2004; Brown et al., 2007). Both amino acids are exposed on the dectin-

1 surface, where they form a shallow groove on the protein surface (Brown et al., 2007). There are

indications that -glucan binding is driven mainly by hydrophobic forces, including stacking with the Trp

and His side chains, although electrostatic interactions would be possible with the nitrogen atoms in

these side chains (Brown et al., 2007).

Figure 1.1 Schematic representation of the structure of murine dectin-1. Dectin-1 consists of an extra-cellular region, which includes the C-type lectin-like carbohydrate recognition domain (CRD) and the stalk region, a transmembrane domain and an ITAM, motif which mediates intracellular signaling (adapted from Brown et al., 2006).


15

1.1.2. Splice variants The full-length dectin-1 gene, CLEC7A, like all other closely related C-type lectin-like receptors in the NK

gene complex, is encoded by six exons (Ariizumi et al., 2000). Mouse dectin-1 is alternatively spliced,

generating at least two major isoforms by the different usage of exon 3. The first isoform is the full-

length dectin-1A, containing a CRD domain, a short stalk region, a transmembrane region and an ITAM

motif while the second dectin-1B isoform lacks a stalk region between the CRD and transmembrane

region and results from omission of exon 3 (Heinsbroek et al., 2006) (Figure 1.2). Comparative studies of

both isoforms in various mouse strains show that the strains either express both transcripts equally or

express predominantly dectin-1B. Human dectin-1 differs from its mouse counterpart in that it is

alternatively spliced in two major and also six minor isoforms (Grunebach et al., 2002; Willment et al.,

2001; Yokota et al., 2001). Only the two major isoforms of both human and mouse are able to bind -

glucans (Heinsbroek et al., 2006; Willment et al., 2001). As these two -glucan binding isoforms are

expressed differently in various cell types, it suggests that the alternative splicing of these two isoforms

can be regulated and that regulation of isoform usage represents a mechanism of control that is

important for the function of dectin-1 in the immune system (Willment et al., 2005). The six other

alternatively spliced human dectin-1 variants consist of variations of the primary isoforms, some with

deletions in the CRD-encoding region (isoforms C and D) and the transmembrane and stalk region

(isoforms E and F), while two isoforms (G and H) possess small insertions. In all the minor isoforms,

except for E, frameshifts would generate premature stop codons and therefore truncated proteins

(Willment et al., 2001). None of these other splice variants has been shown to bind -glucans, although

they may have a regulatory role (Willment et al., 2001). Among the six minor isoforms, isoform E

(hDectin-1E) is structurally unique, containing a complete C-type lectin-like domain as well as an ITAM-

like sequence (Willment et al., 2001). It is retained in the cytoplasm where it associates with a Ran-

binding protein, RanBP. RanBP is a cytosolic endogenous ligand that can interact with the GTPase Ran

and may act as a scaffolding protein to coordinate signaling from cell surface receptors (Xie et al., 2006).

The major difference among the functional isoforms of murine and human dectin-1 is the number and

position of the N-linked glycosylation sites. Both murine isoforms have two N-linked glycosylation sites in

their CRD (Ariizumi et al., 2000). In contrast, human isoform A has only one N-linked glycosylation site in

its stalk region, while the stalkless isoform B has no glycosylation site (Yokota et al., 2001) (Figure 1.2).


16

Figure 1.2 N-glycosylation profile of mouse and human dectin-1 A and B isoforms. All have an extracellular

-glucan binding domain and intracellular ITAM-motif, but the position and number of the N-linked glycosylation sites are different (adapted from Kato et al., 2006).

1.1.3. Dectin-1 expression Dectin-1 was originally thought to be a DC-specific receptor, from which its name ‘dendritic-cell-

associated C-type lectin-1’ was derived (Ariizumi et al., 2000), but the receptor is now known to be

expressed by many other cell types. At portals of pathogen entry, such as the lung (Taylor et al., 2002)

and intestine (Reid et al., 2004), dectin-1 is expressed at high levels, which is consistent with a potential

role for this receptor in immune surveillance.

As such, mouse dectin-1 mRNA is present in immune-rich tissues, such as the thymus, spleen, lung, small

intestine and kidney, and in other organs such as the liver and the stomach (Ariizumi et al., 2000; Taylor

et al., 2002). Dectin-1 expression is found at high levels on CD11clow and CR3+ splenocytes and

macrophage subpopulations of splenic red and white pulp (Carter et al., 2006; Reid et al., 2004; Taylor et

al., 2002). Furthermore, dectin-1 is also expressed on all conventional DC subsets in the spleen and the

lymph nodes (Leibundgut-Landmann et al., 2008). In the thymus, dectin-1 is expressed on

subpopulations of macrophages and DCs in the medullary and corticomedullary regions (Reid et al.,

2004). Peripheral blood neutrophils and monocytes, alveolar macrophages, macrophages and DCs of the

lamina propria and Kupffer cells also express dectin-1 (Reid et al., 2004; Taylor et al., 2002). Resident

peritoneal macrophages express relatively lower amounts of mouse dectin-1 and the receptor has not


17

been detected on eosinophils, macrophages or DCs in the kidney, heart, brain or eye (Meyer-Wentrup et

al., 2007; Reid et al., 2004; Taylor et al., 2002).

In humans, dectin-1 mRNA has also been detected in lymphoid-rich tissues such as spleen, bone marrow

and thymus. Furthermore, dectin-1 has been reported in monocytes, macrophages, neutrophils and

mast cells (Olynych et al., 2006; Willment et al., 2001; Yokota et al., 2001). Different from mice, the

receptor is also expressed by eosinophils, B-cells and some subsets of CD4+ T-cells (Willment et al.,

2005); the significance of this species difference is unknown. Dectin-1 is also present in immature and

mature monocyte-derived DCs, peripheral blood DCs and plasmacytoid DCs. Downregulation of surface

dectin-1 protein, but not its mRNA transcripts, has been observed on mature monocyte-derived DCs

relative to immature monocyte-derived DCs (Meyer-Wentrup et al., 2007; Willment et al., 2005).

Besides human and mice, two major dectin-1 isoforms have also been identified in rat, cow and sheep

(Kato et al., 2008; Willcocks et al., 2006; Zhou et al., 2010).

The levels of dectin-1 expression can be significantly modulated by cellular maturation, cytokines and

other biological response modifiers, including steroids and -glucans (Ozment-Skelton et al., 2006;

Willment et al., 2003; Willment et al., 2005). As such, cytokines that skew the immune response toward

a Th2 profile, such as IL-4 and IL-13, upregulate the expression of mouse dectin-1 on resident

macrophages. Together with IL-4, also GM-CSF induces upregulation of the cell surface expression of

dectin-1 (Willment et al., 2003). This is important as both cytokines are required for in vitro

differentiation of immature human monocyte-derived DCs. Furthermore, IFN-, a Th1-cytokine, and

transforming growth factor- had little direct effect on the expression of dectin-1, while the anti-

inflammatory cytokine IL-10 downregulates surface expression (Willment et al., 2003). Biological

response modifiers, such as LPS, dexamethasone and -glucans, downregulate surface expression on

macrophages and peripheral leukocytes (Ozment-Skelton et al., 2006; Willment et al., 2003). Regulation

of human dectin-1 mRNA expression in monocyte- derived DCs has also been reported. Dectin-1B mRNA

increases with time in culture, whereas dectin-1A mRNA remains consistently low (Weck et al., 2008).

1.1.4. Dectin-1 signaling Dectin-1 is the first pattern recognition receptor (PRR) outside of the TLR family that can mediate its own

intracellular signals (Brown, 2006). Signaling from this receptor following ligand binding is mediated

through the cytoplasmic ITAM-like motif, that resembles the tandem repeat sequences found in other

activation molecules, such as DAP12, Fc receptors and lymphocyte antigen receptor complexes (Lanier et

al., 1998; Pitcher and van Oers, 2003; Van den Herik-Oudijk et al., 1995). It is reported that only


18

phosphorylation of the membrane-proximal YxxxL/I within the ITAM-like motif of dectin-1 is required for

syk signaling (Fuller et al., 2007; Rogers et al., 2005). This single tyrosine based motif is now known as a

ITAM-like motif or a hemITAM (Marakalala et al., 2010). However, in traditional ITAM sequences, the

tandem repeat of YxxL/I sequences (see 1.1.1 structure) become both phosphorylated by Src-homology 2

(SH2) domain-containing spleen tyrosine kinase (syk). Taking into account that ITAMs usually occur in

clusters, it is proposed that a dimer of dectin-1 may be required for inducing the signaling (Figure 1.3)

(Brown, 2006; Brown and Gordon, 2005; Fuller et al., 2007; Gordon, 2002; Rogers et al., 2005). Syk

further activates phospholipase-C2 (PLC2), leading to the engagement of the caspase recruit domain

(CARD)-containing protein, CARD9, which together with Bcl10 and MALT1, signal for activation of NF-B.

NF-B is a master transcription factor, regulating the expression of many inflammatory genesAlthough

the signaling events that link PLC2 activation to CARD9 recruitment are still not known, CARD9 has been

identified as an essential downstream adaptor linking Syk-coupled receptors to the classical or canonical

pathway. This pathway is initiated by activation of an IB kinase (IKK) complex which catalyses the

phosphorylation of IBs (inhibitor of B) resulting in their ubiquitylation and subsequent degradation.

The released NF-Bdimers translocate to the nucleus and activate gene transcription. In addition to

activating NF-Bvia the classical pathway,dectin-1 is the first PRR which can also induce the non-

canonical NF-Bpathway (Gringhuis et al., 2009). This pathway identifies the serine-threonine kinase

Raf-1 as a novel component of a syk-independent signaling pathway (Gringhuis et al., 2009). Although

this pathway is independent of the syk pathway, it integrates with it at the level of NF-Bactivation for

regulation of cytokine production (Gringhuis et al., 2009).

There is also evidence of syk-dependent, but CARD9-independent pathways. For example, in DCs, there

is a syk-dependent activation of extracellular signal regulated kinase (ERK), a mitogen-activated protein

(MAP) kinase, through a CARD9 independent pathway (Slack et al., 2007). Both the syk and Raf-1

pathways are important for driving dectin-1 mediated adaptive responses (Gringhuis et al., 2009;

Leibundgut-Landmann et al., 2008). Syk is involved in inducing most of the dectin-1 mediated cellular

responses, including cytokine production and the respiratory burst (LeibundGut-Landmann et al., 2007;

Rogers et al., 2005; Underhill et al., 2005). Apart from activation of NF-Bin DCs, ligation of dectin-1 also

triggers Nuclear Factor of Activated T-cells (NFAT) activation in macrophages and DCs (Goodridge et al.,

2007; Xu et al., 2009). Dectin-1 mediated activation of NFAT regulates cyclooxygenase-2 and

prostaglandin production in macrophages, and the induction of IL-2, IL-10 and IL-23 in DCs (Goodridge et

al., 2007). The differential signaling way of dectin-1 is depending on the cell type, state of activation and

nature of the downstream response. For example, phagocytosis is independent of syk and CARD9 in


19

macrophages (Goodridge et al., 2009a; Gow et al., 2007; Herre et al., 2004b; Underhill et al., 2005).

CARD9 is also not required for dectin-1 mediated phagocytosis by some DC types, but is necessary for

the production of TNF- in resident peritoneal and alveolar macrophages and primed DCs.

Post-translational modifications, such as glycosylation, have important roles in stabilizing glycoproteins,

molecular folding and intracellular trafficking (Helenius and Aebi, 2001, 2004). It was demonstrated that

N-linked glycosylation modulates cell surface expression of dectin-1. As such, human dectin-1B, which

undergoes no glycosylation, showed reduced cell surface expression and consequently reduced binding

to -glucan compared with human dectin-1A and both glycosylated dectin-1 isoforms of mice (Kato et

al., 2006).

Figure 1.3 Signaling pathways induced by dectin-1. Upon ligand binding, dectin-1 becomes tyrosine-phosphorylated by Src kinases, thereby providing a docking site for syk which initiates downstream signaling. It is proposed that syk bridges two receptor molecules. The downstream signaling is effected by molecules such as

CARD9, Bcl10, and MALT1, which lead to NF-B activation and cytokine production. Dectin-1 can also activate NFAT

and non-canonical NF-B in a CARD9-independent manner. Stimulation of dectin-1 with -glucans can also induce a second Syk-independent signaling pathway mediated by the serine-threonine kinase Raf-1 (adapted from Marakalala et al., 2010).


20

1.1.5. Ligands of dectin-1 Dectin-1 is the first example of an NK cell receptor-like C-type lectin whose main ligand is a

polysaccharide. Dectin-1 specifically recognizes soluble and particulate -1,3- and/or -1,6-linked glucans

(Brown and Gordon, 2001). The primary role of -glucan in primates appears to be the initiation of

immune responses for the control of pathogens. Through its recognition of cell-wall -glucan, dectin-1

binds several fungal species, including Candida (Brown and Gordon, 2003; Gantner et al., 2005),

Aspergillus (Steele et al., 2005), Coccidioides (Viriyakosol et al., 2005), Pneumocystis (Steele et al., 2003)

and Saccharomyces (Brown and Gordon, 2003). However, -glucan exposure at the surface also varies

with fungal morphotype, and consequently different forms of the same fungus can induce different

responses (Goodridge et al., 2009b). The ability to switch between different forms is a key virulence

strategy, permitting the fungi to evade host immune responses (Calderone and Fonzi, 2001; Lo et al.,

1997; Saville et al., 2003). For example, in Candida albicans, -glucan is exposed at discrete patches on

the surface, corresponding to bud and birth scars, areas of altered cell wall structure resulting from

separation of mother and daughter yeast cells. During filamentous growth however, the core -glucan

layer is obscured by the outer layer (mostly mannan) (Gantner et al., 2005; Goodridge et al., 2009b).

Dectin-1 has also recently been implicated in the interaction with a number of mycobacteria, which do

not express -glucans, suggesting that there are other, so far unidentified, exogenous ligands for this

receptor (Lee et al., 2009; Rothfuchs et al., 2007; Shin et al., 2008; Yadav and Schorey, 2006).

Although best known for its ability to recognize-glucans, dectin-1 was originally identified as a receptor

recognizing an endogenous ligand on T-cells (Ariizumi et al., 2000; Hermanz-Falcon et al., 2001; Willment

et al., 2001). However, there has been little progress in identifying the nature and physiological

significance of this T-cell ligand. It is possible that dectin-1 recognizes a proteinaceous endogenous

ligand, a suggestion which is supported by evidence that the T-cell ligand is sensitive to proteases

(Ariizumi et al., 2000). The interaction of dectin-1 with its endogenous ligand has been proposed to

stimulate the activation and proliferation of T-cells (Ariizumi et al., 2000; Grunebach et al., 2002).

Interestingly, the soluble -glucan laminarin doesn’t block T-cell binding, suggesting that dectin-1 may

have two ligand-binding sites (Willment et al., 2001).

1.1.6. Regulation of dectin-1 expression and function As already mentioned before, ligand binding of dectin-1 is already sufficient to activate signalization,

leading to the induction of numerous cellular responses. However, dectin-1 not only regulates the


21

expression of innate response genes, but can also function as a phagocytic receptor (Gantner et al.,

2005; Herre et al., 2004b; Underhill et al., 2005). Recently, it has been demonstrated that dectin-1

mediated phagocytosis of -glucans attenuates dectin-1 signaling (Hernanz-Falcon et al., 2009; Rosas et

al., 2008), suggesting that -glucans, which are too large to be ingested, may have enhanced signaling

compared to small, readily phagocytable -glucans (Hernanz-Falcon et al., 2009; Leal et al., 2010). These

results suggest that dectin-1 signals primarily from the cell surface for induction of gene expression,

although, for alternative responses like ROS production, dectin-1 signalization may still occur inside the

endosome (Gantner et al., 2005; Underhill et al., 2005). The explanation for this phenomenon remains

unclear, but it’s possible that phosphatases involved in attenuation of the signal have better access to

dectin-1 once the receptor is internalized, or that the assembly or stability of signaling complexes is

physically disrupted by receptor endocytosis (Hernanz-Falcon et al., 2009).

When ligands are phagocytosed by dectin-1, the intracellular fate of dectin-1 is dependent on the nature,

solubility and size of the ligand (Herre et al., 2004b). Whereas particulate ligands cause dectin-1 to be

retained within phagosomal compartments, inducing de novo synthesis of the receptor, small soluble -

glucans, like laminarin, allow for receptor recycling. On the other hand, glucan-phosphate, also a soluble

but considerably larger -glucan than laminarin, retained dectin-1 within the cell without significant

receptor recovery on the cell surface (Herre et al., 2004b). Furthermore, it was shown that after systemic

administration of -glucan, the cell surface levels of dectin-1 on monocytes and neutrophils decreased

remarkably and stayed low up to 7 days after a single injection, a period well beyond the clearance half-

life of glucan and longer than the normal circulating lifespan of neutrophils and monocytes (Ozment-

Skelton et al., 2006).

1.2. Other -glucan receptors

Although dectin-1 has emerged as the primary receptor for -glucans, also three other PRRs have been

identified for the recognition of and response to these carbohydrates, including scavenger receptors,

complement receptor 3 (CR3) and lactosylceramide.

1.2.1. Scavenger receptors Scavenger receptors are a family of cell surface glycoprotein PRRs that are structurally diverse and have

a range of cellular functions, being involved in both homeostasis and immunity (Pluddemann et al.,

2007). Most of the scavenger receptors are restricted to myeloid and certain endothelial cells, although

some scavenger receptors are also expressed on epithelial cells (Pluddemann et al., 2007). The range of


22

ligands they bind is very diverse and includes modified lipoproteins, selected polyanionic molecules and

a number of microbes (Mukhopadhyay and Gordon, 2004; Pluddemann et al., 2007). These receptors

have been implicated in -glucan recognition in several studies (Dushkin et al., 1996; Rice et al., 2002;

Vereschagin et al., 1998) but until recently, no specific scavenger member has been identified with this

activity. Recent data have shown that CD5 is able to bind and aggregate several fungal species by

recognition of -glucans (Vera et al., 2009). CD5 is a member of group B (four Cys bridges per domain)

scavenger receptor cysteine rich (SRCR) superfamily of proteins (Freeman et al., 1990; McAlister et al.,

1998). It is thought that scavenger receptors recognize the basic -glucan structure, but that the affinity

of these interactions can be influenced by the polymer charge as well as other structural characteristics

that remain to be defined (Rice et al., 2002).

1.2.2. Complement receptor 3

Complement receptor 3 (CR3), also known as CD11b/CD18, M2-integrin or MAC1, is a type I membrane

glycoprotein made up of two noncovalently linked and subunits known as CD11b or M (165 kD) and

CD18 or 2 (95 kD). This heterodimer is expressed on eosinophils, natural killer cells, dendritic cells,

neutrophils, monocytes, macrophages as well as minor subsets of B- and T-cells (Ross, 2000). CR3

recognizes an array of various ligands and is therefore one of the most promiscuous of the PRRs. The

ability of CR3 to bind diverse ligands is mainly contributed to a consensus binding site within CD11b

(Yakubenko et al., 2002). Ligands for the domain of CD11b include complement activation component

iC3b, intercellular adhesion molecule-1 (ICAM-1), fibrinogen, factor X and heparin (Diamond et al., 1995;

Diamond et al., 1993). CR3 is structurally unique among the integrins in that the subunit not only

contains a binding site for the previously mentioned ligands, but also a spatially separate binding domain

for carbohydrates (Ross, 2002; Thornton et al., 1996; Vetvicka et al., 1996). This lectin domain binds with

highest affinity to -1,3-glucans with -1,6-linked branches (Ross, 2002). There is no detectable binding

of linear -1,6-glucans or -mannans, and there is reduced binding to glucans with mixed -1,3- and -

1,4-linkages (Ross, 2002). CR3 has been implicated in mediating a number of responses to -glucans,

including neutrophil chemotaxis, adhesion and transendothelial migration (Harler et al., 1999; LeBlanc et

al., 2006; Tsikitis et al., 2004; Xia et al., 2002). However, CR3 is best known for its involvement in the

antitumorogenic properties of -glucans (Li et al., 2006a; Yan et al., 1999). Combined therapy of-

glucans with anti-tumor mAbs has been shown to prime leukocytes for CR3-dependent cytotoxicity of

iC3b-coated tumor cells (Liu et al., 2009; Ross, 2000; Vetvicka et al., 1996; Xia et al., 1999). The

involvement of CR3 in mediating the biological activities of -glucans led to the proposal that it was the


23

principal leukocyte receptor for -glucans, but since the discovery of dectin-1, the role of CR3 has

become less clear. CR3, however, may be involved in the recognition of low-molecular-weight -glucans,

that are generated following the dectin-1-mediated uptake of larger polymers, in macrophages and

other cells (Hong et al., 2004; Li et al., 2007).

1.2.3. Lactosylceramide

Lactosylceramide (LacCer), also known as CDw17 or Gal4Glc1Cer, is a neutral glycosphingolipid PRR

found in the plasma membrane of many cells. It consists of a hydrophobic ceramide lipid and a

hydrophilic sugar moiety and is mostly abundant on human neutrophils where it makes up two-thirds of

the total glycolipids, with approximately 20% found in the cell membrane (Iwabuchi et al., 2008; Kniep

and Skubitz, 1998; Symington et al., 1985). Interactions of -glucans with CDw17 have been reported to

induce a number of cellular responses in vitro. In alveolar epithelial cells, it has been shown that -

glucans stimulated the production of macrophage-inflammatory protein-2 (MIP-2) and TNF-, in part

through a protein kinase C (PKC) signaling pathway that resulted in the activation of NF-(Evans et al.,

2005; Hahn et al., 2003; Wang et al., 2005). LacCer forms lipid rafts coupled with the Src family kinase

Lyn on the plasma membrane of human neutrophils. This enzyme becomes activated after ligand binding

and results in superoxide generation and migration of neutrophils (Iwabuchi and Nagaoka, 2002; Sato et

al., 2006).

1.3. Collaboration between dectin-1 and other innate immune receptors Microorganisms are complex and present a wide variety of surface antigens that may be recognized by

different PRRs. It is the complex interaction of the responses mediated by these receptors which directs

the resultant innate responses and ultimately the development of pathogen-specific immunity (Trinchieri

and Sher, 2007). Dectin-1 has the potential to collaborate with different PRRs like TLR2 and TLR4, and

non-PRRs, like the tetraspanins CD63 and CD37 (Ferwerda et al., 2008; Mantegazza et al., 2004; Meyer-

Wentrup et al., 2007; Shin et al., 2008; Valera et al., 2008).

Dectin-1 has been shown to cooperate with TLRs particularly for inflammatory cytokine and chemokine

production (Underhill, 2007). However, this collaboration is cell type dependent. In macrophages,

collaborative signaling by TLRs and dectin-1 was shown to be required for TNF- production in response

to fungi (Brown et al., 2003; Gantner et al., 2003). In DCs, dectin-1 signaling alone can trigger TNF-,IL-

6, IL-23 and IL-10 production, although collaborative signaling with TLRs enhances these responses

(Dennehy et al., 2008; Dennehy et al., 2009; LeibundGut-Landmann et al., 2007). In addition to syk,


24

integration of TLR and dectin-1 signaling requires the adapter Raf-1 (Dennehy et al., 2008; Gringhuis et

al., 2009) (Figure 1.3). If TLRs are also involved in dectin-1 mediated phagocytosis and ROS production,

has not yet been fully established, although it was demonstrated that MyD88-/- and TLR2-/- macrophages

could still bind and internalize zymosan (Ozinsky et al., 2000; Underhill et al., 1999). Moreover, the

zymosan-induced ROS production was normal (Gantner et al., 2003), suggesting no role for TLR signaling

in enhancing dectin-1 mediated phagocytosis and ROS production (Underhill, 2007). However, although

TLR signaling triggers little or no ROS production in macrophages on its own, it has been shown that pre-

exposure to TLR2 agonists primes macrophages for an enhanced dectin-1 mediated respiratory burst

(Gantner et al., 2003).

In addition to PRRs, dectin-1 can interact with tetraspanins. Tetraspanins are members of the

transmembrane four superfamily that influence a wide variety of fundamental biological processes

including adhesion, proliferation, antigen presentation, endocytosis and exocytosis (Hemler, 2005;

Wright et al., 2004). They have been shown to modulate signal transduction by organizing other

transmembrane proteins to form functional microdomains in the plasma membrane (Boucheix and

Rubinstein, 2001; Charrin et al., 2009; Yanez-Mo et al., 2009). Dectin-1 associates with the tetraspanins

CD37 and CD63 in the membrane of antigen presenting cells (Mantegazza et al., 2004; Meyer-Wentrup

et al., 2007). Interaction of dectin-1 with CD37 results in stabilization of dectin-1 in the cell membrane

and inhibition of dectin-1 mediated IL-6 production in response to zymosan (Meyer-Wentrup et al.,

2007). Furthermore, it was also demonstrated that CD37 regulates the anti-fungal humoral response by

inhibiting the formation of IgA+ plasma cells, which is critically dependent on IL-6 (van Spriel et al., 2009).

Tetraspanin CD63 makes part of a signaling complex influencing dectin-1 mediated phagocytosis.

Although the functional significance of a dectin-1-CD63 interaction has not yet been elucidated, it was

shown that phagocytosis of yeast particles by DCs was accompanied by a decrease in CD63 expression,

which was inhibitable by the soluble -glucan, laminarin (Mantegazza et al., 2004). Furthermore, there

have been speculations that the dectin-1 tetraspanin interactions are also related to the collaboration

between dectin-1 and TLR2 (Figdor and van Spriel, 2010; Meyer-Wentrup et al., 2007).

Chapter 2 : β-glucans

25

Chapter 2 : -glucans Immunostimulants have been used for many years in animal husbandry and -glucans may be the one

with the longest track record. Homo-polysaccharides, consisting of only one sugar, are described by use

of the suffix “an”. So, a poly-glucose (poly-glucopyranose) is termed “glucan”. Glucans are broadly

classified by their intrachain linkages as either - or -linked (Dalmo and Bogwald, 2008). Two

stereoisomers of glucose exist, with the naturally occurring D-glucose predominating over L-glucose and

the molecule can adapt a cyclic form as a glucopyranosyl. (1,3)-D-glucans (-glucans) are the most

commonly used terms for homo-polysaccharides that have (1,3)-D linkages in the backbone.

Sometimes, they may also possess -D-glucosidic linkages at position 6 of varying distribution and length.

-glucans are naturally occurring polysaccharides that are major structural components of the cell walls

of fungi (Chihara et al., 1969), yeast (Manners et al., 1973), some bacteria (Harada et al., 1968),

seaweeds (Elyakova and Zvyagintseva, 1974) and cereals such as oat and barley (Wood, 2004). In

seaweeds, they serve as storage carbohydrates (Miyanishi et al., 2003), while in bacteria, fungi, yeast

and cereals, when located within the inner cell wall, they can be useful for their structural behavior. As

-glucans are derived from different sources, they may have some differences in their structure and

biological activity. As such, the oat and barley cell walls contain unbranched -glucans with 1,3- and 1,4-

-linked glycopyranosyl residues (Figure 2.1a), whereas -glucans from bacterial origin are unbranched

1,3--linked glycopyranosyl residues (Brown and Gordon, 2003; Brown et al., 2003; Estrada et al., 1997).

In contrast, cell wall -glucans of yeast and fungi consist of 1,3--linked glucopyranosyl residues with

small numbers of 1,6--linked branches (Figure 2.1b). -glucans are considered to be classic microbe-

associated molecular patterns (MAMPs) (Janeway, 1992) as they are not found in mammals, and

consequently, they are recognized by the immune cells of vertebrates and invertebrates. Although -

glucans are most well known for their immune-modulating activities, their mechanism of action are

largely unknown. The literature on immune responses to -glucans can be quite confusing as what is

observed for one preparation of -glucan is often inappropriately extrapolated to all -glucans. These

contradictions have stemmed from a lack of understanding of the underlying molecular mechanisms, the

use of very different and often impure -glucans, and the analysis of different cell types and model

systems.


26

Figure 2.1 Examples of the chemical structure of β-glucans. -glucans from grains such as oats or barley are mostly unbranched polysaccharides with semi-random alternating β-(1,3) and (1,4)-linkages (a). β-glucans from yeast and fungi are polysaccharides with a regular sugar backbone and a higher degree of branching. An example is

shown with a β-(1,3) backbone and a β-(1,6) branch point (b) (adapted from Vos et al., 2007).Modulation of the

immune system by -glucans

The first evidence for biological activity of -glucans arose when zymosan was observed to generate

hyperplasia and hyperfunctionality in macrophages (Benacerraf et al., 1959). Subsequently, -glucans

were identified as the immunostimulating component (Riggi and Di Luzio, 1961) and classified as

biological response modifiers (Novak and Vetvicka, 2009).2.1.1. Effects on the innate immune system

-glucans are structural components in the cell wall of pathogenic and non-pathogenic microorganisms.

Therefore, there is increasing evidence that -glucans are involved in initiating many aspects of the

immune response. The recognition of pathogens occurs through both opsonic (mainly complement) and

nonopsonic mechanisms, and as conserved structural components, -glucans, along with mannans and

other cell wall components, play an important role in the nonopsonic recognition of these pathogens.

-glucans can enhance the functional activity of macrophages and activate the antimicrobial activity of

mononuclear cells and neutrophils (Tzianabos, 2000; Williams, 1997; Zekovic et al., 2005). This enhanced

immune response is accomplished by an increased pro-inflammatory cytokine production (Adachi et al.,


27

1994; Olson et al., 1996; Young et al., 2001), oxidative burst and chemokine production (Figure 2.2)

(Williams, 1997).

Figure 2.2 Immune activation induced by -glucans. -glucans may bind a range of receptors including dectin-1, CR3, lactosylceramide, Toll-like receptors and scavenger receptors, which are found on macrophages, monocytes, neutrophils and dendritic cells. These activate various immune pathways of both the innate and adaptive immune system, including the secretion of a range of cytokines enhancing the development of innate and adaptive immune responses to pathogens, including the generation of plasma cells and the differentiation of effector T cells (Th1/Th2/Th17) (adapted from Thompson et al., 2010).


28

Certain -glucans, including zymosan and lentinan, appear to be internalized by phagocytosis (Brown et

al., 2002; Kurashige et al., 1997; Ladanyi et al., 1993), permitting their destruction by lytic enzymes in the

acidic environment of the phagolysosome (Goodridge et al., 2009b). Several studies have established

that dectin-1 is the primary and the most efficient phagocytic receptor on macrophages and DC for

phagocytosis of yeast with -glucan-rich cell walls (Brown et al., 2002; Saito et al., 1991; Taylor et al.,

2007). The phagosome is also the site of assembly of the phagocyte oxidase complex, which is

responsible for the production of reactive oxygen species (ROS) upon detection and internalization of -

glucans (Kennedy et al., 2007; Rogers et al., 2005; Underhill et al., 2005).

In addition, -glucans also stimulate macrophages to produce cytokines and chemokines, which in turn

activate the adaptive immunity (Figure 2.2). As such, zymosan can stimulate the production of cytokines

such as IL-2, IL-10 and IL-12 (Brown, 2006; Du et al., 2006) and in vitro studies show that a (1,3)-glucan

derived from Saccharomyces cerevisiae, is rapidly taken up by peritoneal macrophages, leading to

upregulation of TNF-, IL-6 and IL-1 expression (Berner et al., 2005).

Besides leukocytes, also epithelial cells can respond to -glucans, as alveolar epithelial cells isolated from

rats secrete MIP-2 after in vitro culturing with Pneumocytis carinii -glucan (Hahn et al., 2003).

2.1.2. Effects on the adaptive immune system

-glucan receptor binding activates various immune pathways enhancing the innate immune response,

resulting in the secretion of a range of cytokines that can in turn enhance the development of an

adaptive immune response to pathogen challenge. The adaptive immune system functions through the

combined action of antigen-presenting cells and lymphocytes. Stimulation of the dectin-1 pathway in DC

using highly purified -glucans, induces the differentiation of CD4+ Th1/Th17 effector cells in mice

(LeibundGut-Landmann et al., 2007). However, the polarization of the adaptive immune response is

mouse-strain dependent (Schofield et al., 2005). As such, Balb/c mice lacking the key Th1 cytokine, IFN-,

demonstrated no requirement for IFN- for protective immunity against systemic C. albicans infection

(Qian and Cutler, 1997), while studies in C57BL/6 mice lacking IFN-showed that the cytokine is essential

for host defense against the same fungi (Balish et al., 1998; Kaposzta et al., 1998; Lavigne et al., 1998). In

addition to Th1, also Th17 have been implied to play a role in the control of fungal infections. Indeed,

mice lacking IL-23p19, which is required for maintenance of Th17 cells, were susceptible to

oropharyngeal Candida infection, while mice lacking IL-12p35, which is required for Th1 polarization,

were protected (Conti et al., 2009). However, when the Candida infection occurred via the intragastric


29

way, IL-23 deletion did not impact survival of the mice (Zelante et al., 2007). These mice rather had

enhanced Th1-mediated inflammation, suggesting that IL-23 serves as a natural break on tissue

damaging Th1 responses (Goodridge et al., 2009b). These and other studies seem to point toward the

importance of a balance between Th1 and Th17 responses for effective control of fungal infections. It is

hypothesized that IL-17 is important for early steps involving the recruitment of efficient numbers of

neutrophils to deal with the pathogen, and that subsequent IFN- produced by Th1 cells is important for

activation of these neutrophils and other phagocytes for killing fungi (Goodridge et al., 2009b).

Additional regulation of the immune response to fungal pathogens is provided by regulatory T-cells

(Tregs), which are thought to help prevent excessive pathology, due to the immune response as well as

contribute to the development of persistent immune memory (Goodridge et al., 2009b).

Stimulation of dectin-1 can also drive CD8+ T cell responses and purified -glucan was found to act in

vivo as a potent adjuvant for the induction of cytotoxic T-lymphocytes (CTL), protecting mice from

experimental tumor challenge (Leibundgut-Landmann et al., 2008). CTL priming by -glucans could partly

be due to activated DCs producing IL-12p70 (Leibundgut-Landmann et al., 2008), which act as a key

polarizing signal to sustain differentiation of naïve CD8+ T-cells into effector cells (Curtsinger et al., 2003;

Valenzuela et al., 2002). In addition, agonists of dectin-1 could promote antibody responses in vivo

(LeibundGut-Landmann et al., 2007).

2.2. Defining factors of the immunomodulatory character of -glucans

The immunological properties of -glucans have been widely studied since decades. However, there is

limited continuity between the reported studies as various -glucans from different sources and with

different molecular weight have been trialed in different cell lines, tissues or animals. This makes

comparison between studies difficult. It is known that the immunomodulatory effects of -glucans are

influenced by their degree of branching, polymer length and tertiary structure, but there is still no

consensus on the basic structural requirements for biological activity.

2.2.1. Molecular structure and size

Studies of the biological activity of the (1,3)-D-glucans are complicated by the fact that both the size

and shape of the molecules might have an influence on the activity. Moreover, few studies have

compared the biological activity of (1,3)-D-glucans having a different molecular weight (Mw), but equal

structure and conformation.


30

In general, it has been shown in vitro that -glucans with a higher molecular weight are more

advantageous as smaller ones (Wasser, 2002). Large Mw or particulate -glucans can directly activate

leukocytes, triggering phagocytosis, antimicrobial activities, including the production of reactive oxygen

and nitrogen intermediates (Brown et al., 2003), and the production of cytokines, chemokines and other

inflammatory mediators (Brown and Gordon, 2005; Czop, 1986; Williams et al., 1996). (1,3)-glucans

with an average or low Mw < 20 000 kDa (such as glucan phosphate), have biological activity in vivo, but

their in vitro effects are less clear (Brown and Gordon, 2003). Moreover, low Mw -glucans from fungi

only modulate the cellular response after cell activation with e.g. cytokines (Volman et al., 2008). Very

short -glucans (Mw < 5000-10 000 kDa), such as laminarin, are generally considered inactive (Bohn and

BeMiller, 1995; Brown and Gordon, 2003), although they can act as -glucan receptor antagonists

(Brown and Gordon, 2001).

Another contributing factor to the biological activities of -glucans is their persistence in mammalian

systems. Vertebrates lack the appropriate glucanases and cannot rapidly degrade these carbohydrates.

Low Mw glucans are secreted through glomerular filtration, while larger glucans are retained primarily in

the liver being degraded by Kupffer cells, a process which may take several weeks (Suda et al., 1996).

2.2.2. Solubility of -glucans

The solubility of -glucans depends on their degree of polymerization and thus their physical

organization (Zekovic et al., 2005). The frequency and mainly the length of side chains determine the

solubility of different (1,3)-glucans (Fleet and Manners, 1976). Soluble -glucans consist of units of

short-branched (1,3)-glucans, whereas particulate glucans are made up of a (1,3) backbone with long

branched (1,6)-glucans (Ishibashi et al., 2002). The long (1,6)-glucan segments influence the physical

properties, such as stabilizing the conformation and biological activities.

Particulate glucans activate leukocytes to a significantly greater extent than the soluble glucans

(Ishibashi et al., 2002). It has been suggested that the difference in biological activity between particulate

and soluble -glucans is due to the specific interaction between the -glucan and the cell surface

receptors of leukocytes. More specific, the inability of soluble -glucans to activate leukocytes can be

attributed to an inability to cross-link membrane -glucan receptors (Ishibashi et al., 2002; Michalek et

al., 1998). However, in case the ligand is phagocytosed, differences in stimulatory capacity between

particulate and soluble -glucans could also be due to differences in intracellular fate of the -glucan

receptor (see 1.1.6).


31

2.2.3. Branching frequency The branches derived from the glycosidic chain core are highly variable and the 2 main groups of side

branching are 1,4 and 1,6 glycosidic chains. These branching assignments appear to be species-specific.

For example, bacteria have mostly (1,4) side branches, while fungi have (1,6) side branches (Chan et

al., 2009). The branching frequencies of (1,3)-(1,6)-glucans are thought to stem their biological activity

and a branching frequency of 0.2 (1 in 5 backbone residues) to 0.33 (1 in 3 backbone residues) was

suggested as being optimal (Bohn and BeMiller, 1995; Chen and Seviour, 2007). Thus, although the

unbranched bacterial -glucan curdlan showed biological activities, chemical addition of (1,6)-linked

glucose residues to the curdlan backbone led to increases in its activity against mice sarcoma cells (Kiho

et al., 1998).

The varying immunomodulatory activity of structural different -glucans can be attributed to the

differential recognition by dectin-1. It has been shown that dectin-1 is highly specific for glucose

polymers with a (1,3)-linked backbone (Adams et al., 2008a; Palma et al., 2006). More specific, a

branched glucan polymer containing at least eight glucose subunits (branched heptasaccharide) and one

full helical turn is required for interaction with dectin-1 (Adams et al., 2008a). In addition, the presence

of even a single (1,6)- linked glucose side-chain can significantly increase the recognition of -glucans by

dectin-1 (Adams et al., 2008a). On the other hand, although the binding affinity of dectin-1 for -glucans

tends to increase as polymer size increases, the presence of already one 4-substituted glucose residue in

the backbone can dramatically decrease the affinity of dectin-1 for -glucans, regardless of the Mw

(Adams et al., 2008a). Furthermore, the receptor doesn’t interact with plant-derived glucans (e.g. barley

glucan) which have a mixed-linkage polymer backbone of alternating regions of (1,3)- and (1,4)

linkages (Aman and Graham, 1987).

2.2.4. Helical conformation

(1,3)-glucans can either adopt a single or triple helical conformation in solution, where hydrogen bonds

hold the individual polymer chains together. In water, most of the (1,3)-glucan molecules exist in a stiff,

triple stranded helical structure, where the side chains are exposed toward the exterior (Sletmoen and

Stokke, 2008). The triple helical structure can be dissociated either by increasing the temperature in

aqueous solutions above 135°C (Yanaki et al., 1985) or dissolving the molecules in either dimethyl

sulfoxide (Kitamura and Kuge, 1989) or in aqueous hydroxide (Tabata et al., 1981). -glucans with a triple

helical configuration have been regarded as being the most powerful immunomodulators (Falch et al.,


32

2000; Maeda et al., 1988; Tabata et al., 1981) and this immunological activity decreased following

alkaline (0.1 M) or thermal treatments (133°C) close to the strand-separation transitions (Falch et al.,

2000). As such, scleroglucan was only biologically active in a linear triple helical arrangement and its

subsequent denaturation reduced its cytokine inducing activity in monocytes (Falch et al., 2000).

However, with shizophyllan the opposite effect was seen: while the single helical conformation

stimulated TNF- acitivity in U937, Th1 and human pheripheral blood monomorphonuclear cells (PBMC),

the native triple helical form was ineffective (Aketagawa et al., 1993; Ohno et al., 1995; Saito et al.,

1991). This demonstrates that the helical conformation by itself is not decisive and other structure

related factors are also important. As mentioned earlier, it was recently speculated that the helical

glucan structure may facilitate the interaction with the dectin-1 binding groove (Adams et al., 2008a).

Molecular modeling indicates that at least six glucose subunits are required for one helical turn of the

glucan polymer (Adams et al., 2008a).

2.2.5. Dose

Several experiments were already conducted in order to determine the optimal concentration of -

glucans in animal feed in view of improving growth performance and the health status. In Table 2.1, an

overview of in vivo experiments with commercial -glucan preparations in pigs is given. It shows that

there is a high variation of incorporation levels of -glucans and that the effects of glucans on immunity

and growth performance are not predictable. Dritz et al. (1995) for instance, concluded that an inclusion

rate to the diet of 250 mg/kg Macrogard, a -(1,3)-(1,6)-glucan derived from the cell wall of

Saccharomyces cerevisiae, resulted in improved growth performance. However, they also established

that pigs fed a diet of 250 mg/kg -glucan were more likely to die after a challenge with Streptococcus

suis, suggesting the existence of a complex interaction between growth performance and disease

susceptibility after -glucan uptake (Dritz et al., 1995). Li et al. (2006b) demonstrated that pigs

supplemented with 100 and 200 ppm of -glucans had a decreased growth performance compared with

pigs fed diets supplemented with 50 ppm of -glucan. Moreover, they observed a decrease of IL-6 and

TNF- production in pigs supplemented with 50 ppm -glucan after a lipopolysaccharide (LPS) challenge.

It has been demonstrated that pro-inflammatory cytokines, such as IL-6 and TNF- are not only

primarily associated with the immune system, but can also directly regulate nutrient metabolism and

cause detrimental effects on animal performance (Spurlock, 1997). Taking this into account, they

hypothesized that diets, supplemented with 100 and 200 ppm of -glucans, promoted a higher secretion


33

of proinflammatory cytokines such as IL-1, IL-6 and TNF-, than diets supplemented with 50 ppm -

glucan and that this could explain the decreased growth performance for pigs fed diets supplemented

with the higher dose of -glucans (Li et al., 2006b).

As also demonstrated in Table 2.1, the effects of in-feed -glucan supplements on systemic immunity are

quite well documented, whereas local intestinal immune responses have only received little attention.

As mucosal responses can occur independently of systemic immunity, the study of systemic immune

responses may not reflect immune functions and dysfunctions occurring in the gut (Cunningham-Rundles

and Lin, 1998; Hannant, 2002). Moreover, it should be highlighted that most of the studies were

performed under experimental conditions and didn’t reflect the real situation in pig production. In the

context of animal production, the ‘desired’ immune function could be defined as the one that offers both

the best growth and the best health status to animals. This requires however, that animals are not kept

under very clean conditions, but are exposed to immune/infectious challenges to assess the impact of in-

feed immunomodulators simultaneously on immunity and on performances and health (Gallois et al.,

2009). It seems that an optimal level of -glucan concentration should be determined for each -glucan

product, thereby not only considering the influences on systemic immunity, but also the development of

immune responses in gut-associated lymphoid tissues (GALT).


34

Table 2.1 Immune parameters and growth performance measured in in vivo experiments where pigs are dietary supplied with -glucans (adapted from Gallois et al., 2009 with modifications)

Glucans Feed content (%)

Weaning age

Time supplementation

PW

Challenge or vaccination

Intestinal Immune response

Systemic immune response

Growth performance

Reference

A 0.05 25-26 d 4 wk None Not measured Blood: Ig titre (no effect) Increase (high IP) No effect (low IP)

Decuypere et al. (1998)

0.1 21 d 4 wk1 None Not measured Blood: neutrophil

function (no effect) No effect Dritz et al. (1995)

0.1 14 d 4 wk None Not measured None No effect Dritz et al. (1995))

0.025-0.05 18 d 6 wk Streptococcus suis (6.5 x 108 CFU i.v.)

4 wk PW

Not measured Blood: neutrophil function (no effect),

haptoglobin Lung: macrophage function (no effect)

Increase Dritz et al. (1995)

B 0.015-0.03 28 d 4 wk PRSS virus, vaccination

1 d PW2

Not measured Blood: Ig titre (no effect) LC proliferation (no

effect), haptoglobin

Increase Hiss and Sauerwein

(2003b)

0.03 27 d 4 wk None Not measured Blood: neutrophil phagocytic activity (no

effect), serum IgG and

IgA , haptoglobin

Slight decrease Sauerwein et al. (2007)

0.3 30 d 4 wk None Not measured Blood: neutrophil phagocytic activity (no

effect), serum IgG and

IgA , haptoglobin

Slight decrease Sauerwein et al. (2007)

1 Starting 1 wk post-weaning

2 PRRSV (Ingelvac PRRS MLV, Boehringer, Ingelheim, Germany)


35

Glucans Feed content (%)

Weaning age

Time supplementation

PW

Challenge or vaccination

Intestinal Immune response

Systemic immune response

Growth performance

Reference

0.03 Fattening pigs

2 wk before slaughter

None Ileum: IgM, IgA, CD4

+ and

CD8+ T-cells:

no effect

Not measured Not measured Sauerwein et al. (2007)

C 0.02-0.04 6.1 kg (? d) 8 wk Atrophic rhinitis vaccination, i.m.

9d PW3

Not measured Blood: Ig titre No effect Hahn et al. (2006)

D 2.5 14 d 2 wk4 LPS (150 µg/kg BW i.v.)

14 d PW Ileum: mRNA

for TNF-,

IL-1,

IL-1 Ra

Blood: TNF-no effect)

Increase Eicher et al. (2006)

E 0.005 28 d 5 wk None Not measured Blood: LC proliferation:

after 14d; no effect after 28d

Increase Li et al. (2006b)

0.005 28 d 31 d LPS (25 µg/kg BW i.p.) 31 d PW

Not measured Blood: TNF-, IL-6 ,

IL-10

Increase Li et al. (2006b)

0.005 28 d 31 d ova (5 µg/kg BW) 14 d PW;

LPS (25 µg/kg BW i.p.) 31 d PW

Not measured Blood: anti-ova Ig ,

TNF-, IL-6 , IL-10

Not measured Li et al. (2005)

PW = post-weaning; d = day; wk = week; PRRS = porcine reproductive and respiratory syndrome; Ig = Immunoglobulin; LC = lymphocyte; LPS =

lipopolysaccharide; TNF- = tumor necrosis factor-; IL = interleukin; IL-1Ra = interleukine-1 receptor antagonist; IP = infection pressure

A: Macrogard-STM

(Biotec-Mackymal A/S, Tromso, Norway; or Provesta Corp., Bartlesville, OK, USA; or Jeil Vet. Chem. Co., Seoul, Korea); B: -(1,3)-(1,6)-glucans

(Antaferm MG, Dr Eckel GmbH, Niederzissen, Germany) – insoluble fraction containing 25% -D-glucans and 10% mannans; C: Glucagen (Enbiotec Company,

Seoul, Korea); D: Energy Plus (Natural Chem Industries, Ltd, Houston, TX, USA); E: From baker’s yeast, manual manufacturing – should contain 86 % of -glucans.

3 Pfizer, Co., Seoul, Korea

4 Treatment also administered in suckling piglets by gavage


36

2.3. Possible mechanisms of -glucan uptake after oral administration

Although most of our knowledge about the immunomodulatory effect of glucans was acquired

through in vitro research or after parenteral administration, a number of reports has shown that oral

administration of -glucans may also have biological effects. As such, Rice et al. (2005) found an

increased survival in mice challenged with Staphylococcus aureus or Candida albicans after oral

administration of glucan-phosphate. Furthermore, in pigs, yeast -glucan was described to reduce the

replication of swine influenza virus after oral administration (Jung et al., 2004). In dogs, oral

administration of Macrogard influenced the systemic humoral immune response by decreasing

temporarily the total IgA response, while the total IgM response increased at the same time (Stuyven et

al., 2010). Those studies show that the addition of -glucans to the diet may be used to modulate

immune function, thereby improving the resistance against invading pathogens. However, although

several reports describe the biological activities of orally administered -glucans, until today, there have

been very few reports on the absorption and pharmacokinetics after oral uptake. Vertebrates do not

possess (1,3)-glucanases making it difficult to digest -glucans. Instead, when orally administered, -

glucans are metabolized slowly by oxidation (Nono et al., 1991), which allows transit from the stomach

to the small intestine, where immunstimulation of the GALT can occur. The intestinal epithelial cells

together with the immune cells of the Peyer’s patches (PP) play an important role in regulating immune

responses. It was demonstrated in mice that a subpopulation of intestinal epithelial cells were capable of

internalizing soluble -glucans independently of dectin-1 (Rice et al., 2005). Although the identity of

these epithelial cells has never been established, it is possible that the uptake of -glucans is mediated

by Microfold (M) cells (Figure 2.3) (Hashimoto et al., 1991; Rice et al., 2005), specialized intestinal

epithelial cells responsible for the transport of macromolecules and particulates into the PP (Sanderson

and Walker, 1993). In the GALT, -glucans interact through dectin-1 with intestinal macrophages and

dendritic cells and this triggers phagocytosis, ROS production and the production of cytokines.

Subsequently, they are transported to lymph nodes, spleen and bone marrow (Hong et al., 2004).

Whether the -glucans enter the systemic circulation depends on their physical state. Rice et al. (2005)

demonstrated that highly purified, water-soluble -glucans can enter the systemic circulation, while

insoluble -glucans were not present in the plasma. Orally administered particulate -glucans are rather

internalized by gastrointestinal macrophages or DCs (Figure 2.3), which transport glucan to various sites

throughout the body and slowly degrade the particulate into a bioactive soluble glucan product (Hong et

al., 2004). The interaction of particulate -glucans with macrophages, which are known to release IL-6,


37

may account for the differences seen in biological activity between orally administered particulate and

soluble -glucans. Indeed, oral administration of soluble -glucan resulted in increased serum IL-12, an

immunomodulatory and pro-inflammatory cytokine, while particulate -glucans increased serum levels

of the pro-inflammatory cytokine IL-6 (Rice et al., 2005).

-glucans are often added to animal feed as prebiotics and are found in many food sources including

edible mushrooms and yeasts. However, the potential role of -glucan fermentation, including the

modulation of the microbiota and their metabolites, receives little attention in studies that focus on oral

applications of these components. Nevertheless, barley-derived -glucans showed clear prebiotic

properties in rats, as they increased the proportion of lactobacilli (Figure 2.3, panel b) (Snart et al., 2006).

In addition, other authors have described that -glucans reach the cecum and the colon where they are

rapidly fermented resulting in the formation of short-chain fatty acids (SCFA) (Dongowski et al., 2002;

Lund and Johnson, 1991). However, prebiotic -glucans might also affect the host and/or the microbiota

on the basis of their carbohydrate structure, for example, by activating and/or blocking cellular receptors

(Figure 2.3, panel a) (Vos et al., 2007).

Additional experiments of well characterized -glucans are needed to more completely understand the

immunomodulatory effects and specific applications for oral use. Such studies will need to focus on the

optimal timing and duration for -glucan ingestion and they will have to question if -glucans should be

consumed continuously, before, at the time of, or after exposure to a pathogen or environmental insult.

Only a few studies have actually investigated the impact of timing of -glucan intake to achieve optimal

benefits and it has been demonstrated that daily feeding of lentinan appears to result in diminished

effects possibly due to tolerance (Hanaue et al., 1989).


38

Figure 2.3 Potential mechanisms of immune modulation by non-digestible carbohydrates (NDC) like -glucans in the gastrointestinal tract. Panel a: The mechanism of NDC-induced activation or blocking of host receptors that are involved in immunological responses by direct binding. Panel b: The mechanism of NDC-induced modulation of the composition of the microbiota and the production of bacterial metabolites. Panel c: The mechanism of direct interaction of NDCs with bacteria, by which adhesion to the mucosa and uptake of bacteria may be modulated. I: Intestinal epithelial cells (E) may play a role in receptor-mediated interaction with NDCs and bacterial adhesion that may be modulated by NDCs. II: Lamina propria DC have been described to sample the gut lumen. Receptor activation or blocking and modulation of bacterial uptake by NDCs may modulate the function of DCs. Because these DCs are known to migrate to mesenteric lymph nodes, mucosal and systemic immune-modulating effects may be induced. III: M cells (M) are known to take up bacteria and soluble antigens from the gut. NDCs might modulate these mechanisms or be taken up by M cells, possibly affecting underlying immune cells including mononuclear cells (MN), macrophages (Mɸ), and DCs. IV: Dietary applied oligosaccharides may facilitate systemic immunomodulatory effects of NDCS (adapted from Vos et al., 2007).

Chapter 3 : Use of -glucans for oral immunization

39


3.1. Challenges of effective oral immunization The vast majority of pathogens gain access to the host at mucosal surfaces. Therefore, a strong immune

response at the site of entry is often necessary to protect and clear pathogens. Oral vaccines represent

an attractive approach for immunization against various mucosal infections due to their advantage in

generating both mucosal and systemic immune responses (Bouvet et al., 2002; Mastroeni et al., 1999).

Moreover, as a result of the interaction of mucosa-specific homing receptors by mucosally primed

lymphocytes and complementary mucosal-tissue specific receptors (addressins) on the vascular

endothelial cells, together with the interaction between chemokine receptors and site-specific secretion

of chemokines , immunological induction at one mucosal site often results in immune responses at distal

mucosal sites (Holmgren and Czerkinsky, 2005; Silin et al., 2007). Other advantages are their relative

ease and safe manner of administration without the need for sterile needles and syringes, and the

simplification of mass immunization (Mestecky et al., 2008; Mestecky et al., 2007). However, despite

many attractive features, developing an oral vaccine has often been difficult. The delivery of antigens to

and through the gastrointestinal barrier remains challenging due to their sensitivity to gastric acid and

proteolytic enzymes, the rapid gastrointestinal transit and also the biological barriers created by tight

epithelial junctions, restricting the transport of macromolecules (Mishra et al., 2010). Moreover, in

comparison with injectable vaccines, the antigen absorption from mucosal surfaces is relatively low,

making it difficult to determine the precise dose of orally administered antigens (Mestecky et al., 2008).

Another major obstacle in the development of effective oral vaccines, is oral tolerance (Poonam, 2007).

Oral tolerance is a default physiological mechanism of the intestinal immune system protecting the body

from unnecessary immune responses to dietary and commensal bacterial antigens (Strobel and Mowat,

1998). The mechanism of tolerance has not been fully elucidated, but understanding the

immunoregulatory mechanisms involved in immune tolerance is critical for the design of effective oral

vaccines. To overcome tolerance, specific targeting to M-cells versus enterocytes (Foster and Hirst, 2005)

or to a particular subtype of dendritic cells (Steinman et al., 2003) is crucial. Therefore, the development

of oral vaccines requires efficient antigen delivery and adjuvant systems. Ideally, such systems should

protect the vaccine from physical elimination and enzymatic digestion, target mucosal inductive sites,

like Peyers patches (PPs )and mesenteric lymph nodes (MLNs), and appropriately stimulate the innate

immune system to generate effective adaptive immunity (Mishra et al., 2010; Poonam, 2007).


40

3.2. Targeting to pattern recognition receptors (PRRs)

3.2.1. -glucans as oral adjuvant in immunization Most antigens are not immunogenic enough and need help to induce a good immune reponse.

Adjuvants can be useful tools to enhance the immunogenicity of the antigen. Classical adjuvants such as

aluminium salts and water-in-oil- emulsions allow the sustained release of antigens, delayed clearance

and better exposure to the immune system (Leroux-Roels, 2010). However, a second role of adjuvants

has become increasingly important: enhancing CD4+ and CD8+ T-cell responses and not only the classical

understanding of antibody production and B-cell memory (Reed et al., 2009). To promote differentiation

of functional CD8+ T-cells, a successful adjuvant must facilitate antigen entry into the MHC class I

processing pathway, trigger DC activation and induce type-I interferon (IFN) production, an effect not

seen with the currently used adjuvants (e.g. aluminium hydroxide) (Coffman et al., 2010). To achieve this

goal, immunostimulants, which are recognized by PRRs and which act directly on the immune system,

have laid the foundation for the development of a series of novel adjuvants/immuno-enhancers (Ishii

and Akira, 2007). Examples include TLR ligands, cytokines, bacterial toxins and -glucans. To achieve the

desired immunological enhancement, these novel adjuvant formulations utilize multiple mechanisms,

including the increased immunological presentation of vaccine antigens by DCs through the engagement

of PRRs (Kono and Rock, 2008) and the induction of CD8+ CTL responses and/or CD4+ T-helper

lymphocyte responses (Th1 or Th2) (Allison and Byars, 1990).

Several studies have demonstrated the adjuvant effect of -glucans when co-administered with either

bacterial, fungal, protozoa or viral antigens (Benach et al., 1982; Hetland et al., 2000; Mohagheghpour et

al., 1995). In all these studies, -glucans were administered via the parental route. As previously

discussed (see Table 2.1), most of the studies describing the adjuvant effect of orally administrated -

glucans, during an in-feed supplementation of several weeks and followed by systemic immunization,

focused only on systemic immune parameters and not on local intestinal immune responses. However,

recently, it was demonstrated that piglets receiving food supplemented -glucans during 2 weeks after

weaning, showed a decreased susceptibility to F4+ enterotoxigenic Escherichia coli (ETEC) (Stuyven et al.,

2009). Furthermore, in pigs fed with the Sclerotium rolfsii -glucan, the amount of F4-specific IgM and

IgA antibody-secreting cells in the lymphoid tissue were lower than in the control pigs (Stuyven et al.,

2009). The improved effect on humoral and cellular immunity could be attributed to the ‘priming’ effect

of -glucans on the immune system.


41

In addition, -glucan has also been used as an immunoadjuvant therapy for cancer since 1980, primarily

in Japan (Kimura et al., 1994; Matsuoka et al., 1997; Takeshita et al., 1991; Yan et al., 1999). More

specific, -glucans have particularly proven their effectiveness in combination with anti-tumor mAbs

(Cheung et al., 2002; Hong et al., 2004). -glucan action is mediated via CR3, which is found on NK-cells,

neutrophils and lymphocytes. Through binding to the lectin domain of CR3, -glucans could prime

neutrophils or NK cells for cytotoxicity against iC3b-opsonized tumors (Vetvicka et al., 1996; Yan et al.,

1999). iC3b opsonization of cancer cells is the result of complement activation by anti-tumor mAbs. Dual

ligation of neutrophil CR3 mediated by the I-domain ligand iC3b, and the lectin-like domain ligand, -

glucan, leads to degranulation and cytotoxic responses (Xia and Ross, 1999; Xia et al., 1999). Although

soluble low Mw -glucans could prime CR3 directly, they are rapidly excreted by the kidneys, thus

limiting their bioactivity and clinical utility (Yan et al., 2000). In contrast, orally delivered particulate -

glucans are expected to have a longer half-life in vivo as they are processed and digested into small

fragments that can prime CR3 (Hong et al., 2004). The uptake of -glucans is dectin-1 dependent.

3.2.2. -glucan-mediated targeting of antigens Another promising approach of targeting antigens to PRRs is the encapsulation of proteins and antigens

in polymeric matrices in the form of nanoparticles/microparticles (Andrianov and Payne, 1998). The

concept of a polymeric carrier system involves delivery of antigens to a specific target site where it is to

be released from the carrier. Ideally, the particle carrier itself should also have an adjuvant activity and

should be capable of immunostimulation. Such carriers have three main characteristics: (i) they deliver a

‘danger’ signal (Gallucci et al., 1999); (ii) they stabilize antigen release over time and (iii) they can target

the antigen to M cells and APCs, like macrophages or DCs (Apostolopoulos and Plebanski, 2000; Brayden

et al., 2005; des Rieux et al., 2007; Gullberg et al., 2000; Jeannin et al., 2000) (Figure 3.1).


42

Figure 3.1 The possible outcome of non-targeted and M cells targeted mucosal vaccines. The uptake by M cells allows the delivery of intact antigen into the immune-inductive environment of PPs. Hence, antigen being taken up by M cells induces potent immune responses, whereas the antigens that are taken up by ordinary enterocytes are more likely to induce tolerance (adapted from Mishra et al., 2010).

Because of their immunomodulating capacities combined with their characteristics to be recognized by

specific DC surface receptors, -glucans form potential candidates for targeting of antigens to APCs. As

-glucans are the predominant molecules in the cell wall of fungi and yeast, such microorganisms are

potential candidates as immunostimulatory antigen-carrier. However, studies using whole fungi or crude

extracts as stimulants, are difficult to interpret because the contributions of the individual cellular

components are not easily to discriminate (Huang et al., 2009). As such, zymosan contains multiple

pathogen-associated molecular patterns (PAMPs), including -glucans, mannans and ligands for TLR2

and TLR4 (Sato et al., 2003; Tada et al., 2002). This complexity greatly hinders the interpretation of

experimental results, as -glucans can modulate the cytokine response to TLR agonists, making it difficult

to use zymosan as a model -glucan. Indeed, depending upon the cytokines studied, additive,

antagonistic and indifferent effects were seen when the combinations of -glucans and other PAMPs


43

were compared with the responses stimulated by individual PAMPs (Huang et al., 2009). To avoid as

much as possible the presence of PAMPs other than -glucans in the yeast cell wall, whole yeast is

subjected to a series of alkali and acid treatments leading to hollow shells consisting of primarily -

glucans and low levels of chitin (Figure 3.2) (Hong et al., 2004; Huang et al., 2009; Soto and Ostroff,

2008). Hollow yeast shells, also called -glucan particles have great potential to be exploited as a

targeting antigen delivery vehicle as it has been demonstrated that they can be efficiently taken up in

vitro by mouse bone marrow-derived DCs (BMDCs) (Huang et al., 2009). Moreover, the hollow porous

yeast shell structure allows for high antigen loading (Aouadi et al., 2009; Soto and Ostroff, 2008). It has

been demonstrated that yeast shells loaded with ovalbumin (ova) stimulated robust Th1- and Th17-

skewed T-cell responses and strong antibody responses in mice following subcutaneous immunization

(Huang et al., 2010).

The use of hollow yeast shells finds also their application as DNA delivery system. As such, they have

been shown to be an effective microparticulate delivery system to encapsulate DNA based on the in situ

layer-by-layer synthesis (Soto and Ostroff, 2008). This encapsulation system is based on the formation of

complexes between negatively charged genetic material and a cationic polymer. Therefore, the yeast

shells are subsequently loaded with transfer RNA (tRNA) and polyethylenimine (PEI) in order to obtain a

positively charged gel matrix within the hollow yeast shells (Figure 3.2) (Hong et al., 2004; Soto and

Ostroff, 2008). The advantages of this DNA delivery system include, besides -glucan receptor targeting,

high DNA capacity and small interfering RNA (siRNA) delivery (Aouadi et al., 2009; Hong et al., 2004; Soto

and Ostroff, 2008).


44

Figure 3.2 Production of fluorescent hollow yeast shells or -glucanparticles. -1,3-glucan particles were purified from baker’s yeast by a series of alkaline and solvent extractions, hydrolyzing outer cell wall and

intracellular components and yielding purified, porous 2-4 µm, hollow -(1,3)-glucan particles (diagram of particles,

left; procedure, middle; microscopy of particles, right). Empty -(1,3)-glucans were then labeled with fluorescein (FL, green). The cores were synthesized by absorbing yeast tRNA, polyethylenimine (PEI) and, in this case, Dy547- siRNA (red fluorescence) in a layer-by-layer format. Instead of siRNA, the cores can also be loaded with a protein or DNA (adapted from Aouadi et al., 2009).


45

As an alternative to the natural -glucan ligands, the application of synthetic -glucan structures offers a

number of advantages. As structure-function relationships of natural -glucans are still a subject of

debate (Descroix et al., 2006; Leung et al., 2006; Sletmoen and Stokke, 2008), synthetic -glucans give

the advantage that the composition of the structure is completely known and controlled, improving

receptor-binding or increasing resistance to enzymatic degradation (Adams et al., 2008b). Furthermore,

synthetic oligosaccharides have low batch-to-batch heterogeneity in contrast to their natural

counterparts, which can show considerable variation among batches in branching frequency as well as

linkage to chitins and mannoproteins (Saraswat-Ohri et al., 2010). Compared with antibody-mediated

targeting, it’s also possible to generate large amounts of synthetic -glucan structures in a relatively

inexpensive way (Adams et al., 2008b; Xie et al., 2010).

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Part II Aims of the study

Part II : Aims of the study

63

Aims of the study Because all commonly-used growth-promoting antibiotics have been banned in the EU member states,

there is a great need for alternatives to in-feed antibiotics. One of the promising alternatives for

antibiotics are -glucans. Recognition of -glucans is thought to be mediated by a combination of cell

surface receptors, but in human and mice, only dectin-1 has been clearly demonstrated to play a role in

the cellular responses of these carbohydrates. Despite the fact that orally administered -glucans are

already used as immunomodulatory agents in humans and animals (Kogan and Kocher, 2007; Sohn et al.,

2000), there is still a lot of confusion about their biological effects and it is still unknown how orally

applied -glucans modulate systemic and intestinal immune parameters. Most studies are focused on

the immunomodulating capacity of only one -glucan and are concentrated on one or at most two

particular doses. It frequently involves raw extracts whose composition and structure is unknown. The

heterogeneity in experiments makes it difficult to draw conclusions about the immunomodulating

potency of -glucans and this can partly explain their discrepancies in their efficacy.

The aim of this thesis was to characterize dectin-1 in the pig and to analyze the dose-effect of various

commercial -glucanpreparations on the innate and the adaptive immunity of the pig in vitro. We

focused on monocytes and neutrophils (innate) as well as lymphocytes and DCs (adaptive) isolated from

blood. The final goal of this thesis was to investigate if the immunomodulating character of -glucans

could be used in antigen delivery.

Following questions were addressed:

1. Is dectin-1, which has been described as the most important -glucanreceptor in different species,

also expressed in pigs? In which tissues and on which blood cell types is this receptor expressed?

2. Which effects have different -glucanpreparations on porcine leukocytes and the maturation of

porcine dendritic cells? Are these effects dose-dependent? Which parameters are important for the

immunomodulating effects of -glucans? Could the observed effects in leukocytes be attributed to

dectin-1?

3. Could the immunomodulating character of -glucans be exploited for enhanced antigen delivery?

Part III : Eperimental studies

64

Part III Experimental studies

Chapter 4 : Identification of the Porcine C-Type Lectin Dectin-1

65

Chapter 4 : Identification of the Porcine C-Type Lectin dectin-1

Eva Sonck, Edith Stuyven, Bruno Goddeeris, Eric Cox

Veterinary Immunology and Immunopathology 130 (2009) 131–134


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4.1. Abstract

-glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria.

They have a backbone of -(1-3)-linked glucose units with -(1-6)-glucan-linkages. Although a number of

receptors are thought to play a role in mediating the biological response to -glucans, dectin-1, a C-type

lectin, was described as the most important receptor. Dectin-1 belongs to the large family of pattern

recognition receptors (PRRs), which recognize conserved pathogen-associated molecular patterns

(PAMPs). Here, we report the identification and characterization of dectin-1 in the pig. We identified two

major isoforms (Gen Bank acc. no. FJ386383 and FJ386384), which differ by the presence of a stalk

region separating the carbohydrate recognition domain from the transmembrane region. Furthermore, a

third minor isoform (acc. no. FJ386385) was identified, which was a variation of the primary isoform with

a deletion in the transmembrane and stalk region. At the nucleotide level, the full length porcine dectin-

1 comprises 744 bp and is 88% identical to the bovine dectin-1 and 82% identical to the human dectin-1.

Messenger RNA transcripts for porcine dectin-1 were detected in the stomach, the small intestine, colon

and rectum, the spleen, the mesenterial lymph nodes and the lungs. The transcript was not expressed in

the liver, the kidneys, the bladder, the heart, the brains and the skin.

4.2. Technical report

The ability of the innate immune system to quickly recognize and respond to an invading pathogen is

essential for controlling the infection. For this purpose, cells of the immune system express receptors,

which recognize conserved structures expressed by various pathogens, but absent from host cells. One

of the most classical PAMPs are -glucans, which are glucose polymers found in the cell wall of fungi,

plants and bacteria. At least four receptors have been identified for the recognition of -glucans:

complement receptor 3, lactosylceramide, scavenger receptors and dectin-1. Among them, dectin-1 is

described as the most important -glucan receptor. The receptor possesses a single C-type lectin-like

carbohydrate recognition domain (CRD) connected to the transmembrane region by a stalk or neck

region and a cytoplasmic tail with an intracellular immunoreceptor tyrosine-based activation motif

(ITAM). Recognition of -glucans by dectin-1 can trigger phagocytosis, which leads to the induction of a

respiratory burst (Gantner et al., 2005; Steele et al., 2003) and to the production of pro-inflammatory

cytokines and chemokines (Steele et al., 2003; Viriyakosol et al., 2005). In mice, two receptor isoforms of

dectin-1 were identified, while in humans two major (A and B) and six minor (C-H) transcripts could be

characterized (Willment et al., 2001). In contrast to the mouse receptor, the human receptor is


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alternatively spliced in a cell specific manner (Willment et al., 2001). The various receptor isoforms differ

in their ability to recognize -glucans. The two major isoforms are the major receptors for -glucans,

while the function of the six minor isoforms, however, remains poorly understood. Given the importance

of dectin-1 as pattern recognition receptor and the lack of information about isoforms, sequence and

expression of this receptor in pigs, we describe here the detailed cloning, characterization and tissue

distribution of dectin-1 in pigs. To begin investigating porcine dectin-1, total RNA was isolated from

Porcine Alveolar Macrophages (PAM) using TRIzol® (Invitrogen, Merelbeke, Belgium) following the

manufacturer’s protocol. The FirstChoice® RLM-RACE kit (Ambion, Austin, TX) was used to amplify cDNA

containing the entire 5’ or 3’ ends of porcine dectin-1 as described by the manufacturer. To obtain the 5’

end, RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase for 1h at 42°C. The

mRNA corresponding to the porcine dectin-1 transcript was then amplified using SuperTaq DNA

polymerase (SphaeroQ, Gorinchem, Netherlands) in two separate PCR reactions. The first PCR reaction

used a 5′RACE outer primer included in the kit and a gene-specific outer reverse primer (5’-

GGAGAGCCTTGGTGGTATCCT-3’), which was based on the full length dectin-1 sequences of human,

bovine and mouse. The PCR products were then re-amplified using an inner forward oligo that was

specific for the RNA adapter and a gene-specific outer reverse primer which was the same as previously

used. RLM-RACE was also used to obtain the 3’ end of porcine dectin-1. Total RNA was reverse

transcribed into cDNA using an oligo dT containing adapter sequence. Subsequently, cDNA was amplified

using an outer reverse oligo, specific for the oligo dT containing primer and a forward outer oligo (5’-

CAACCACAGGAGTTCTTTCTAG-3’) corresponding to a gene-specific outer sequence derived from the

human, bovine and mouse dectin-1 sequences. PCR was performed at 94°C for 30 sec, 65°C for 30 sec

and 72°C for 30 sec for a total of 35 cycles followed by a final extension at 72°C for 10 min. Aliquots of

the PCR reactions were visualized by ethidium bromide agarose gel electrophoresis and subsequently

extracted from the gel using the Wizard SV-gel kit (Promega), according to the manufacturer’s protocol.

The products were then cloned into pCR2.1-TOPO cloning vector (Invitrogen, Merelbeke, Belgium).

Escherichia coli (Top10, Invitrogen) transformed with the constructs were selected with ampicillin (100

µg/ml, Roche) and 5 positive clones were identified and sequenced. We identified 3 different isoforms of

porcine dectin-1 (Figure 4.1).


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Figure 4.1 Total RNA isolated from alveolar macrophages was reverse transcribed into cDNA and analyzed for the presence of dectin-1 mRNA transcripts. The different fragments were confirmed to include most of the encoding sequence, lacking only a few nucleotides at the 5’ and 3’ end.

The resulting sequences were deposited in the Genbank (acc. no. FJ386383, FJ386384 and FJ386385).

The full length dectin-1 has a length of 744 nucleotides and codes for a protein of 247 amino acids, which

has the same length as isoform A of bovine (Willcocks et al., 2006) and human dectin-1 (Willment et al.,

2001). It consists of a cytoplasmic region on the N-terminal end, a transmembrane region, a stalk region

and a single C-terminal carbohydrate recognition domain. Similar to the human (Willment et al., 2001)

and bovine (Willcocks et al., 2006) homologue, the second major isoform (isoform B), which has a length

of 606 bp, lacks a stalk region, resulting in a truncated protein. The third isoform is a minor transcript

(507 bp) which consists of a variation of the primary isoform with a deletion in the transmembrane and

stalk region. The PCR analysis also indicated the presence of 2 other, minor isoforms, which are yet to be

characterized. In humans, beside two major isoforms (isoform A and B), 6 other minor isoforms, which

consisted of variations of the primary isoforms, could be detected (Willment et al., 2001). Isoform E of

dectin-1 in humans shows strong similarities with the third minor transcript of 507 bp in the pig. Human

dectin-1E is not secreted and resides mainly in the cytoplasm of cells functioning as a Ran-binding

protein (Xie et al., 2006).


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To find similarity between porcine dectin-1 and the dectin-1 sequences of bovine, human and mouse, a

multiple sequence alignment was carried out (http://www.ebi.ac.uk/Tools/clustalw2/). At the nucleotide

level, porcine dectin-1 isoform A is 88 % identical to bovine dectin-1 A (long), while it shares 82 % with

human dectin-1 A and only 72 % identity with murine dectin-1 A. Porcine dectin-1 A shows 82 % identity

at the amino acid level with the bovine dectin-1, but only 73 % and 58 % with human and murine dectin-

1A, respectively. When more particularly the extracellular domain of isoform A is considered, which

consists of the stalk region and the carbohydrate recognition domain, the similarity between the

different species is nearly the same as within the complete dectin-1 sequence (Figure 4.2). The second,

shorter major isoform of dectin-1 (isoform B), shows a similarity of 87% and 82% with dectin-1B of

bovine and human, respectively, while, at the amino acid level, it shares 83% (bovine) and 75% identity

(human) with the dectin-1B homologue. Trp221 (W) and His223 (H) are highly conserved in all the dectin-

1 homologues of the different species (Figure 4.2). As mutations of both amino acids results in reduced

-glucan binding (Adachi et al., 2004), these side chains play a crucial role in the formation of the dectin-

1 -glucan binding site where they most likely participate directly in ligand binding (Brown et al., 2007).

Figure 4.2 Multiple sequence alignment of the exctracellular domain (stalk + carbohydrate recognition domain (CRD)) of porcine, bovine, human and murine dectin-1. * exact match : amino acids with a strong similarity . amino acids with a weak similarity

http://www.ebi.ac.uk/Tools/clustalw2/


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Expression profiles of porcine dectin-1 were determined in tissues as well as some cell types in the

blood. For the isolation of blood cells, blood was taken from healthy pigs. Peripheral blood

monomorphonuclear cells (PBMC) and neutrophils were isolated by density gradient centrifugation on

respectively Lymphoprep (NYCOMED Pharma AS, Life Technologies, Belgium) and a discontinuous Percoll

gradient (GE healthcare, Sweden) (68% and 75%). CD172a+ cells and CD3+ cells were isolated from PBMC

by magnetic activated cell separation (MACS; Miltenyi-Biotec, Bergisch Gladbach, Germany) using anti-

CD172a monoclonal antibody (mAb) (74-12-15A; Pescovitz et al., 1984 ) or anti-CD3c mAb (FY1H2; Yang

et al., 1996), and anti-mouse IgG microbeads together with LS separation columns (Miltenyi-Biotec)

following the manufacturer’s protocol. For purification of T cell subsets, CD3+ cells were sorted by flow

cytometry using a FACSariaIII cell sorter (Becktin Deckinson). Hereto, cells were stained with anti-CD4

and anti-CD8 mAb, followed by anti-mouse IgG2b PE (CD4) (molecular probes) and anti-mouse IgG2a

AF647 (CD8) (molecular probes). Monocyte-derived dendritic cells (MoDCs) were generated from

CD172a+ cells as previously described (Carrasco et al., 2001). After isolation, both tissues and cells were

washed with PBS and immediately submerged in TRIzol® for RNA preservation, after which they were

frozen in liquid nitrogen and stored at -80°C until total RNA extraction. All cDNAs were prepared by

reverse transcription polymerase chain reaction (RT-PCR). Three microgram total RNA was pre-incubated

with 1 µl (50 µM) random hexanucleotide primer (Perkin Elmer, Brussels, Belgium) for 10 min at 72°C.

After chilling on ice, 5 mM of MgCl2, 1x PCR buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl), 1 mM dNTP-

mix (Roche, Mannheim, Germany), 20 units of RNAsin® Ribonuclease inhibitor (Promega, Leiden, the

Netherlands) and 200 units of Superscript II Rnase H- reverse transcriptase (Invtirogen, Merelbeke,

Belgium) were added. The reaction mix was incubated for 10 min at 42°C followed by heating for 5 min

at 99°C to inactivate the reverse transcriptase. The resulting cDNA served as a template for PCR. -actin,

an essential expressed reference gene (Nygard et al., 2007), was used as a control for the uniformity of

the reverse transcription reaction. The oligonucleotide primers used for detection of dectin-1 were 5’-

TCAGCTCTCACAACCTCACCA-3’ (sense) and 5’-TGAAGGCACACTGCAAAGTTG-3’ (antisense). The reaction

mix constisted of 1x SuperTaq-buffer (100 mM Tris-HCl, pH 9.0; 15 mM MgCl2; 500 mM KCl; 1% Triton X-

100, 0.1% (w/v) stabilizer) (SphaeroQ, Gorinchem, Netherlands), 0.2 µM of each sense and antisense

primer, 0.2 mM dNTP-mix (Roche, Mannheim, Germany) and 2.5 units of SuperTaq DNA polymerase. PCR

was performed at 94°C for 30 sec, 65°C for 30 sec and 72°C for 30 sec for a total of 35 cycles followed by

a final extension at 72°C for 10 min. All the three dectin-1 mRNA transcripts were widely transcripted in a

variety of immune and non-immune tissues (Figure 4.3). This transcription pattern is comparable with

the dectin-1 transcription in tissues of mouse and human with exception of the liver, where no


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expression was found (Reid et al., 2004). Furthermore, dectin-1 mRNA expression of the three isoforms

was found in different blood cell types (Figure 4.4). As such, dectin-1 is highly expressed in monocytes,

neutrophils as well as MoDCs. Also in CD3+ T-cells, dectin-1 expression was found. As dectin-1 mRNA was

detected in none of the T-cell subsets, probably NK-cells are responsible for the dectin-1 mRNA

expression profile. Which cell types are expressing porcine dectin-1 in the digestive tract have yet to be

defined, but given that dectin-1 is highly expressed on dendritic cells and monocytes in the blood, it’s

likely that these cells are responsible for the expression profile of dectin-1 in the digestive tract.

Figure 4.3 Expression profile of porcine dectin-1 mRNA in different tissues. Total RNA isolated from the

indicated tissues was examined for mRNA expression of dectin-1 and -actin by reverse transcription polymerase chain reaction. Amplification products were size-fractionated on a 1% agarose gels and stained with ethidium bromide.


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Figure 4.4 Expression profile of porcine dectin-1 mRNA in different blood cell types. Total RNA isolated from the cells was examined for mRNA expression of dectin-1 by reverse transcription polymerase chain reaction. Amplification products were size-fractionated on a 1% agarose gels and stained with ethidium bromide.

4.3. Acknowledgements This research was funded by a PhD grant of the Institute for the Promotion of Innovation through

Science and Technology in Flanders (IWT-Vlaanderen).

4.4. References Adachi, Y., Ishii, T., Ikeda, Y., Hoshino, A., Tamura, H., Aketagawa, J., Tanaka, S., Ohno, N., 2004, Characterization of beta-glucan recognition site on C-type lectin, dectin 1. Infect Immun 72, 4159-4171. Ariizumi, K., Shen, G.L., Shikano, S., Xu, S., Ritter, R., 3rd, Kumamoto, T., Edelbaum, D., Morita, A., Bergstresser, P.R., Takashima, A., 2000, Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning. J Biol Chem 275, 20157-20167. Brown, G.D., Taylor, P.R., Reid, D.M., Willment, J.A., Williams, D.L., Martinez-Pomares, L., Wong, S.Y., Gordon, S., 2002, Dectin-1 is a major beta-glucan receptor on macrophages. J Exp Med 196, 407-412.


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Brown, J., O'Callaghan, C.A., Marshall, A.S., Gilbert, R.J., Siebold, C., Gordon, S., Brown, G.D., Jones, E.Y., 2007, Structure of the fungal beta-glucan-binding immune receptor dectin-1: implications for function. Protein Sci 16, 1042-1052. Gantner, B.N., Simmons, R.M., Underhill, D.M., 2005, Dectin-1 mediates macrophage recognition of Candida albicans yeast but not filaments. EMBO J 24, 1277-1286. Hermanz-Falcon, P., Arce, I., Roda-Navarro, P., Fernandez-Ruiz, E., 2001, Cloning of human DECTIN-1, a novel C-type lectin-like receptor gene expressed on dendritic cells. Immunogenetics 53, 288-295. Nygard, A.B., Jorgensen, C.B., Cirera, S., Fredholm, M., 2007, Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol 8, 67. Pescovitz, M.D., Lunney, J.K., Sachs, D.H., 1984, Preparation and characterization of monoclonal antibodies reactive with porcine PBL. J Immunol 133, 368-375. Reid, D.M., Montoya, M., Taylor, P.R., Borrow, P., Gordon, S., Brown, G.D., Wong, S.Y., 2004, Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions. J Leukoc Biol 76, 86-94. Steele, C., Marrero, L., Swain, S., Harmsen, A.G., Zheng, M., Brown, G.D., Gordon, S., Shellito, J.E., Kolls, J.K., 2003, Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor. J Exp Med 198, 1677-1688. Taylor, P.R., Brown, G.D., Reid, D.M., Willment, J.A., Martinez-Pomares, L., Gordon, S., Wong, S.Y., 2002, The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J Immunol 169, 3876-3882. Viriyakosol, S., Fierer, J., Brown, G.D., Kirkland, T.N., 2005, Innate immunity to the pathogenic fungus Coccidioides posadasii is dependent on Toll-like receptor 2 and Dectin-1. Infect Immun 73, 1553-1560. Willcocks, S., Yamakawa, Y., Stalker, A., Coffey, T.J., Goldammer, T., Werling, D., 2006, Identification and gene expression of the bovine C-type lectin Dectin-1. Vet Immunol Immunopathol 113, 234-242. Willment, J.A., Gordon, S., Brown, G.D., 2001, Characterization of the human beta -glucan receptor and its alternatively spliced isoforms. J Biol Chem 276, 43818-43823. Xie, J., Sun, M., Guo, L., Liu, W., Jiang, J., Chen, X., Zhou, L., Gu, J., 2006, Human Dectin-1 isoform E is a cytoplasmic protein and interacts with RanBPM. Biochem Biophys Res Commun 347, 1067-1073. Yang, H., Oura, C.A., Kirkham, P.A., Parkhouse, R.M., 1996, Preparation of monoclonal anti-porcine CD3 antibodies and preliminary characterization of porcine T lymphocytes. Immunology 88, 577-585.

Chapter 5 : The effect of β-glucans on porcine leukocytes

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Eva Sonck, Edith Stuyven, Bruno Goddeeris, Eric Cox

Veterinary Immunology and Immunopathology 135 (2010) 199–207


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5.1. Abstract

-glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria.

They have the capacity to activate innate immunity, thereby enhancing defence barriers. Besides

differences in type of linkage and branching, β-glucans can vary in solubility, molecular mass, tertiary

structure, polymer charge and solution conformation. All these characteristics may influence their

immunomodulating effects. In this study, the effect of seven -glucans that differed in origin (fungi,

yeast, seaweed, bacteria or algae) and structure (linear or branched; soluble, gel or particulate) were

tested on peripheral blood monomorphonuclear cells (PBMC) and neutrophils of the pig. We looked at

lymphocyte proliferation, reactive oxygen species (ROS) production by neutrophils and monocytes and

cytokine production. The soluble -glucans laminarin and scleroglucan did not activate ROS production

of monocytes and neutrophils, while the particulate -glucans (-glucan from algae (Euglena gracilis) and

glucan preparations from baker’s yeast (Macrogard, Saccharomyces cerevisiae and zymosan)) had a

stimulating effect. The highest stimulation of lymphocyte proliferation occurred by curdlan (bacteria),

zymosan and the -glucan of Euglena gracilis, especially at high concentrations (200 and 800 µg/ml).

TNF- was particularly stimulated by Macrogard and Saccharomyces cerevisiae, while all -glucans

(except laminarin) induced IL-1 Furthermore, it was interesting that all -glucans and in particular

curdlan, gave rise to IL-10 secretion, whereas any -glucan induced the release of IL-8, IL-4, IL-12, IL-6 or

IFN-

5.2. Introduction In animal husbandry the control and prevention of infectious diseases is of great economic importance.

Antibiotics have been used in pig production for many years both as prophylactic agents and growth

promotors. Because of the European ban on growth-promoting antibiotics in animal production, the

necessity of controlling animal health to preserve the competitiveness of European pig production has

brought the need for alternatives to in-feed antibiotics. One of the promising alternatives of antibiotics

are -glucans. -glucans are indigestible oligosaccharides in the cell wall of yeast, fungi and bacteria.

They consist of β(1,3)-linked β-D-glucopyranosyl units that form a backbone containing randomly

dispersed β(1,6)-linked side chains. In humans and mice, β-glucans have been shown to stimulate both

specific (vaccine adjuvants) and non-specific immune responses (Abel and Czop, 1992; Adachi et al.,

1994; LeBlanc et al., 2006; Mucksova et al., 2001; Williams et al., 1989). As such, -glucans stimulate

phagocytosis and production of inflammatory cytokines by macrophages (Brown, 2006; Brown and


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Gordon, 2001). Furthermore, -glucans have the ability to stimulate neutrophils and monocytes to the

production of reactive oxygen species (ROS) (Rubin-Bejerano et al., 2007; Thornton et al., 1996; Vetvicka

et al., 1996; Xia et al., 1999).

The recognition of fungi and yeast derived particles has been attributed to a variety of pattern

recognition receptors (PRRs), including complement receptor 3 (CR3), lactosylceramide and a β-glucan-

specific receptor, namely dectin-1. Recently, dectin-1 was identified in the pig (Sonck et al., 2009). Only a

few studies have been undertaken to analyze the effect of in-feed -glucan supplementation in pigs,

however, sometimes with different results ((Chae et al., 2006; Dritz et al., 1995; Eicher et al., 2006; Li et

al., 2006c; Stuyven et al., 2009). Besides the use of different concentrations in these studies, also

physicochemical parameters such as solubility, primary structure, molecular weight, branching and

polymer charge, could explain the differences in immune response (Vetvicka et al., 2008). In the present

study, we aimed to increase our insights into the direct effects of -glucans on monocytes, neutrophils

and lymphocytes of pigs by testing the dose response of different -glucans preparations in vitro.

5.3. Materials and Methods

5.3.1. -glucans

Seven -glucans were evaluated. A description and comparison of these carbohydrates is given in Table

5.1a, while the sugar composition of the preparations is shown in Table 5.1b. Laminarin, curdlan and the

glucans from baker’s yeast (Saccharomyces cerevisiae) and Euglena gracilis were all purchased from

Sigma (Bornem, Belgium), just as lipopolysaccharide (LPS), which served as a control. Scleroglucan and

Macrogard were kindly provided by INVE (Dendermonde, Belgium) and Biotec Pharmacon ASA (Norway),

respectively. To prevent additional contamination with endotoxins, all material coming into contact with

the -glucans was made pyrogen-free. As such, clean glassware and material was rendered endotoxin-

free by heating at 250°C for 4 hours. All -glucan solutions were made in 0.9% NaCl. Particulate -

glucans were solubilized as much as possible by sonicating them. Endotoxin-concentrations, already

present in the -glucan preparations, were determined by the Chromogenic Limulus Amebocyte Lysate

(LAL) Test (Cambrex Bio Science Walkersville, Inc.) and were, with exception of curdlan and scleroglucan,

consistently lower than 0.5 endotoxin units/µg -glucan.


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Table 5.1a An overview of the source, solubility and structure of the -glucans used in this study.

Name Origin Source type Solubility Structure MW (kDa)

laminarin Laminaria digitata Algae e.g. brown seaweeds

Soluble -(1,3) with some -(1,6) branching (30:1).

The -(1,6 )side chains are composed of 2 glucose-units.

7,7

scleroglucan Sclerotium rolfsii Fungus Soluble -(1,3)-(1,6) branched (6:1). The -(1,6) side chains are composed of 2 glucose-units.

1020

curdlan Alcaligenes faecalis Gram negative bacteria

Particulate -(1,3) unbranched 100

glucan from Euglena gracilis

Euglena gracilis Algae Particulate -(1,3) unbranched 500

glucan from Saccharomyces cerevisiae

Saccharomyces cerevisiae

Yeast Particulate -(1,3)-(1,6) branched (30:1). 200

Macrogard Saccharomyces cerevisiae

Yeast Particulate -(1,3)-(1,6) branched (10:1 or 20:1). The -(1,6) side chains are composed of 2 or 3 glucose-units.

Not known

zymosan Saccharomyces cerevisiae

Yeast Particulate Crude extract with -glucan, mannan and proteins; non-uniform branches and

backbone units. -(1,3)-(1,6) branched

Not known


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Percentage (%) of dry weight

Name Glucosamine Glucose Mannose Sugar (%) dry weight

laminarin 0 87 0 87 scleroglucan 8.6 77 3.6 89.2 curdlan 0 80.1 0 80.1 glucan from Euglena gracilis

0 77.3 0 77.3

glucan from Saccharomyces cerevisiae

1.9 81.3 7 90.2

Macrogard 1.1 79.5 8.2 88.7 zymosan 0.6 70.9 16.4 87.9

Table 5.1b Sugar composition of the β-glucan preparations as determined by high performance anionic exchange chromatography (HPAEC) analysis.


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5.3.2. Animals and blood samples Peripheral blood was collected on heparin from the jugular vein of four weaned pigs (Piétrain boar x

Hypor Libra hybrid sow) of 8-10 weeks old. All animal experiments have been approved by the animal

care and ethics committee of the Faculty of Veterinary Medicine, Ghent University.

5.3.3. Isolation of peripheral blood monomorphonuclear cells (PBMC) Peripheral blood monomorphonuclear cells were isolated by density gradient centrifugation (800g at

18°C for 25 min) on Lymphoprep (NYCOMED Pharma AS, Life Technologies, Merelbeke, Belgium). After

lysis of erythrocytes in ammonium chloride (74.7%) and subsequent centrifugation (350 g at 18°C for 10

min), the pelleted cells were washed three times and resuspended at 107 cells/ml in leukocyte medium

(RPMI-1640 (GIBCO BRL, Life Technologies, Merelbeke, Belgium) containing foetal calf serum (10%),

nonessential amino acids, Na-pyruvate (100 µg/ml), L-glutamine (292 µg/ml), penicillin (100 IU/ml),

streptomycin (100 µg/ml) and kanamycin (100 µg/ml)).

5.3.4. Isolation of neutrophils Heparinized blood was layered onto a discontinuous Percoll gradient (68% and 75%) and centrifugated

(500g at 4°C for 30 min) to separate the neutrophils from lymphocytes, monocytes and platelets. After

lysis of erythrocytes in ammonium chloride (74.7%) and subsequent centrifugation (350 g at 18°C for 10

min), the pelleted cells were washed three times and resuspended at 106 cells/ml in RPMI-1640 without

phenol-red.

5.3.5. Determination of ROS production by porcine neutrophils and monocytes exposed to structurally different -glucans

Reactive oxygen species production was measured using a chemiluminescence (CL) assay as described by

Donne et al. (2005), with some modifications. The chemiluminescent response of porcine neutrophils or

monocytes stimulated with different -glucans, was compared to the response obtained after

stimulation with 20 µg/ml phorbol myristate acetate (PMA) (Sigma Aldrich, Bornem, Belgium).

Neutrophils were seeded in a 96-well plate at 2.105 cells/well in 200µl RPMI without phenol-red, while

the PBMC were seeded at a concentration of 2.106 cells/well. The plates were incubated at 37°C for 2 h

in a humidified atmosphere with 5% CO2, so that the neutrophils or the monocytes could adhere to the

plastic surface. The supernatant was removed and 175 µl luminol was added to a final concentration of

0,5 mM. After 5 minutes of background measurement at 37°C, 25 µl of the -glucans were added as well


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as the control agents (PMA as positive control and HBSS with Ca2+/Mg+ as negative control). Different

concentrations (800 µg/ml, 400 µg/ml, 200 µg/ml and 100 µg/ml) of each -glucan were tested after

which ROS production was measured during 120 minutes in the integration mode. Each stimulation was

performed in duplicate. ROS production was expressed as relative light units (RLU).

5.3.6. Lymphocyte proliferation assay

To investigate the proliferating effect of different -glucans on PBMC, cells were stimulated with

different concentrations of -glucans (800 µg/ml, 200 µg/ml, 50 µg/ml and 5 µg/ml) and a proliferation

assay was performed as described by Van der Stede et al. (2003). Different concentrations of -glucans

or the control agents (1 µg/ml Concanavalin A (ConA) (Sigma Aldrich, Bornem, Belgium) as positive

control or medium as negative control) were added to the wells of a 96-well flat-bottom microtitre plate

(NUNC®) containing 5×105 PBMC per well (final volume of 200 μl). Each stimulation was performed in

duplicate. The cells were incubated at 37°C in a humidified atmosphere with 5% CO2. After 72 h the cells

were pulse-labelled with 1 μCi of *3H]-thymidine (Amersham ICN, Bucks, UK) per well and 18 h later the

cells were harvested onto glass fibre filters (Perkin-Elmer, Life Science, Brussels, Belgium). The

radioactivity incorporated into the DNA was measured using a β-scintillation counter (Perkin-Elmer, Life

Science, Brussels, Belgium). The results are presented as the mean counts per minute (cpm).

5.3.7. Cytokine production

PBMC (1.107) were stimulated with 150 µg/ml of different -glucans for 24h and the supernatant was

harvested and stored at -20°C. The concentrations of IL-1, IL-8, IL-4, IL-12, IL-6, IFN-TNF- or IL-10

were measured using commercially available porcine-specific ELISA kits (R&D Systems Inc.; Minneapolis,

MN, USA) according to the manufacturer’s recommended protocols. IL-1 concentrations were also

determined in the supernatant of PBMC stimulated with 800, 200, 50 and 5 µg/ml of the different -

glucans, as we wanted to know to what extent variation in IL-1secretion is linked with lymphocyte

proliferation.

5.3.8. Statistics All experiments were performed in duplicate with cells obtained from four different pigs. Data are given

as mean ± SEM. Statistical analyses were performed using SPSS 16. For each experiment and each

concentration, differences between the -glucans were investigated using ANOVA mixed-effects model

(with -glucan as fixed factor and pig as random factor) and Scheffé’s multiple comparison range.


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Levene’s test was used to assess the homogeneity of the variances. Data were considered significant at p

< 0.05.

5.4. Results

5.4.1. ROS production by neutrophils and monocytes

The soluble -glucans laminarin and scleroglucan didn’t activate ROS production of monocytes (Table

5.2) and neutrophils (Table 5.3), while all particulate -glucans, except for curdlan, had a stimulating

effect. The ROS production was strongly dependent on the concentration of the particulate -glucan. For

the particulate -glucans originating from S. cerevisiae, the optimal concentration-range was mostly

situated between 200 and 400 µg/ml, except for Macrogard, which showed increased stimulation of

monocytes at 100 µg/ml. Indeed, the optimal concentration of Macrogard to stimulate ROS production

differed between the two cell types. As such, while it stimulated monocytes stronger at lower

concentrations (100 and 200 µg/ml), neutrophils are stimulated rather at high concentrations (400

µg/ml). Whereas the optimal concentration of Macrogard seemed to be 200 µg/ml and 400 µg/ml for

ROS production of monocytes and neutrophils, respectively, the opposite is true for the glucan from S.

cerevisiae. The ROS production mediated by glucan from Euglena gracilis and zymosan, however,

seemed to increase with increasing concentrations. Glucan from Euglena gracilis in its highest

concentration (800 µg/ml) induced a significantly higher ROS production in both cell types than all the

other tested -glucans (p < 0,05). Curdlan only had a weak stimulating effect on monocytes while it

didn’t stimulate ROS production by neutrophils at all.

5.4.2. Lymphocyte proliferation

As seen in Table 5.4, most particulate -glucans clearly stimulated lymphocyte proliferation at low

concentrations (50 µg/ml). For Macrogard and the glucan from S. cerevisiae, the effect decreased with

higher concentrations. Curdlan, the glucan from Euglena gracilis and zymosan however, showed an

increased proliferating capacity at higher concentrations. Curdlan and zymosan could stimulate

lymphocyte proliferaton significantly more than all other -glucans at concentrations of 200 and 800

µg/ml (p < 0,05). Laminarin did not induce lymphocyte proliferation, while scleroglucan caused a weak

proliferation at concentrations between 50 and 800 µg/ml.


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Table 5.2 The ROS response of porcine monocytes stimulated with different concentrations of different -glucans determined by chemiluminescence. Data are shown as the means ± SEM of 4 pigs. PMA (20 µg/ml) and HBSS were used as a positive and negative control respectively and gave values of 115,2 ±

14,7 (PMA) and 16,4 ± 2,8 (HBSS) RLU. As the -glucan preparations could be contaminated with endotoxins, also the influence of LPS (10 and 1 µg/ml) was tested on the ROS production of monocytes and gave values of 13,1 ± 2,9 (10 µg/ml) and 17,3 ± 3,1 (1 µg/ml) RLU. Values in the same column showing the same superscript letter are not significantly different (p > 0.05).

-glucans ROS production (relative light units) following stimulation of monocytes with different

concentrations of - glucans

800 µg/ml 400 µg/ml 200 µg/ml 100 µg/ml

laminarin 13,1 ± 2,9a 10,2 ± 2,4a 14,4 ± 3,1a 14,9 ± 3,2a

scleroglucan 27,5 ± 4,6a 28,1 ± 4,9a 24,2 ± 3,6a 21,2 ± 3,5a

curdlan 188,7 ± 32,7b 188,2 ± 37,3 155,6 ± 34,4a 79,6 ± 15,1a

glucan from Euglena gracilis 1076,6 ± 161,0 941,4 ± 164,2 787,5 ± 107,7b 625,0 ± 115,9

glucan from Saccharomyces cerevisiae 271,5 ± 35,1b,c 638,1 ± 76,5b 561,4 ± 25,6c 390,7 ± 62,5b

Macrogard 357,9 ± 36,6b,c 589,5 ± 74,9b 880,0 ± 127,3b 899,0 ± 114,7

zymosan

616,9 ± 54,7 603,7 ± 24,2b 494,4 ± 48,2c 351,9 ± 68,1b


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-glucans ROS production (relative light units) following stimulation of neutrophils with different

concentrations of - glucans


laminarin 22,9 ± 2,6a 20,2 ± 2,8a 27,3 ± 3,0a 35,0 ± 4,0a

scleroglucan 23,6 ± 4,0a 24,3 ± 3,1a 22,0 ± 3,1a 18,5 ± 3,2a

curdlan 37,8 ± 5,0a 29,8 ± 4,8a 28,3 ± 3,5a 21,4 ± 4,5a

glucan from Euglena gracilis 1389,9 ± 217,0 1183,5 ± 135,6b 691,7 ± 76,9 353,9 ± 40,4

glucan from Saccharomyces cerevisiae 335,1 ± 123,5b 1155,1 ± 281,3b 1252,6 ± 321,6 820,1 ± 107,8b

Macrogard 757,6 ± 155,2 1246,1 ± 105,3b 969,6 ± 126,3 740,8 ± 130,1b

zymosan

279,1 ± 15,8a,b 255,0 ± 44,2 93,3 ± 21,0a 51,5 ± 19,0a

Table 5.3 The ROS response of porcine neutrophils stimulated with different concentrations of different -glucans determined by chemiluminescence. Data are shown as the means ± SEM of 4 pigs. PMA (20 µg/ml) and HBSS were used as a positive and negative control respectively and gave values of 937,8 ±

50,3 (PMA) and 21,0 ± 3,0 (HBSS) RLU. As the -glucan preparations could be contaminated with endotoxins, also the influence of LPS (10 and 1 µg/ml) was tested on the ROS production of neutrophils and gave values of 9,8 ± 6,9 (10 µg/ml) and 7,4 ± 5,6 (1 µg/ml) RLU. Values in the same column showing the same superscript letter are not significantly different (p > 0.05).


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Table 5.4 Influence of different -glucans on the lymphocyte proliferation. Data are shown as the mean counts per minute (cpm) ± SEM of 4 pigs. ConA (1 µg/ml) and cell medium were used as a positive and negative control respectively and gave values of 119647,9 ± 11251,64 (ConA) and 823,0 ± 265,5 (cell medium) cpm. Values in the same column showing the same superscript letter are not significantly different (p > 0.05).

-glucans Lymphocyte proliferation (cpm) following stimulation of PBMC with different concentrations

of - glucans


laminarin 1566,3 ± 162,4a,b 1377,0 ± 428,2a,b,c 737,6 ± 165,4a 1085,5 ± 287,8a,d

scleroglucan 2405,5 ± 247,9a,b 2127,1 ± 172,6a,b,c 3830,6 ± 759,7a,b,c 1459,1 ± 348,3a,b,c,d

curdlan 20942,5 ± 1933,8 14813,8 ± 1560,6 5663,1 ± 561,7b,c 2760,9 ± 477,6b,c

glucan from Euglena gracilis 4153,5 ± 1113,2b 3491,4 ± 858,4b 2718,0 ± 452,6a,c 1279,8 ± 197,8a,c,d

glucan from Saccharomyces cerevisiae 96,9 ± 13,9a 9,6 ± 3,1c 6347,5 ± 1057,6b,c 5199,8 ± 521,8

Macrogard 36,3 ± 10,2a 3663,8 ± 666,5a,b 6496,3 ± 1111,0a 2247,0 ± 290,6d

zymosan

9623,1 ± 1792,7 9679,8 ± 1590,7 7482,0 ± 965,9b 3029,1 ± 403,0b


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5.4.3. Cytokine production

Since, for most -glucans, an optimum response for proliferation and ROS production was seen with a

dose between 50 and 200 µg/ml, an intermediate -glucan dose (150 µg/ml) was tested for cytokine

analysis. None of the -glucans induced the release of IL-8, IL-4, IL-12, IL-6 or IFN-data not shown). All

-glucans, except for laminarin, gave rise to TNF- secretion (Figure 5.1A). Macrogard and the glucan

from Saccharomyces cerevisiae stimulated TNF- secretion significantly more (p < 0,05) than laminarin

and LPS. Furthermore, all -glucans except laminarin, gave a much stronger TNF- secretion than LPS.

Although all -glucans gave rise to IL-10 secretion, the highest secretion of IL-10 was found following

treatment with curdlan (Figure 5.1B). This secretion was significantly higher than this by all other -

glucans, except for Macrogard and scleroglucan (p < 0,05). While this latter -glucan had no influence on

ROS production and almost didn’t induce proliferation, surprisingly, it did had an effect on cytokine

production in contrast with the other soluble -glucan, laminarin.

IL-1secretion was determined in the supernatant of PBMC following stimulation with different

concentrations of -glucans (Figure 5.2A-D). Except for laminarin, all -glucans induced the release of IL-

1. However, at 800 µg/ml, no IL-1 could be detected in the supernatant of PBMC stimulated with

Macrogard (Figure 5.2A), while the other concentrations did give rise to IL-1 secretion (Figure 5.2B-D).

The glucan of Euglena gracilis induced the highest IL-1concentration, whereas for curdlan, IL-1

secretion increases as the concentration of the -glucan decreases.


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Figure 5.1 TNF-(A) and IL-10 (B) production after stimulation of PBMC (1.107) with 150 µg/ml of the

different -glucans or 1 µg/ml LPS. After 24h, the supernatant was harvested and cytokine concentrations were measured using commercially available ELISA kits. Data are shown as the means ± SEM of 4 pigs. Letters indicate a significant difference (p<0.05).

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Figure 5.2 IL-1 secretion by PBMC upon -glucan stimulation at different concentrations. PBMC were stimulated with different concentrations of -

glucans (800 µg/ml (A), 200 µg/ml (B), 50 µg/ml (C) and 5 µg/ml (D)). After 24h the supernatant was harvested and IL-1concentrations were measured using commercially available ELISA kits. Data are shown as the means ± SEM of 2 pigs.

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5.5. Discussion

Previous in vivo studies conducted in our laboratory indicated that -glucans supplemented at 500

mg/kg feed or 500 ppm could have a protective effect on an enterotoxigenic E. coli (ETEC) infection

(Stuyven et al., 2009). Furthermore, also other studies done in pigs, have shown that the optimal

inclusion level of -glucan is situated between 250 and 500 ppm, although most studies done in pigs

focused on growth performance (Dritz et al., 1995; Hahn et al., 2006; Hiss and Sauerwein, 2003a) and

only two research groups studied also the effect on immune parameters (Hahn et al., 2006; Hiss and

Sauerwein, 2003a). Knowing that this concentration had a beneficial effect, different -glucans in a

concentration range between 5 and 800 ppm were used in the present in vitro study to determine

whether they have a stimulatory effect on neutrophils, monocytes and lymphocytes. There are few

studies comparing the immunomodulatory effect of different -glucans at different concentrations

(Kudrenko et al., 2009; Ohno et al., 1996). Moreover, to our knowledge, this is the first study where this

is tested on immune cells of the pig.

The results obtained in the present work show that there’s a great difference in stimulatory capacity

between the -glucans. Indeed, the soluble -glucan laminarin showed almost no activity in the assays

used in our study, while all the particulate -glucans, like the -glucan from Euglena gracilis and all the -

glucans originating from Saccharomyces cerevisiae, do have a stimulatory effect. Many researchers

showed that biological activity by -glucans in vitro or in vivo, in terms of the synthesis of TNF- and H202

by macrophages, is related to solubility (Ishibashi et al., 2001; Michalek et al., 1998; Vassallo et al., 2000).

The observed differences in stimulatory capacity between soluble and particulate -glucans could be

explained by the need of cross-linking of membrane receptors. As such, Michalek and coworkers showed

that Betafectin, a soluble (1,3)-(1,6)glucan manufactured from Saccharomyces cerevisiae, requires

cross-linking of membrane receptors to stimulate respiratory burst in rat macrophages (Michalek et al.,

1998). The main receptor for -glucans is dectin-1, which was recently identified in the pig (Sonck et al.,

2009). Also other receptors have been implicated in -glucan recognition such as complement receptor 3

(CR3), Toll-like receptor (TLR) 2 and 6, lactosylceramide and scavenger receptors.

In our study, the soluble scleroglucan is immunostimulatory for several but not all tests. However, in

high concentrations, scleroglucan has gelforming characteristics in solution. The tertiary structure, which

this -glucan assumes in solution, is a rigid triple helix, a highly ordered solution conformation, while the

other soluble -glucan (laminarin) has a single helical solution conformation (Mueller et al., 2000).

Furthermore, scleroglucan has a much higher molecular weight than laminarin (Table 5.1). It has been


89

demonstrated that -glucans with a higher molecular weight exhibit higher binding affinities for the -

glucan receptors (Mueller et al., 2000). Those two reasons could be an explanation for the fact that

scleroglucan did stimulate lymphocyte proliferation and TNF- production in contrast with laminarin.

However, Adams and coworkers demonstrated that scleroglucan has backbone regions that are not

(1,3)-linked. They hypothesized that the presence of other than (1,3)-substituted glucose residues in

the backbone could limit interaction with dectin-1 molecules and could result in lower affinity (Adams et

al., 2008a). Besides their difference in solution conformation, this may be an explanation for the fact that

scleroglucan shows less immunological activity than all particulate -glucans, which are all glucose

polymers with only (1,3)-linked backbones (Table 5.1).

The immunological potency of -glucans is not only dependent on molecular mass, solution

conformation and backbone structure, also the degree of branching is an important factor. As such,

particulate -glucans, like curdlan and the -glucan from Euglena gracilis, are both unbranched (1,3)-

linked glucans, while the -glucans originating from Saccharomyces cerevisiae, namely -glucan from

Saccharomyces cerevisiae, Macrogard and zymosan have 1,6)-linked side-chain branches (Table 5.1).

It’s remarkable that, allthough not all cell types became stimulated, the stimulatory capacity of curdlan

and the -glucan from Euglena gracilis increased with increasing concentrations, while the stimulation by

Macrogard and the -glucan from Saccharomyces cerevisiae seemed to decrease for the highest

concentration. Trp221 and His223, two amino-acids which are highly conserved in all the dectin-1

homologues of different species, including the pig, play a crucial role in the formation of the dectin-1 -

glucan binding site (Adachi et al., 2004; Brown et al., 2007). Adams and coworkers suggested that (1,3)-

glucans with a backbone composed of seven or more glucose repeat subunits, assume a helical

conformation, which facilitates the interaction with the Trp221/His223 groove in dectin-1. Nevertheless,

because of the increased affinity of dectin-1 for glucans with (1,6) side-chain branches, they suggested

that the binding groove preferentially interacts with the branched glucan (Adams et al., 2008a). We

speculate that more dectin-1 molecules become bound when the concentration of linear (1,3)-linked

glucans increases, and as a result, biological activity is increased. As the presence of a single (1,6)- side

chain branch on a 1,3)-glucan polymer, can already dramatically increase recognition by dectin-1

(Adams et al., 2008a; Graham et al., 2006), we suspect that small concentrations of large 1,6)-

branched glucan polymers bind to many dectin-1 molecules resulting in a high avidity binding. In too high

concentrations, however, it’s possible that (1,6)-linked glucans stereochemically interfere with each

other, and as a result less dectin-1 molecules become bound. To examine this, 1,6)-side chain branches


90

should be removed from, for instance Macrogard, and the effect on binding to dectin-1 should be

measured using a BIAcoreT100TM surface plasmon resonance instrument (Uppsala, Sweden).

Not only the nature and the concentration of the -glucan determines the stimulatory effect, but also

the cell type that is targeted. As such, while curdlan doesn’t seem to activate monocytes or neutrophils,

it does stimulate lymphocyte proliferation and cytokine production. The high concentration of IL-10

induced by curdlan (Figure 5.1B), makes us suspect that this is probably caused by the LPS

contamination, as the endotoxin concentrations for curdlan were higher than 1 unit/ml. Several studies

have already shown that (1,3)-glucans in combination with LPS can result in lower concentrations of

inflammatory cytokines and higher concentrations of IL-10, an anti-inflammatory cytokine (Engstad et al.,

2002; Hetland et al., 2000; Vereschagin et al., 1998). Another -glucan, which has a variable effect on the

different tested cell types, is the -glucan from Euglena gracilis. In contrast with curdlan, the

proliferating effect of the -glucan from Euglena gracilis is less pronounced over the different

concentrations, while it does activate ROS production of neutrophils and monocytes. This stimulatory

effect on superoxide anion production by both cell types is mainly seen at high concentrations. For both

cell types, ROS production was significantly different for all other -glucans at a concentration of 800

µg/ml. It may be possible that higher concentrations could have even a stronger effect on respiratory

burst activity than already seen for 800 µg/ml.

Both products originating from Saccharomyces cerevisiae (Macrogard and -glucan from Saccharomyces

cerevisiae) seem to have an inflammatory effect. Besides the proliferation of lymphocytes, they

stimulate also neutrophils and monocytes and give rise to TNF- and IL-1 production, two pro-

inflammatory cytokines. It’s however remarkable that at high concentrations, both -glucans don’t

stimulate lymphocyte proliferation at all. As a lot of cell debris was seen after the 72h incubation period

and as confirmed by the MTT test, it seems that both -glucans do have cytotoxic effects at high

concentrations.

Presence of excess IL-1 can be an indication of enhanced activation of macrophages. This may lead to

suppression of proliferation (Albina et al., 1991; Ganguly et al., 2001; Keller, 1975; Metzger et al., 1980).

To test this negative correlation between IL-1 production and T-lymphocyte proliferation, Il-1

concentrations were also determined in the supernatant of PBMC stimulated with the same

concentrations of -glucans as used in the proliferation tests (800 µg/ml, 200 µg/ml, 50 µg/ml and 5

µg/ml). There was only a concentration-dependent effect for zymosan and the -glucan of

Saccharomyces cerevisiae, as such that IL-1 secretion decreased with increasing concentrations of -


91

glucan (except for 5 µg/ml). However, when the proliferation tests were repeated with a monoclonal

neutralizing -porcine IL-1 antibody, no variation in proliferation was seen (data not shown), suggesting

that IL-1 is not the only cytokine which has an influence on proliferation after stimulation of PBMC with

-glucans.

In summary, we observed that particulate -glucans do stimulate different porcine cell types, in contrast

with soluble ones. The immunostimulating capacity however is strongly dependent on structural factors

such as backbone structure and degree of branching.



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Mucksova, J., Babicek, K., Pospisil, M., 2001, Particulate 1,3-beta-D-glucan, carboxymethylglucan and sulfoethylglucan--influence of their oral or intraperitoneal administration on immunological respondence of mice. Folia Microbiol (Praha) 46, 559-563. Mueller, A., Raptis, J., Rice, P.J., Kalbfleisch, J.H., Stout, R.D., Ensley, H.E., Browder, W., Williams, D.L., 2000, The influence of glucan polymer structure and solution conformation on binding to (1-->3)-beta-D-glucan receptors in a human monocyte-like cell line. Glycobiology 10, 339-346. Ohno, N., Egawa, Y., Hashimoto, T., Adachi, Y., Yadomae, T., 1996, Effect of beta-glucans on the nitric oxide synthesis by peritoneal macrophage in mice. Biological & Pharmaceutical Bulletin 19, 608-612. Rubin-Bejerano, I., Abeijon, C., Magnelli, P., Grisafi, P., Fink, G.R., 2007, Phagocytosis by human neutrophils is stimulated by a unique fungal cell wall component. Cell Host Microbe 2, 55-67. Sonck, E., Stuyven, E., Goddeeris, B., Cox, E., 2009, Identification of the porcine C-type lectin dectin-1. Vet Immunol Immunopathol 130, 131-134. Stuyven, E., Cox, E., Vancaeneghem, S., Arnouts, S., Deprez, P., Goddeeris, B.M., 2009, Effect of beta-glucans on an ETEC infection in piglets. Vet Immunol Immunopathol 128, 60-66. Thornton, B.P., Vetvicka, V., Pitman, M., Goldman, R.C., Ross, G.D., 1996, Analysis of the sugar specificity and molecular location of the beta-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18). J Immunol 156, 1235-1246. Van der Stede, Y., Cox, E., Verdonck, F., Vancaeneghem, S., Goddeeris, B.M., 2003, Reduced faecal excretion of F4+-E coli by the intramuscular immunisation of suckling piglets by the addition of 1alpha,25-dihydroxyvitamin D3 or CpG-oligodeoxynucleotides. Vaccine 21, 1023-1032. Vassallo, R., Standing, J.E., Limper, A.H., 2000, Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses. J Immunol 164, 3755-3763. Vereschagin, E.I., van Lambalgen, A.A., Dushkin, M.I., Schwartz, Y.S., Polyakov, L., Heemskerk, A., Huisman, E., Thijs, L.G., van den Bos, G.C., 1998, Soluble glucan protects against endotoxin shock in the rat: the role of the scavenger receptor. Shock 9, 193-198. Vetvicka, V., Thornton, B.P., Ross, G.D., 1996, Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J Clin Invest 98, 50-61. Vetvicka, V., Vetvickova, J., Frank, J., Yvin, J.C., 2008, Enhancing effects of new biological response modifier beta-1,3 glucan sulfate PS3 on immune reactions. Biomed Pharmacother 62, 283-288. Williams, D.L., Yaeger, R.G., Pretus, H.A., Browder, I.W., McNamee, R.B., Jones, E.L., 1989, Immunization against Trypanosoma cruzi: adjuvant effect of glucan. Int J Immunopharmacol 11, 403-410. Xia, Y., Vetvicka, V., Yan, J., Hanikyrova, M., Mayadas, T., Ross, G.D., 1999, The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J Immunol 162, 2281-2290.

Chapter 6 : Role of dectin-1 in porcine leukocytes

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Chapter 6 : The specific dectin-1 inhibitor laminarin doesn’t block the -glucan induced effects in porcine leukocytes

Eva Sonck, Bruno Goddeeris, Eric Cox


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6.1. Abstract

-glucans are natural occurring carbohydrates that form the major cell wall component of fungi, yeast,

some bacteria and seaweed. These widely studied glucose polymers can induce direct receptor-mediated

immunomodulating activities. Different -glucan binding receptors have been described in humans and

mice, including dectin-1, which is considered as the main -glucan receptor. In the pig, it is still unknown

through which receptors -glucans can exert their immunomodulating effects. A better understanding of

the mechanism of -glucan recognition and subsequent immune activation is necessary to optimize their

use as feed additive in pig industry. In the present study, we investigated whether laminarin, one of the

most effective inhibitors of dectin-1, could block the immunomodulating effects of different -glucans on

porcine leukocytes. Dectin-1 is probably not involved in -glucanrecognition and –signaling in porcine

monocytes as well as neutrophils, as laminarin couldn’t block ROS production by both cell types. The role

of dectin-1 in -glucan induced lymphocyte proliferation is less clear, as laminarin could only inhibit

proliferation induced by all -glucans, after stimulation with 200 µg/ml. For the other concentrations,

the inhibiting effect of laminarin was -glucan dependent.

6.2. Introduction

-glucans are a heterogeneous group of conserved glucose polymers, which are abundantly present in

the cell wall of fungi (Chihara et al., 1969), algae (Elyakova and Zvyagintseva, 1974), yeast (Manners et

al., 1973) and some bacteria (Harada et al., 1968). Particularly -glucans with a main chain of (1,3)-

linked glucose units and (1,6)-linked branches have been shown to activate immune cells. Their

immunomodulating effects are attributed to their ability to bind different pattern recognition receptors

(PRRs), including complement receptor 3 (CR3) (Ross et al., 1985), lactosylceramide (Zimmerman et al.,

1998), scavenger receptors (Rice et al., 2002) and dectin-1 (Brown and Gordon, 2001). Although it has

been demonstrated that each of these receptors can recognize and respond to -glucans, it is possible

that still other receptors remain to be identified. Since the discovery of dectin-1 as -glucan binding

receptor, most of the studies focused on and attributed the immunomodulating effects of -glucans to

this receptor (Brown and Gordon, 2001). Nevertheless, controversy remains on the relative importance

of each of the receptors and it is likely that the different aspects of the -glucan induced immune

response are the result of the binding to different PRRs, depending on the cell type and on the nature of

the -glucan preparation (Murphy et al., 2010; Sonck et al., 2010). In the pig, we have previously shown


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that -glucans have an immunomodulating effect on monocytes, neutrophils and lymphocytes (Sonck et

al., 2010). Given the importance of dectin-1 in humans and mice, we wanted to study whether the

observed effects on porcine leukocytes could be attributed to this receptor. Hereto, -glucan binding to

dectin-1 was blocked by laminarin, a specific dectin-1 inhibitor.


6.3.1. -glucans

Laminarin, curdlan, zymosan and the -glucan purified from Euglena gracilis and Saccharomyces

cerevisiae, were purchased from Sigma (Bornem, Belgium). Scleroglucan and Macrogard were kindly

provided by INVE (Belgium) and Biotec Pharmacon ASA (Norway), respectively. A description and

comparison of the carbohydrate structures, just as the preparation and storage of these -glucans, has

been published (Sonck et al., 2010). The endotoxin concentration present in each -glucan preparation,

was determined by the Chromogenic Limulus Amebocyte Lysate (LAL) Test (Cambrex Bio Science

Walkersville, Inc.) and, with exception of curdlan (47 endotoxin units/µg -glucan), were consistently

lower than 0.5 endotoxin units/µg -glucan.




6.3.3. Isolation of PBMC and neutrophils Peripheral blood monomorphonuclear cells (PBMCs) and neutrophils were isolated by density gradient

centrifugation on respectively Lymphoprep (NYCOMED Pharma AS, Life Technologies, Belgium) and a

discontinuous Percoll gradient (GE healthcare, Sweden) (68% and 75%) as described previously (Sonck et

al., 2010).

6.3.4. Influence of laminarin on the -glucan induced ROS production by porcine neutrophils and monocytes

ROS production was measured using a chemiluminescence assay as described by Donne et al. (2005),

with some modifications. Neutrophils were seeded in a 96-well plate at 2 *105 cells/well in 200 ml of

RPMI without phenol-red, while the PBMCs were seeded at a concentration of 2 *106 cells/well. The

plates were incubated at 37°C for 2 h in a humidified atmosphere with 5% CO2, so that the neutrophils or


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the monocytes could adhere to the plastic surface. To investigate if laminarin could reduce or inhibit the

effect of -glucan on ROS production, first laminarin (1 mg/ml) was added or not to the cells and the

plates were placed back for 1h in the CO2 incubator. Subsequently, the supernatant was removed and

replaced by 175 µl luminol (0.5 mM). After 5 minutes of background measurement at 37°C, 25 µl of the

-glucans was added as well as the positive control agent phorbol myristate acetate (PMA) (20 µg/ml).

Different concentrations (400 µg/ml, 200 µg/ml and 100 µg/ml) of each -glucan were tested after which

ROS production was measured during 120 min in the integration mode. Each stimulation was performed

in duplicate. ROS production was expressed as relative light units (RLU).

6.3.5. Influence of laminarin on the-glucan induced PBMC proliferation

PBMC were seeded in a 96-well plate at 5*105 cells/well (100 µl/well) in leukocyte-medium (RPMI-1640

(Gibco BRL, Life Technologies, Belgium) containing foetal calf serum (FCS) (10%), nonessential amino-

acids, Na-pyruvate (100 µg/ml), L-glutamine (292 µg/ml), penicillin (100 IU/ml), streptomycin (100

µg/ml) and kanamycin (100 µg/ml)). To investigate the influence of laminarin on the -glucan induced

PBMC proliferation, laminarin (50 µl; final concentration of 1 mg/ml) or leukocyte-medium (50 µl) was

added to the cells. Subsequently, different concentrations of the -glucans (800 µg/ml, 200 µg/ml, 50

µg/ml and 5 µg/ml) or the control agents (Concanavalin A (ConA) (Sigma-Aldrich, Belgium) as positive

control or medium as negative control) were added. The plates were incubated during 72 h at 37°C in a

humidified atmosphere with 5% CO2. Then, the cells were pulse-labelled with 1 µCi of [H3]-thymidine

(Amersham ICN, UK) per well for 18 h at 37 °C and 5% CO2, where after the cells were harvested onto

glass fibre filters (PerkinElmer, Life Science, Belgium). Each stimulation was performed in duplicate and

the results are presented as counts per minute (cpm).

6.3.6. Influence of laminarin on the -glucan induced cytokine production

PBMC were seeded in a 24-well plate at 1*107 cells. Laminarin (1 mg/ml) or medium was added

simultaneously with 150 µg/ml of the different -glucans to the cells. After an incubationperiod of 24h at

37 °C in a humidified atmosphere with 5% CO2 , the supernatant was harvested and stored at -20°C. The

concentrations of IL-1, IL-6, TNF- and IL-10 were measured using commercially available porcine-

specific ELISA kits (R&D Systems Inc, USA) according to the manufacturer’s protocols.


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6.3.7. Statistics All experiments were performed with cells of four different pigs. Data are presented as the mean ± SEM.

Data from the proliferation tests were analyzed with SPSS16 using an independent-Samples T-test to

compare conditions with or without laminarin. Levene’s test was used to assess the homogeneity of the

variances. Data were considered significant at p < 0.05 (two-sided test).

6.4. Results and discussion Laminarin, together with glucan phosphate, has shown a high affinity interaction for dectin-1 (Adams et

al., 2008a) and it has been demonstrated that both -glucans are the most effective inhibitors of

zymosan-binding by dectin-1 (Herre et al., 2004a). The inhibiting effect of laminarin does not only apply

to zymosan, as also dectin-1 mediated binding and signaling of other -glucans could be inhibited by

laminarin (Rothfuchs et al., 2007). In order to explore the potential role of dectin-1 in -glucan induced

ROS production in the pig, monocytes and neutrophils were pre-incubated for 1 hour with 1 mg/ml

laminarin to allow blocking of dectin-1. Subsequently, -glucans at different concentrations (400, 200

and 100 µg/ml) were added to the cells and ROS production was measured and compared to the non-

laminarin treated cells. The results are presented in Figure 6.1 (monocytes) and Figure 6.2 (neutrophils).

Laminarin could not block ROS production for none of the -glucans and for none of both cell types,

demonstrating that dectin-1 is not, or to a limited extent, involved in -glucan recognition and –signaling

in monocytes as well as neutrophils. Although porcine monocytes and neutrophils express dectin-1

mRNA, protein expression still has to be shown (Sonck et al., 2009). However, as the receptor is

expressed on the surface of human monocytes and neutrophils (van Bruggen et al., 2009; Willment et al.,

2001), most likely expression of dectin-1 does also occur on the surface of both cell types in the pig.

Recently, the function of dectin-1 in -glucan activation of human neutrophils was seriously questioned,

as it was demonstrated that the phagocytosis of zymosan as well as whole yeast was completely

dependent on CD11b/CD18, also known as complement receptor 3 (CR3) (van Bruggen et al., 2009). Also

for human monocyte-derived macrophages, no role for dectin-1 could be established yet, although

phagocytosis of Saccharomyces cerevisiae by these cells was not only mediated via CR3, but also through

other not yet identified pattern recognition receptors (PRRs) (van Bruggen et al., 2009). These findings

seem to be in conflict with the fact that dectin-1 was shown to be the major receptor for zymosan on

murine macrophages and DCs (Brown, 2006). The reason for these species-dependent differences is not

clear and although nothing is known yet about the expression levels of dectin-1 on primary murine

neutrophils, van Bruggen et al. (2009) suggested that the low expression levels on human neutrophils


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may explain the minor or even the absence of a role of human dectin-1 in yeast phagocytosis. Another

explanation may be that -glucans have a higher affinity for murine dectin-1 than for the human variant.

By performing multiple sequence alignment, the inter-species homology of the carbohydrate recognition

domain (CRD) of dectin-1 could be determined. Porcine dectin-1 showed a similarity of only 58% with

murine dectin-1, while the identity between human and mice was only 61%. The similarity between pig

and human was 73%. So it is possible that the larger differences with the murine variant can lead to

reduced -glucan affinity for human as well as porcine dectin-1, although Trp221 and His223, two amino

acids which have been shown to play a crucial role in -glucan binding, are highly conserved in all dectin-

1 homologues of the different species (Adachi et al., 2004; Brown et al., 2007). In this respect, it would

be interesting to study the binding affinity between a well-characterized -glucan and recombinant

dectin of human, mouse and pig using a BIAcore surface plasmon resonance instrument (Uppsala,

Sweden).


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Figure 6.1 Influence of laminarin on the -glucan induced ROS production by monocytes. Monocytes were pre-incubated with 1 mg/ml laminarin, after which the cells were stimulated with 400 µg/ml (A), 200 µg/ml (B) and

100 µg/ml (C) of the indicated -glucan preparations. ROS production was determined by chemiluminescence. Data are shown as the mean ± SEM of four pigs. PMA (20 µg/ml) was used as a positive control and gave values of 257 RLU. Laminarin (1 mg/ml) gave values of 6 RLU.

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Figure 6.2 Influence of laminarin on the -glucan induced ROS production by neutrophils. Neutrophils were pre-incubated with 1 mg/ml laminarin, after which the cells were stimulated with 400 µg/ml (A), 200 µg/ml (B) and

100 µg/ml (C) of the indicated -glucan preparations. ROS production was determined via chemiluminescence. Data are shown as the mean ± SEM of four pigs. PMA (20 µg/ml) was used as a positive control and gave values of 520 RLU. Laminarin (1 mg/ml) gave values of 7 RLU.

To investigate the potential role of dectin-1 on the -glucan induced PBMC proliferation as well as

cytokine production, laminarin and -glucans were added simultaneously to the cells. For lymphocyte

proliferation, the results are presented in Figure 6.3. For all tested concentrations (800, 200, 50 and 5

µg/ml), lymphocyte proliferation induced by scleroglucan, curdlan and zymosan is reduced when

laminarin was added, although this reduction was only significant for slceroglucan after stimulation with

800 µg/ml (p=0.022 < 0.05). Also after stimulation with 200 µg/ml of Macrogard and the glucan from

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Euglena gracilis, laminarin reduced lymphocyte proliferation and for Macrogard, this effect was

significant (p=0.012 < 0.05) (Figure 6.3B).

Figure 6.3 Influence of laminarin on the -glucan induced PBMC proliferation. PBMC were incubated simultaneously with laminarin (1 mg/ml) and different concentrations (800 µg/ml (A), 200 µg/ml (B), 50 µg/ml (C)

and 5 µg/ml (D)) of the indicated -glucans. Data are shown as the mean ± SEM of four pigs. ConA (1 µg/ml) was used as a positive control and gave values of 52*10

3 without laminarin and 56*10

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used as a negative control and gave values of 418 cpm without laminarin and 378 cpm with laminarin. The asterisk (*) indicate a significant difference between the condition with and without laminarin (p<0.05).

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In humans and mice, the extracellular domain of dectin-1 could, besides to -glucan, also bind to a yet

unidentified ligand on the surface of T-cells and could promote their proliferation. As the latter effect

was only seen in the presence of anti-CD3, it was suggested that dectin-1 could deliver a costimulatory

signal to T-cells (Ariizumi et al., 2000; Brown and Gordon, 2001; Grunebach et al., 2002; Willment et al.,

2001). It is possible that porcine dectin-1 could also deliver a costimulatory signal for the -glucan

induced T-cell proliferation and that this effect is reduced when adding laminarin. If the hypothesis of

costimulation by dectin-1 holds true, one should expect that -glucan induced cytokine production is

affected. However, as demonstrated in Figure 6.4, laminarin doesn’t have an effect on the -glucan

induced production of IL-1, TNF- and IL-10 and only has a small effect on the production of IL-6 by

some of the -glucans (curdlan, glucan from Saccharomyces cerevisiae and zymosan) (Figure 6.4). In

addition to these observations, it is remarkable that only the T-cell proliferation induced by Macrogard

and the glucan from Euglena gracilis after stimulation with 200 µg/ml and 5 µg/ml is reduced by

laminarin, while for the other -glucans, the inhibiting effect was not concentration dependent. The

reasons for these discrepancies are currently unclear, but it shows that other yet unidentified factors

could play a role in the suppressive effect of laminarin on -glucan induced T-cell proliferation. It is

possible that laminarin inhibits -glucan induced T-cell proliferation through another mechanism

independent of dectin-1. Indeed, it has been demonstrated that the T-cell binding capacity of human

dectin-1 couldn’t be blocked by laminarin, suggesting that the T-cell binding site is different from the -

glucan binding site (Willment et al., 2001).

In humans, dectin-1 expression was also found on a subset of CD4+ T-cells (CD3lowCD4low) as well as B-

cells. It has been suggested that these cells may be involved in interaction with other lymphocytes

resulting in T-cell proliferation, through recognition of the unidentified endogenous ligand (Willment et

al., 2001). However, in pigs, this hypothesis is very unlikely as dectin-1 mRNA expression wasn’t detected

in porcine T-cells.


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Figure 6.4 Influence of laminarin on the -glucan-induced IL-1, IL-6, TNF- and IL-10 production. PBMC were

stimulated simultaneously with laminarin (1 mg/ml) and 150 µg/ml -glucans for 24h. Cytokine-production was measured by ELISA.

In summary, we observed that dectin-1 is probably not involved in -glucanrecognition and –signaling in

porcine monocytes as well as neutrophils. The role of dectin-1 in -glucan induced lymphocyte

proliferation is less clear. Further studies need to be done to clarify which -glucan receptors do play an

important role in the pig.

6.5. Acknowledgements

This research was funded by a PhD grant of the Institute for the Promotion of Innovation through


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6.6. References Adachi, Y., Ishii, T., Ikeda, Y., Hoshino, A., Tamura, H., Aketagawa, J., Tanaka, S., Ohno, N., 2004, Characterization of beta-glucan recognition site on C-type lectin, dectin 1. Infect Immun 72, 4159-4171. Adams, E.L., Rice, P.J., Graves, B., Ensley, H.E., Yu, H., Brown, G.D., Gordon, S., Monteiro, M.A., Papp-Szabo, E., Lowman, D.W., Power, T.D., Wempe, M.F., Williams, D.L., 2008, Differential high-affinity interaction of dectin-1 with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and side-chain branching. J Pharmacol Exp Ther 325, 115-123. Ariizumi, K., Shen, G.L., Shikano, S., Xu, S., Ritter, R., 3rd, Kumamoto, T., Edelbaum, D., Morita, A., Bergstresser, P.R., Takashima, A., 2000, Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning. J Biol Chem 275, 20157-20167. Brown, G.D., 2006, Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat Rev Immunol 6, 33-43. Brown, G.D., Gordon, S., 2001, Immune recognition. A new receptor for beta-glucans. Nature 413, 36-37. Brown, J., O'Callaghan, C.A., Marshall, A.S., Gilbert, R.J., Siebold, C., Gordon, S., Brown, G.D., Jones, E.Y., 2007, Structure of the fungal beta-glucan-binding immune receptor dectin-1: implications for function. Protein Sci 16, 1042-1052. Chihara, G., Maeda, Y., Hamuro, J., Sasaki, T., Fukuoka, F., 1969, Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) sing. Nature 222, 687-688. Donne, E., Pasmans, F., Boyen, F., Van Immerseel, F., Adriaensen, C., Hernalsteens, J.P., Ducatelle, R., Haesebrouck, F., 2005, Survival of Salmonella serovar Typhimurium inside porcine monocytes is associated with complement binding and suppression of the production of reactive oxygen species. Vet Microbiol 107, 205-214. Elyakova, L.A., Zvyagintseva, T.N., 1974, A study of the laminarins of some Far-Eastern, brown seaweeds. Carbohydr Res 34, 241-248. Grunebach, F., Weck, M.M., Reichert, J., Brossart, P., 2002, Molecular and functional characterization of human Dectin-1. Exp Hematol 30, 1309-1315. Harada, T., Misaki, A., Saito, H., 1968, Curdlan - a Bacterial Gel-Forming Beta-1 3-Glucan. Archives of Biochemistry and Biophysics 124, 292-&. Herre, J., Gordon, S., Brown, G.D., 2004, Dectin-1 and its role in the recognition of beta-glucans by macrophages. Mol Immunol 40, 869-876. Manners, D.J., Masson, A.J., Patterson, J.C., Bjorndal, H., Lindberg, B., 1973, The structure of a beta-(1--6)-D-glucan from yeast cell walls. Biochem J 135, 31-36. Murphy, E.A., Davis, J.M., Carmichael, M.D., 2010, Immune modulating effects of beta-glucan. Curr Opin Clin Nutr Metab Care 13, 656-661. Rice, P.J., Kelley, J.L., Kogan, G., Ensley, H.E., Kalbfleisch, J.H., Browder, I.W., Williams, D.L., 2002, Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-D-glucans. J Leukoc Biol 72, 140-146. Ross, G.D., Thompson, R.A., Walport, M.J., Springer, T.A., Watson, J.V., Ward, R.H., Lida, J., Newman, S.L., Harrison, R.A., Lachmann, P.J., 1985, Characterization of patients with an increased susceptibility to bacterial infections and a


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genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1. Blood 66, 882-890. Rothfuchs, A.G., Bafica, A., Feng, C.G., Egen, J.G., Williams, D.L., Brown, G.D., Sher, A., 2007, Dectin-1 interaction with Mycobacterium tuberculosis leads to enhanced IL-12p40 production by splenic dendritic cells. J Immunol 179, 3463-3471. Sonck, E., Stuyven, E., Goddeeris, B., Cox, E., 2009, Identification of the porcine C-type lectin dectin-1. Vet Immunol Immunopathol 130, 131-134. Sonck, E., Stuyven, E., Goddeeris, B., Cox, E., 2010, The effect of beta-glucans on porcine leukocytes. Vet Immunol Immunopathol 135, 199-207. van Bruggen, R., Drewniak, A., Jansen, M., van Houdt, M., Roos, D., Chapel, H., Verhoeven, A.J., Kuijpers, T.W., 2009, Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for beta-glucan-bearing particles. Mol Immunol 47, 575-581. Willment, J.A., Gordon, S., Brown, G.D., 2001, Characterization of the human beta -glucan receptor and its alternatively spliced isoforms. J Biol Chem 276, 43818-43823. Zimmerman, J.W., Lindermuth, J., Fish, P.A., Palace, G.P., Stevenson, T.T., DeMong, D.E., 1998, A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J Biol Chem 273, 22014-22020.

Chapter 7 : The effect of -glucans on the maturation of porcine MoDCs

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Chapter 7 : Varying effect of different β-glucans on the maturation of porcine monocyte-derived dendritic cells

Eva Sonck, Bert Devriendt, Bruno Goddeeris, Eric Cox

Submitted to Clinical and Vaccine Immunology


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7.1. Abstract Beta-glucans are well known for their immunomodulatory capacities in human and mice. For this reason

together with the European ban on growth-promoting antibiotics, -glucans are intensively used in pig

feed. However, as described in the present study, there is much variation in the stimulatory capacity

between -glucans of different sources. As dendritic cells (DCs) are the first cells which are encountered

after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of

two concentrations (5 and 10 µg/ml) of five commercial -glucanpreparations, differing in structure and

source, on porcine monocyte-derived dendritic cells (MoDCs). Although all -glucans gave rise to a

significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypical

maturation. Besides Macrogard, also zymosan, another -glucan derived from Saccharomyces cerevisiae,

and curdlan significantly improved the T cell stimulatory capacity of MoDCs. Most interesting however, is

the cytokine secretion profile of curdlan-stimulated MoDCs as only curdlan induced significant higher

expression levels of IL-1, IL-6, IL-10 and IL-12/IL-23p40. As the cytokine profile of DCs influences the

outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful

choice is necessary when -glucans are used as dietary supplement.

7.2. Introduction Dendritic cells (DCs) are the directors of the immune system and form the messengers between the

innate and the adaptive immune system. Immature DCs identify pathogens by recognizing signatures

present in microbes, so-called pathogen-associated molecular patterns (PAMPs), through the expression

of pattern-recognition receptors (PRRs). Recognition of PAMPs by these PRRs results in the activation

and maturation of DCs. This maturation process is associated with a loss of the phagocytic activity and an

upregulation of MHC and costimulatory molecules, such as CD80, CD86 and CD40. Besides functional and

phenotypical changes, the exposure of DCs to microbial components results in the production of

cytokines that modulate the T-cell polarization and their functions. Upon interaction with DCs, CD4+ T-

cells can differentiate into a variety of effector subsets, including Th1, Th2 cells and the more recently

identified Th17 cells, and regulatory T-cells (Iwasaki and Medzhitov, 2010; Manicassamy and Pulendran,

2009; Zhou et al., 2009). Furthermore, DCs have been shown to trigger B-cell growth and differentiation

(Dubois et al., 1998; Jego et al., 2003). DCs are thus capable of modulating the nature of immune

responses, which in turn is dependent on the type of PRR that is activated. Phagocytes, such as DCs,

express a wide variety of PRRs, such as Toll-like receptors (TLRs), nucleotide-binding oligomerization


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domain (NOD)-like receptors (NLRs) and C-type lectin receptors (CLRs) on their cell surface (Lee and Kim,

2007; Robinson et al., 2006; Trinchieri and Sher, 2007). Dectin-1, a C-type lectin receptor and the most

important PRR recognizing -glucans, is expressed by various antigen-presenting cells (APCs), including

immature DCs (ImDCs) and macrophages (Willment et al., 2005).

-glucans are one of the most abundant forms of polysaccharides found inside the cell wall of bacteria,

fungi and yeasts (Novak and Vetvicka, 2009). All -glucans are glucose polymers linked by a 1,3 linear -

glycosidic chain core and, depending on the source, they differ in length and branching structures. -

glucans have a number of beneficial effects on the immune system, making them interesting for the

development of -glucan-based therapeutics and food supplements (Chan et al., 2009; Zekovic et al.,

2005). In humans, -glucans are regularly used as prebiotic supplement to increase the stability of the

gut flora, to augment innate immune responses and to orchestrate healthy immune responses (Vos et

al., 2007). In the pig industry, -glucans are also applied as dietary supplements. However, the molecular

mechanisms through which different -glucans exert their effects, are not well known (Vetvicka and

Vetvickova, 2007). Nevertheless, it is important to know and understand the effect of different -glucans

on the immune system to use -glucans efficiently in practice. In a previous study, we tested the direct

effect of different -glucanpreparations on porcine monocytes, neutrophils and lymphocytes (Sonck et

al., 2010). In the present study, we focused on the effects they have on DC maturation as these cells are

the most important antigen-presenting cells and key players in the initiation and stimulation of the

adaptive immunity.


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7.3.1. -glucans and LPS

Laminarin, curdlan, zymosan and the -glucan purified from Euglena gracilis were purchased from Sigma

(Bornem, Belgium), just as LPS (serotype 055:B5), which served as a control. Macrogard, which is

currently used as dietary supplement in pig industry, was kindly provided by Biotec Pharmacon ASA

(Norway). A description and comparison of the carbohydrate structures, just as the preparation and

storage of these -glucans, has previously been published (Sonck et al., 2010). The endotoxin

concentration present in each -glucan preparation, was determined by the Chromogenic Limulus

Amebocyte Lysate (LAL) Test (Cambrex Bio Science Walkersville, Inc.) and, with exception of curdlan (47

endotoxin units/µg -glucan), were consistently lower than 0.5 endotoxin units/µg -glucan.




7.3.3. Generation of monocyte-derived dendritic cells Porcine monocyte-derived dendritic cells (MoDCs) were generated from peripheral blood

monomorphonuclear cells (PBMC). Briefly, PBMC were isolated by density gradient centrifugation on

Lymphoprep (NYCOMED Pharma AS, Life Technologies, Merelbeke, Belgium). CD172a+ cells were

isolated from the PBMC fraction by positive magnetic activated cell separation (MACS; Miltenyi-Biotec,

Bergisch Gladbach, Germany) using anti-CD172a monoclonal antibody (mAb) (74-12-15A; Pescovitz et al.,

1984 ) and anti-mouse IgG microbeads together with LS separation columns (Miltenyi-Biotec). The

obtained cells were cultured in 24-well plates at a density of 5.105 cells in phenol-red free Dulbecco’s

modified Eagle’s medium (DMEM, Gibco, Merelbeke, Belgium) containing fetal calf serum (FCS) (10%)

(Greiner), penicillin (100 IU/ml) (Gibco), streptomycin (100 µg/ml) (Gibco), recombinant porcine (rp) GM-

CSF (Inumaru et al., 1998) and rpIL-4 (R&D systems) and incubated at 37°C in a humidified atmosphere at

5% CO2 to generate monocyte-derived dendritic cells as previously described (Carrasco et al., 2001).

After three days, the cultures were supplemented with fresh cytokines. After another day, cells were

stimulated for 24 h with 5 or 10 µg/ml of the different -glucans or LPS.


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7.3.4. Phenotyping of MoDCs The surface expression of various DC maturation markers after stimulation was assessed by flow

cytometry (FACSCanto). Upon stimulation with 5 and 10 µg/ml of the different -glucans or 1 and 10

µg/ml of LPS, MoDCs were harvested, washed with RPMI-1640 + 1% FCS and labeled with a primary

mouse mAb for 20 minutes at 4°C. The following primary mAbs were used to identify the maturation

markers: anti-MHCII (MSA3 (Lunney et al., 1994), anti-CD40 (G28-5) (Bimczok et al., 2007) and a human

CTLA4-muIg fusion protein (Ancell, Bayport, MN, USA) to detect the expression of CD80 and CD86. Cells

stained with isotype-matched irrelevant mAbs were used as a negative control. After incubation, cells

were washed and stained with FITC conjugated F(ab’)2 fragments of sheep anti-mouse IgG antibodies

(Sigma) for another 20 minutes at 4°C. Next, the cells were washed and propidium iodide (PI) was added

to the cells to exclude dead cells from the flow cytometer analysis. Data were acquired on a FACSCanto

flow cytometer with a minimum event count of 20 000 and were analyzed with FACSDiva software

(Becton Dickinson, Erembodegem, Belgium).

7.3.5. Antigen uptake

The phagocytic activity of -glucan stimulated MoDCs was evaluated with ovalbumin-dQ (ova-dQ;

Invitrogen, Molecular Probes). Upon incubation with the different -glucans (5 and 10 µg/ml) or LPS (1

and 10 µg/ml) for 24 h, MoDCs were harvested, washed with RPMI-1640 + 1% FCS and incubated for 1 h

with 10 µg/ml ova-dQ at 37°C in a humidified atmosphere at 5% CO2. To analyze the background

fluorescence, the uptake at 4°C was measured. The uptake of ova-dQ by stimulated MoDCs was analyzed

by flow cytometry as described above.

7.3.6. Allogeneic mixed leukocyte reaction Mixed leukocyte reactions were performed in 96-well round-bottomed culture plates (Nunc) with

CD172a-depleted cells (2.105 cells/well) as responder cells. Allogeneic MoDCs, stimulated for 24 h with

the different -glucans (5 and 10 µg/ml) or LPS (1 and 10 µg/ml), were added to the cultures as

stimulator cells at ratios of 1:30, 1:90 and 1:270. Cocultures were performed in triplicate and maintained

in DMEM, 10% FCS, 1% penicillin/streptomycin and 50 µM 2-mercapto-ethanol at 37°C in a humidified

atmosphere at 5% CO2. After 5 days of culture, the cells were pulse-labeled with 1 µCi of [3H]-methyl

thymidine (Amersham ICN, Bucks, UK) per well and 18 h later the cells were harvested onto glass fibre

filters (PerkinElmer, Life Science, Brussels, Belgium). The radioactivity incorporated into the DNA was


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measured using a -scintillation counter (PerkinElmer). The results are presented as the mean counts per

minute (cpm).

7.3.7. Cytokine ELISA

MoDCs were stimulated with -glucans as mentioned above and the culture supernatant was harvested

after 24 h and stored at -20°C. The concentrations of IL-1, IL-8, IL-12p40, IL-6, TNF- and IL-10 were

measured using commercially available ELISA kits (R&D Systems Inc.; Minneapolis, MN, USA) according

to the manufacturer’s recommended protocols. The cytokine concentrations were calculated using

DeltaSOFT JV 2.1.2 software (BioMetallics, Princeton, NJ, USA) with a 4-parameter curve-fitting

algorithm.

7.3.8. Statistics All experiments were performed with cells of four different pigs. Statistical analyses were performed

using SPSS16. One-way ANOVA with least significant difference (LSD) post hoc test was performed.

Levene’s test was used to assess the homogeneity of the variances. A p-value < 0.05 was considered

statistically significant.

7.4. Results

7.4.1. -glucans enhance the upregulation of DC activation markers Optimal antigen presentation and subsequent T-cell responses require DC maturation. This process

includes the upregulation of costimulatory molecules and major histocompatibility complex class II

(MHCII). To investigate the effect of -glucans on the phenotypical DC maturation, immature MoDCs

were stimulated for 24 h with 5 or 10 µg/ml of the different -glucan preparations or 1 or 10 µg/ml of

LPS, and the cell surface expression of CD80/86, CD40 and MHCII was assessed by flow cytometry. Figure

7.1 shows that Macrogard, both at 5 and 10 µg/ml, induced a significant upregulation of MHCII

expression, compared to the untreated immature cells (ImDCs) (p < 0.01), while CD80/86 expression was

only significantly upregulated at the 10 µg/ml dose (p < 0.01). Both doses of LPS (1 and 10 µg/ml) failed

to induce an increased marker expression.


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Figure 7. 1 Analysis of the expression ofCD80/86 (A), CD40 (B) and MHCII (C) after stimulation of immature

MoDCs with 5 µg/ml (grey bars) or 10 µg/ml (black bars) of -glucans or LPS (1 and 10 µg/ml). The expression of the maturation markers was assayed by flow cytometry. Data are shown as the means ± SEM of 4 pigs. Asterisks (*)

indicate a significant difference between -glucan stimulated MoDCs and immature MoDCs (ImDCs) (p < 0.01).

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7.4.2. -glucan treatment downregulates the phagocytic capacity of MoDCs To evaluate DC maturation other than analyzing the phenotype switch associated with DC activation,

functional maturation of the DCs upon treatment can be assessed. In terms of phagocytosis, it is known

that ImDCs can efficiently engulf antigens, but upon maturation, DCs lose this phagocytic ability. In our

studies, the phagocytic capacity of -glucan stimulated MoDCs was determined through the uptake of

ova-dQ (Figure 7.2). At 10 µg/ml, all -glucans significantly reduced the capacity of MoDCs to endocytose

antigen compared to the untreated MoDCs (for all -glucans p < 0.01 except for the -glucan from

Euglena gracilis: p=0.02 < 0.05). Stimulation of MoDCs with 5 µg/ml curdlan also significantly reduced

their phagocytic ability (p=0.005 < 0.01). While there was a reduced, although not significant uptake of

ova-dQ after stimulation of MoDCs with 10 µg/ml LPS, no effect was seen at the lowest concentration.

Figure 7.2 Analysis of the phagocytic activity of -glucan stimulated MoDC. Immature MoDCs were

stimulated with 5 µg/ml (grey bars) or 10 µg/ml (black bars) of -glucans or LPS (1 and 10 µg/ml) and the uptake of ova-dQ was assayed by flow cytometry. MFI values were calculated by subtracting MFI values obtained at 4°C from those at 37°C. Data are shown as the means ± SEM of 4 pigs. Asterisks (*) indicate a significant difference between

-glucan stimulated MoDCs and immature MoDCs (ImDCs) (for all -glucans p < 0.01 except for the -glucan from Euglena gracilis: p < 0.05).


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7.4.3. -glucan stimulated MoDCs induce T cell proliferation As a result of maturation, MoDCs become potent stimulators of immune responses resulting in an

increased T-cell stimulatory capacity. In order to determine whether MoDCs stimulated with -glucans

are able to induce T-cell proliferation, different ratios of -glucan stimulated MoDCs were cocultured

with CD172a-depleted cells (Figure 7.3). When stimulated with 5 µg/ml, Macrogard-, curdlan- as well as

zymosan-stimulated MoDCs significantly increased T-cell proliferation compared to ImDCs (p < 0.01).

MoDcs stimulated with a higher dose of curdlan and zymosan (10 µg/ml), tended to induce a weaker T-

cell proliferation than after stimulation with 5 µg/ml, whereas MoDCs stimulated with 10 µg/ml

Macrogard significantly increased T-cell proliferation more than after stimulation with 5 µg/ml (p < 0.01).

Stimulation of MoDCs with the higher dose of LPS (10 µg/ml) also gave a significantly stronger T-cell

proliferation (p= 0.001 < 0.05) than the lower dose (1 µg/ml) as compared to the ImDCs.

a

Figure 7.3 Analysis of the ability of -glucan stimulated MoDC to enhance T cell proliferation. Porcine MoDC

were left untreated or stimulated for 24 h with 5 µg/ml (grey bars) or 10 µg/ml (black bars) of different -glucans or LPS (1 and 10 µg/ml) and then added to CD172a-depleted PBMC (lymphocytes) at the indicated ratios. After 5 days, cultures were pulsed with 1µCi [

3H]-methyl thymidine. T cell proliferation was measured after an additional co-

culture of 18h. Data are shown as the means ± SEM of 4 pigs. Asterisks (*) indicate a significant difference between

-glucan stimulated conditions (1:30 ratio) and the untreated condition (ImDCs) (p < 0.01 except for LPS (10 µg/ml): p=0.019 < 0.05). The proliferative responses of the CD172a- lymphocytes (no MoDCs were added) was below 300 cpm.


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7.3.4. -glucans stimulate cytokine secretion by MoDCs Cytokine expression is another parameter to evaluate the functional maturation of DCs and is

responsible for the differentiation and polarization of T-cells. To investigate whether the observed

phenotypical and functional maturation of MoDC induced after -glucan treatment correlates with a

certain cytokine expression profile, the IL-1, IL-6, IL-8, IL-12/IL-23p40, TNF- and IL-10 concentrations

were determined in the culture supernatant of -glucan stimulated MoDCs (Figure 7.4). Compared to

ImDCs, stimulation with all -glucans, except laminarin, could significantly increase TNF- secretion.

Interestingly, while the -glucan from Euglena gracilis and Macrogard induced a significantly higher TNF-

production at 10 µg/ml and zymosan induced a significantly higher production at both concentrations,

curdlan only induced a significant increase in TNF-secretion at the lowest concentration (5 µg/ml) (p <

0.05). By contrast, only stimulation with 10 µg/ml curdlan resulted in a significantly higher production of

IL-1, IL-6, IL-10 and IL-12/IL-23p40 in comparison with ImDCs and all the other stimulated MoDCs (p <

0.01). Macrogard and zymosan also enhanced the production of these cytokines, while zymosan and to a

lesser degree, the -glucan from Euglena gracilis and curdlan, gave rise to IL-8 secretion, but these

increases were not significant.


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Figure 7.4 Cytokine expression pattern of MoDCs treated with the different -glucans. MoDCs were left untreated (ImDCs) or stimulated with 5 µg/ml

(grey bars) and 10 µg/ml (black bars) of different -glucans or LPS (1 and 10 µg/ml). After 24 h the culture supernatant was harvested and the IL-1, IL-6, IL-8, IL-

10, IL-12/IL-23p40 and TNF cytokine concentrations were measured using commercially available ELISA kits. Data are shown as the means ± SEM of 4 pigs.

Asterisks (*) indicate a significant difference (p < 0.05) between -glucan stimulated MoDCs and immature MoDCs, while the triangle () indicates a significant

difference (p < 0.01) between MoDCs stimulated with 10 µg/ml of Curdlan and all the other -glucan stimulated (10 µg/ml) MoDCs or immature MoDCs.


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7.5. Discussion

In human and mouse, there is an extensive amount of information about -glucans and their

immunomodulatory effects. However, the effects are not always consistent and it’s not clear whether

these differences are due to the use of different -glucan preparations or due to variation among

experimental models. In pig industry, dietary -glucan supplementation is often used, especially during

periods of stress and immune challenge like weaning. However, the beneficial effect of -glucan

supplementation may be influenced by several factors, like structural features and the dose of -glucan

used in the diets. In a previous study, we described already the dose-effect of different -glucan

preparations on porcine leukocytes (Sonck et al., 2010). In the current study, we focused on the action of

different -glucans on porcine dendritic cells. For this study, we used much narrow doses of -glucans

(namely 5-10 µg/ml for DCs compared to 50-800 µg/ml for leukocytes), as preeliminary experiments with

higher concentrations gave unclear results because of the presence of too much particulate -glucans in

the MoDC population during flow cytometry analysis. DCs have not only been described to sense the

luminal environment at epithelial surfaces and sample luminal pathogens, but have also been shown to

be the key players in the initiation and differentiation of the immune response (Iwasaki and Medzhitov,

2010; Lanzavecchia and Sallusto, 2001; Pulendran et al., 2001).

The results of the present study demonstrate that there is a large variation in terms of DC maturation

inducing properties between the different -glucans. Assuming the current experiments, Macrogard,

zymosan and curdlan have the greatest impact on the functional and phenotypical maturation of porcine

DCs. Moreover, those three -glucans have a strong effect on the T-cell proliferative capacity of MoDCs.

Macrogard and zymosan are both -(1,3)-(1,6) glucans isolated from Saccharomyces cerevisiae. The

main difference between both preparations is that zymosan also contains mannans and proteins, while

Macrogard is a purfied -(1,3)-(1,6) glucan (Sonck et al., 2010). Curdlan, on the other hand, is an

unbranched -(1,3)-glucan derived from the gram-negative bacteria Alcaligenes faecalis. Apparently, the

presence of -(1,6) branches is less important for the immunostimulatory capacity of -glucans on DCs,

since laminarin, which is also a -(1,3)-glucan with some -(1,6)-branching (30:1), failed to induce

porcine DC maturation. These findings are in line with studies with both human and murine DCs (Nisini et

al., 2007). Since laminarin is a soluble, low MW -glucan, and all other tested -glucans are particulate,

high MW -glucans, this suggests that the molecular complexity together with solubility are important

factors in the immunostimulating capacity. The immunomodulatory effect of -glucans has been

attributed to dectin-1-mediated signaling, which is the primary PRR for -glucans (Brown et al., 2003). In


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human and murine DCs, dectin-1 is the major -glucan receptor responsible for downstream signaling

and the uptake of -glucans by DCs (Backer et al., 2008). Recently, we demonstrated dectin-1 expression

in porcine intestinal tissues (Sonck et al., 2009) and as in humans (Willment et al., 2003; Willment et al.,

2005), we found that dectin-1 mRNA transcripts are abundantly present in porcine MoDC (data not

sshown). This allows us to assume that recognition of -glucans by porcine DCs is primarily mediated via

dectin-1. However, in addition to dectin-1, other -glucan receptors, such as complement receptor 3

(CR3) (Ross et al., 1985), lactosylceramide (Zimmerman et al., 1998) and scavenger receptors (Rice et al.,

2002) may be involved in the recognition of -glucans and their immunostimulatory effects.

It should be noted that the difference in immunostimulating capacities between the different -glucan

preparations, could be attributed to the presence of LPS. As such, the -glucan from Euglena gracilis,

which is, like curdlan, also an unbranched -(1,3)-glucan (Sonck et al., 2010; Wismar et al., 2010), did

contain much lower levels of LPS (0.2 EU/µg vs. 47 EU/µg for curdlan) and displayed a much lower

immunomodulatory capacity than curdlan. Based on the approximation that 1 EU correlates to 0.2 ng

LPS5, we estimated the LPS contamination in curdlan to correspond to the administration of

approximately 10 ng LPS/µg -glucan, which is 100x less than the LPS control, representing a minor but

possibly stimulatory dose. The varying immunomodulatory activity of both linear -(1,3)-glucans was

earlier reported in mice DC by Wismar et al. (2010) and they also attributed the different effects

between both -glucans (curdlan and the -glucan from Euglena gracilis) to the presence of LPS in the

curdlan preparation (Wismar et al., 2010). In line with their results, we also determined a higher

production of IL-10, IL-6 and IL-12p40 after stimulation with curdlan. However, in contrast with human

and mice and in line with earlier reports (Devriendt et al., 2010; Guzylack-Piriou et al., 2006; Pilon et al.,

2009), porcine MoDCs are less responsive towards LPS itself. LPS could not upregulate the expression of

maturation markers and did not significantly increase the proinflammatory cytokines IL-1, IL-6 and TNF-

, nor the Th1 cell inducing IL-12, the chemoattractant IL-8 or the anti-inflammatory cytokine IL-10.

Although we cannot completely rule out an additive effect of LPS on DC maturation upon simultaneous

stimulation of these cells with LPS and curdlan, preliminary experiments showed only a very small

additive effect of costimulation of Macrogard or curdlan with 1 µg/ml LPS on the expression of

maturation markers.

5 US Department of Health and Human Services, Public Health Service Food and Drug Administration. Guideline on

validation of the Limulus Amebocyte Lysate Test as an endproduct endotoxin test for Human and Animal parenteral drugs, biological products, and medical devices. 1987.


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The difference in immunostimulatory capacity between species is also noticed for zymosan. In human

and mice, it is suggested that zymosan induces regulatory-type DCs, characterized by the production of

few proinflammatory cytokines but abundant levels of IL-10 (Dillon et al., 2006). By contrast, in the pig,

zymosan-stimulated DC produced low amounts of IL-10 and high amounts of IL-12p40, so we can assume

that no tolerogenic nor regulatory DCs were induced. The induction of pro-inflammatory cytokines such

as IL-1, TNF-, IL-6, IL-8 and IL-12p40 together with the upregulation of costimulatory molecules after

curdlan stimulation, could direct T-cells towards a Th1 or Th17 response. However, the finding that DCs

secrete particularly large quantities of TNF together with IL-12p40 and IL-6 is notable, as all three

cytokines have synergistic effects on the induction of Th17 cells (Veldhoen et al., 2006). IL-23, which is a

member of the IL-12 superfamily, is a heterodimer composed of a unique p19 subunit and a p40 subunit,

which is shared with the IL-12p70 cytokine (p35/p40 heterodimer). It was previously demonstrated that

DC activation through the dectin-1-syk-CARD9 signaling pathway induced Th17 skewing (Gross et al.,

2006; Hara et al., 2007; LeibundGut-Landmann et al., 2007; Rogers et al., 2005) and that IL-1, in synergy

with IL-23, plays an essential role in the induction or expansion of murine and human Th17 cells (Agrawal

et al., 2010). Th17 cells are characterized by the expression of IL-17, and have been implicated in the

protection against a variety of pathogens at mucosal surfaces (Abbas et al., 1996; Guglani and Khader,

2010). In line with the results we obtained with porcine DCs, Kankkunen and colleagues (2010)

demonstrated that particularly curdlan was the most potent IL-1 inducer in human DCs. Moreover, they

reported that IL-1 production by curdlan is triggered by the activation of the NLRP3 inflammasome and

that this pathway is required for innate responses as well as B cell-mediated humoral immunity

(Kankkunen et al., 2010; Kumar et al., 2009). Following stimulation of porcine MoDCs with curdlan, we

could detect a significant increase in IL-12/IL-23p40 secretion. However, as no commercial ELISA kits are

available to measure porcine IL-12p35 or IL-23 and the p40 subunit alone gives few information about IL-

12/IL-23 bioactivity, it’s difficult to speculate on Th1 or Th17 skewing by curdlan-stimulated DCs.

In summary, we have shown that stimulation of porcine MoDCs with -glucans, especially curdlan and

the -glucans derived from Saccharomyces cerevisiae, enhance the DC maturation and the DC induced T-

cell proliferation. Particularly curdlan seems to imprint DC with the ability to skew the T-cell polarization

towards a Th1 or Th17 phenotype. Since Th17 cells have been implicated in the induction of mucosal

immunity, further research is necessary towards the mechanisms of curdlan and its potential as mucosal

adjuvant.


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Science and Technology in Flanders (IWT-Vlaanderen). We acknowledge Prof. Dr. S. Inumaru (Institute of

Animal Health, Ibaraki, Japan) for kindly providing rpGM-CSF and Prof. Dr. H.J. Rothkötter (Institute of

Anatomy, Magdeburg, Germany) for the anti-CD40 hybridoma SN.

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Zhou, L., Chong, M.M., Littman, D.R., 2009, Plasticity of CD4+ T cell lineage differentiation. Immunity 30, 646-655. Zimmerman, J.W., Lindermuth, J., Fish, P.A., Palace, G.P., Stevenson, T.T., DeMong, D.E., 1998, A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J Biol Chem 273, 22014-22020.

Chapter 8 : Yeast cells as potential antigen carriers

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Chapter 8 : β-glucan rich Yeast shells as potential carriers for antigen targeting to dendritic cells Eva Sonck, Bert Devriendt, Bruno De Geest, Bruno Goddeeris, Eric Cox


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8.1. Abstract By their unique capacity to internalize antigens together with their antigen-presenting capacities to naïve

CD4+ or CD8+ T-cells, dendritic cells (DCs) form the ultimate candidate for antigen targeting. However,

developing suitable carriers for efficient antigen targeting to DCs is an intensive area of research. To

protect weaning piglets against F4+ Escherichia coli infections, it has been shown that oral immunization

with F4 fimbriae is the most effective strategy. However, new formulations of the F4 antigen are

necessary to reduce the antigen dose and to allow oral immunization in the presence of interfering

maternal antibodies or other milk components. In the present study, it was examined if -glucan-rich

yeast shells could be used to target antigen to DCs. Preliminary results in the present study demonstrate

that F4-loaded yeast shells are efficiently taken up by DCs, thereby inducing a phenotypical as well as a

functional DC maturation.

8.2. Introduction Antigen-presenting cells, such as dendritic cells (DCs), use pattern recognition receptors (PRRs) to

recognize specific molecular signatures on pathogens called pathogen-associated molecular patterns

(PAMPs) (Gordon, 2002). DCs are the key antigen-presenting cells (APCs) of the immune system,

expressing a wide variety of PRRs, including Toll-like receptors (TLRs) and C-type lectin receptors (CLR)

(Robinson et al., 2006; Trinchieri and Sher, 2007). Since they have the unique capacity to initiate and

regulate adaptive immune responses, strategies for targeting those cells in vivo have the potential for

use in immunizations. However, there are a number of prerequisites for effective DC targeting. The

carriers must deliver the antigen efficiently to the DCs, activate them, and then release the antigen so

that antigen processing and presentation can occur (Jones, 2008). Several approaches for DC-targeting

vaccines have been proposed, including antigen-conjugated antibodies specific for DC-expressed

molecules as well as nano- or microparticulate delivery vehicles (Bonifaz et al., 2002; O'Hagan and Singh,

2003; O'Hagan et al., 2006). To enhance the immune response, an immunomodulator can be coupled to

the particle or an encapsulant with potent immunomodulatory effects can be used.

-glucans, which are found to be the major component of the outer cell wall of different

microorganisms, including yeast, are referred to as PAMPs, although they are not exclusively present in

pathogens. They consist of a backbone of (1,3)-linked D-glucose units, often with several (1,6)-linked-

D-glucose branches. Different PRRs have been associated with -glucan binding, including complement

receptor 3 (CR3) (Ross et al., 1985), lactosylceramide (Zimmerman et al., 1998), scavenger receptors


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(Rice et al., 2002) and dectin-1 (Brown and Gordon, 2001). Dectin-1 is a C-type lectin receptor, originally

thought to be DC specific, but subsequently demonstrated to be expressed by other innate immune cells

including neutrophils, monocytes and macrophages (Goodridge et al., 2009b). Dectin-1 is abundantly

expressed at portals of pathogen entry, such as the lungs and the intestine (Reid et al., 2004; Taylor et

al., 2002), making this receptor also an attractive target for mucosal immunization. Recently, we have

demonstrated that -glucans derived from Saccharomyces cerevisae are able to enhance porcine DC

maturation and DC induced T-cell proliferation (Sonck et al., submitted). Based on these results, we

wanted to investigate if hollow yeast shells (YC), purified from Saccharomyces cerevisiae and rich in -

glucans, could serve as a potential antigen carrier, using F4 fimbriae as model antigens.

F4 fimbriae are long, filamentous polymeric proteins, present on the surface of enterotoxigenic

Escherichia coli (ETEC). F4+ ETEC infections are an important cause of diarrhea in neonatal and recently

weaned pigs (Jones and Rutter, 1972). Through their fimbrial antigens, ETEC adhere to the intestinal

epithelial surface allowing the bacteria to multiply and to transfer their enterotoxins directly to the

intestinal epithelial cells. For the control of neonatal and post-weaning ETEC diarrhea of piglets, no

commercial vaccine is yet available. Developed parenteral vaccines against neonatal ETEC infections

predominantly stimulate systemic immune responses rather than protective mucosal immune responses

(Bianchi et al., 1996; Moon and Bunn, 1993; Van der Stede et al., 2003). In our lab, it was demonstrated

that orally administered F4 fimbriae induce a protective intestinal immune response in weaned piglets,

protecting them against a subsequent F4+ ETEC challenge (Van den Broeck et al., 1999a). However, since

the required dose needed to protect the pigs is still high, a more efficient F4 formulation is searched for.

Moreover, to be protected against postweaning diarrhea, piglets need to be vaccinated during the

suckling period, so that the fimbriae have to be protected against neutralization by F4-specific maternal

antibodies in the milk (Rutter and Jones, 1973). By encapsulation of F4 fimbriae into -glucan shells (F4-

YC), we wanted to investigate if the uptake of F4 by DCs and the ensuing maturation process of dendritic

cells could be enhanced. Therefore, uptake efficiency and maturation of porcine monocyte-derived

dendritic cells (MoDCs) was compared between encapsulated and free F4 fimbriae.


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8.3.1. Preparation of yeast shells

-glucan rich yeast shells (YC) were prepared from Saccharomyces cerevisiae (Sigma-Aldrich) as

previously described (Aouadi et al., 2009; Soto and Ostroff, 2008). Briefly, to remove the cytoplasm and

other non--glucan cell wall polysaccharides, S. cerevisiae were treated with a series of hot alkali and

acid extractions and were successively washed with water, isopropanol and acetone. Subsequently, the

yeast shells were dried to obtain a fine, white powder. To obtain a cationized core allowing incorporation

of the F4 antigen, the dry yeast shells were subsequently loaded with the anionic core polymer tRNA

(derived from torula yeast; Sigma-Aldrich) and cationic polyethylenimine (PEI, Sigma-Aldrich) as

previously described (Soto and Ostroff, 2008). Briefly, dry yeast shells were mixed with tRNA (10 mg/ml

in PBS) and incubated during 2 h to allow the particles to swell and to absorb the tRNA solution.

Thereafter, the tRNA loaded yeast shell were mixed with a solution of PEI (2 mg/ml in PBS) for at least 1h

to form a cationic complex within the yeast shells. After centrifugation, the particles were sterilized in

70% ethanol, washed three times in 0.9% saline and stored at -20°C.

8.3.2. Purification of F4 fimbriae and fluorochrome labeling F4 fimbriae were purified as previously described by Van den Broeck et al. (1999a). Briefly, fimbriae were

isolated by homogenizing the bacterial suspension of the IMM01 strain using an Ultra Turax (Janke &

Kunkel, IKA Labortechnik, Staufen, Germany). After ammonium sulphate (40% w/v) precipitation, the

fimbrial proteins were dialyzed, the protein concentration was determined by the bicinchoninic acid

assay (BCA) and their purity was assessed by SDS-PAGE and Western blotting.

F4 was either labeled with Alexa Fluor 488 or FITC using the Alexa Fluor® Protein Labeling kit (Molecular

Probes, Invitrogen) or fluorescein Protein Labeling kit (Roche Applied Science), respectively .

8.3.3. Encapsulation of F4-fimbriae into yeast shells Yeast shells modified with tRNA + PEI were incubated with F4 fimbriae in a 1:1 ratio, mixed thoroughly,

centrifuged, washed and resuspended in PBS. To calculate the amount of F4 fimbriae encapsulated into

the yeast shells, the unincorporated F4 in the PBS wash was measured by BCA. Typically, the amount of

incorporated F4 varied between 0.1-0.3 µg F4/µg yeast shells.


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8.3.4. Generation of monocyte-derived dendritic cells Porcine monocyte-derived dendritic cells (MoDCs) were generated from peripheral blood

monomorphonuclear cells (PBMCs) as previously published (Bimczok et al., 2009; Devriendt et al., 2010).

Briefly, peripheral blood was collected on heparin from the jugular vein of three 8-12 weeks old pigs

(Piétrain boar x Hypor Libra hybrid sow) and PBMCs were isolated by density gradient centrifugation on

Lymphoprep (NYCOMED Pharma AS, Life Technologies, Merelbeke, Belgium). CD172a+ cells were

isolated from the PBMC fraction by positive selection with magnetic activated cell separation (MACS;

Miltenyi-Biotec, Bergisch Gladbach, Germany) using anti-CD172a monoclonal antibody (mAb) (74-12-

15A; Pescovitz et al., 1984) and anti-mouse IgG microbeads together with LS separation columns

(Miltenyi-Biotec). To generate MoDCs, monocytes were cultured in 24-well plates at a density of 5.105

cells in phenol-red free Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Merelbeke, Belgium)

containing fetal calf serum (FCS) (10%) (Greiner), penicillin (100 IU/ml) (Gibco), streptomycin (100 µg/ml)

(Gibco), recombinant porcine (rp) GM-CSF (Inumaru et al., 1998) and rpIL-4 (R&D systems) and incubated

at 37°C in a humidified atmosphere at 5% CO2 (Carrasco et al., 2001). After three days, the cultures were

supplemented with fresh cytokines. On day 4, MoDCs were treated for 24 h with F4 fimbriae (3 µg/ml),

yeast shells (10 µg/ml), F4 encapsulated yeast shells (10 µg/ml) or were left untreated. After this

incubation period, cells were harvested and further processed for phenotyping of MoDCs and evaluation

of the phagocytic capacity as described below.

8.3.5. Evaluation of the uptake of F4 encapsulated yeast shells by MoDCs To analyze binding and internalization, immature MoDCs were harvested, washed and incubated with 1

µg F4-FITC or F4- FITC encapsulated in yeast shells (YC/F4-FITC) for 30 min at 4°C or 37°C. After

incubation, cells were washed and propidium iodide (PI) was added to exclude dead cells from the flow

cytometric analysis. Data were acquired on a FACSCanto flow cytometer with an event count of 20 000

and were analyzed with FACSDiva software (Becton Dickinson, Erembodegem, Belgium).

Live cell imaging was used to visualize the spatio-temporal interactions between immature MoDCs and

the F4 encapsulated yeast shells. Therefore, CD172a+ cells were seeded at a density of 5.105 cells in a

chambered coverglass (Nunc) and differentiated to MoDCs at described above. After 4 days, when fully

differentiated, MoDCs were placed on ice and different dilutions of ice-cold fluorescent F4-yeast shells

were added to the cells. Subsequently, cells were placed in a custom-made chamber maintained at 37°C

and 5% CO2, and the uptake was recorded during 50 minutes by Live cell imaging with one record frame


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every 2 minutes. Live cell imaging experiments were performed on a IX81 Olympus fluorescence

microscope (magnification 60x) linked to a Cell*M imaging system (Olympus).

8.3.6. Phenotyping of MoDCs The surface expression of various DC maturation markers was assessed by flow cytometry (FACSCanto).

Hereto, harvested MoDCs were washed with RPMI-1640 + 1% FCS and incubated for 20 minutes at 4°C

with a primary mouse mAb against MHCII (MSA3; Lunney et al., 1994), CD25 (K231.3B2; Bailey et al.,

1992), CD40 (G28-5; Bimczok et al., 2007) or with a human CTLA4-muIg fusion protein (Ancell, Bayport,

MN, USA) to detect the expression of CD80 and CD86. Cells stained with isotype-matched irrelevant

mAbs were used as a negative control. After incubation, cells were washed and stained with FITC

conjugated F(ab’)2 fragments of sheep anti-mouse IgG antibodies (Sigma) for another 20 minutes at 4°C.

Next, the cells were washed and propidium iodide (PI) was added to the cells to exclude dead cells from

the flow cytometric analysis. Data were acquired on a FACSCanto flow cytometer with a event count of

20 000 and were analyzed with FACSDiva software (Becton Dickinson).

8.3.7. Antigen uptake capacity To evaluate the phagocytic activity of the treated MoDCs, cells were harvested, washed with RPMI-1640

+ 1% FCS and incubated for 1 h with 10 µg/ml ova-dQ at either 4°C or 37°C. The uptake of ova-dQ was

analyzed by flow cytometry as described above.

8.3.8. Statistics Data from the phenotypical and functional maturation were analyzed with SPSS16 to compare

differences between the different treatments and the immature MoDCs. One-way ANOVA with least

significant difference (LSD) post hoc test was performed. A p-value < 0.05 was considered statistically

significant.


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8.4. Results

8.4.1. Uptake of F4 encapsulated yeast shells To determine whether the uptake of F4 by MoDCs could be enhanced by encapsulation into yeast shells,

MoDCs were incubated for 30 minutes with either fluorescent F4 (F4-FITC) or fluorescent F4

incorporated into yeast-shells (F4-FITC/YC) and their uptake was analyzed by flow cytometry. As

demonstrated in Figure 8.1, encapsulation of F4 into yeast shells increases the uptake by MoDCs as

compared to free F4.

Figure 8.1 The uptake of F4 and F4-loaded yeast shells by MoDCs, analyzed by flow cytometry. Data are

shown as the mean ± SEM of two pigs (MFI = mean fluorescence intensity).

To visualize and to determine the time needed for uptake of the encapsulated F4 by MoDCs, the

interaction between MoDCs and the fluorescent F4-yeast shells was followed by live cell imaging. In 25

minutes, migrating immature MoDCs reached a cluster of fluorescent F4-loaded yeast shells after which

the particles were engulfed using dendrites (Figure 8.2A-D).

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Figure 8.2 Uptake of fluorescent F4 loaded yeast shells by MoDCs as visualized by live cell imaging. Images show the uptake at different timepoints (A-D). A: t = 10 min; B: t = 12 min; C: t = 18 min; D: t = 32 min (arrow indicates the endocytosed F4 encapsulated yeast shell).

8.4.2. F4 encapsulated yeast shell induce upregulated expression of DC activation markers

The enhanced capacity of DCs for antigen-presentation is accompanied by the upregulation of cell-

surface markers (i.e. CD25), costimulatory molecules (i.e. CD80/86 and CD40) and major

histocompatibility complex class II (MHCII). To investigate whether encapsulation of F4 in yeast shells or

the yeast shells themselves had an effect on these maturation markers, immature MoDCs were

incubated for 24 h with either 3 µg/ml F4, 10 µg/ml F4 loaded yeast shell (with 0.3µg F4/µg yeast shell)

or 10 µg/ml yeast shell (Figure 8.3). For MHCII, differences in expression were significantly higher for

both hollow (YC) and F4-encapsulated yeast (F4-YC) as compared to the untreated as well as the F4-

stimulated MoDCs (p<0.05). Compared to the untreated MoDCs, CD25 expression was also significantly

upregulated for both hollow and F4-encapsulated yeast, while the latter was additionally significant with

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the F4-stimulated MoDCs (p<0.05). For CD40, there was only a tendency towards increased expression

after stimulation with both hollow and F4-encapsulated yeast shell, while the CD80/86 expression was

slightly upregulated after treatment with either F4, hollow or F4-encapsulated yeast shell (p< 0.05).

Figure 8.3 Phenotypical maturation of MoDCs stimulated during 24 h with F4, hollow yeast shells (YC) and F4-loaded yeast shells (F4-YC). Data are shown as the mean ± SEM of 3 pigs. The letters indicate a significant difference compared to (a) ImDCs (p < 0.05) or (b) F4 stimulated DCs (p < 0.05) (MFI = mean fluorescence intensity).

8.4.3. Antigen uptake A typical feature of immature DCs is their proficient ability to take up antigens. To allow efficient

presentation of the internalized antigen to T-cells, DCs need to mature, which reduces their ability to

take up new antigen. Therefore, the capacity of the treated MoDCs to phagocytose ova-dQ was

assessed. As shown in Figure 8.4, MoDCs treated with the hollow and those treated with the F4 loaded

yeast shell displayed a reduced, although not significant, uptake of ova-dQ compared to the untreated

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MoDCs (p< 0.05). Surprisingly, the endocytic capacity of MoDCs treated with the free F4-fimbriae was

even higher than the untreated MoDCs.

Figure 8.4 Functional maturation of MoDCs stimulated with F4, hollow yeast shells (YC) and F4-loaded yeast shells (F4-YC). Immature MoDCs were treated during 24 h with the different stimuli and the uptake of ova-dQ was assayed by flow cytometry. Mean fluorescence intensity (MFI) values were calculated by subtracting MFI values obtained at 4°C from those at 37°C. Data are shown as the mean ± SEM of 3 pigs.

8.5. Discussion To optimize oral immunization against F4+ ETEC infections, encapsulation of F4 fimbriae would be an

interesting strategy, since it can protect the antigen from degradation and from neutralization by milk

antibodies, and could therefore result in a reduction of the dose needed for immunization. Encapsulation

in yeast shells has several advantages. At first, yeast shells are predominantly composed of -glucans,

which can play the role of an adjuvant. Secondly, -glucans can bind to -glucan receptors including

dectin-1 on DCs, offering the potential to target DCs. Recently, it has been shown that yeast shells can

facilitate oral delivery of siRNA to mouse macrophages in vivo and in vitro (Aouadi et al., 2009).

Moreover, systemic immunization of mice with ovalbumin encapsulated into yeast shells, induced robust

Th1- as well as Th17-biased immune responses (Huang et al., 2010). Given the promising results obtained

in mice, we wanted to investigate whether yeast shells could also be used in pigs for the delivery of a

relevant antigen, namely F4, to dendritic cells. The most direct assay to evaluate the immunopotentiator

properties of new antigen delivery systems is assessing their capacity to mature DCs in vitro.

0

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The preliminary results in these study demonstrate that F4-loaded yeast shells, mainly consisting of -

glucans, are internalized more efficiently by MoDCs than the naked soluble F4 fimbriae. Most likely, their

enhanced uptake is due to the particulate nature of the carrier combined with the characteristics of -

glucans to be recognized by -glucan specific DC surface receptors. Indeed, it has been demonstrated

that micro- and nanoparticles are more efficiently internalized by DCs as they mimic the dimensions of

respectively bacteria and viruses. Although particles can be efficiently internalized by DCs and even

encourage them to undergo functional maturation (De Koker et al., 2011), the presence of PRR-ligands

like -glucans could further enhance particle endocytosis and DC maturation through their inherent

adjuvant capacity. -glucans are recognized by several -glucan binding receptors and dectin-1 has been

described as the most important one. Recently, we demonstrated dectin-1 mRNA expression in several

porcine tissues (Sonck et al., 2009). As dectin-1 is the primary and the most efficient phagocytic receptor

on DCs for phagocytosis of yeasts with -glucan-rich cell walls (Brown et al., 2002; Saito et al., 1991;

Taylor et al., 2007) and as dectin-1 mRNA transcripts are abundantly present in porcine MoDCs (data not

shown), we assume that recognition of -glucan rich yeast-shells by porcine MoDCs is partly mediated by

dectin-1. However, the exact role of dectin-1 in porcine MoDCs should further be established by

investigating the effect of inhibiting dectin-1 by laminarin or a specific blocking monoclonal antibody.

To induce optimal T-cell activation, antigen-presentation by DCs must be accompanied by the expression

of costimulatory molecules such as CD80, CD86 and CD40. Lack of costimulation normally leads to

tolerance (Bachmann et al., 1998; Perez et al., 1997; Schwartz, 2003). Recently, we demonstrated that

Macrogard, a (1,3)-(1,6)-glucan extracted from the cell wall of Saccharomyces cerevisiae, was a strong

inducer of porcine DC maturation, significantly upregulating the expression of costimulatory molecules

(CD40 and CD80/86) and MHCII, when compared to immature MoDCs (Chapter 7). In the present study,

when compared with immature MoDCs, the yeast shell gave a significantly stronger upregulation of

MHCII and CD25 and an tendency towards increased expression of the costimulatory molecule CD40 (p<

0.05). The finding that yeast shells are able to induce DC maturation is also reflected in the low endocytic

capacity of MoDCs stimulated with both hollow yeast shells as well as F4-loaded yeast shells. However,

so far, the incorporation of F4 in the yeast shells didn’t result in an enhanced MoDC maturation

compared to the hollow yeast shells. Indeed, phenotypic maturation markers were, with the exception

of CD25, equally upregulated in MoDCs stimulated with either F4-loaded or hollow yeast shells and also

the effect on functional MoDC maturation was similar for both agents. As F4 is encapsulated within the

yeast shell, the initial recognition and uptake by MoDCs is presumably the same for the hollow as for the

Ag-loaded yeast shell. It has been described that endocytosed (or internalized) antigens are retained in a


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retention compartment for up to 24 h (Rescigno et al., 1997). These large storage vesicles are

characterized by a mildly acidic pH, necessary for the preservation of antigens (Rescigno et al., 1997) and

making the release of F4 from the yeast shell very unlikely within the 24 h time period used in our assays.

As a result, the additive effect of F4 on DC maturation will be limited.

Unexpectedly, the endocytic capacity of DC stimulated with free F4 was higher than the untreated

MoDCs, despite the slightly upregulated expression of some maturation markers by free F4-stimulated

MoDCs. These results are in contrast with previous findings (Devriendt et al., 2010). The reason for our

results are unclear but, could possibly, although unlikely, be explained by a dose-dependent effect.

In conclusion, the preliminary results in this study demonstrate that F4-loaded yeast shells are efficiently

taken up by MoDCs and have the capacity to induce their maturation. As these in vitro data imply that

yeast shells are a promising candidate for the delivery of F4, further studies have to be undertaken to

determine whether the encapsulated F4 will be efficiently processed within DCs and presented to CD4+

or CD8+ T-cells. Subsequently, in order to evaluate the efficacy/efficiency of yeast shells as oral antigen

delivery systems, their behavior in the gastrointestinal tract has to be analyzed and their interaction with

intestinal APC such as DCs assessed. Finally, in vivo studies are required to evaluate whether orally

administered F4-loaded yeast shells induce a protective immune response in pigs against an F4+ ETEC

challenge infection.



8.7. References Aouadi, M., Tesz, G.J., Nicoloro, S.M., Wang, M., Chouinard, M., Soto, E., Ostroff, G.R., Czech, M.P., 2009, Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 458, 1180-1184. Bachmann, M.F., Zinkernagel, R.M., Oxenius, A., 1998, Immune responses in the absence of costimulation: viruses know the trick. J Immunol 161, 5791-5794. Bailey, M., Stevens, K., Bland, P.W., Stokes, C.R., 1992, A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors. J Immunol Methods 153, 85-91. Bianchi, A.T., Scholten, J.W., van Zijderveld, A.M., van Zijderveld, F.G., Bokhout, B.A., 1996, Parenteral vaccination of mice and piglets with F4+ Escherichia coli suppresses the enteric anti-F4 response upon oral infection. Vaccine 14, 199-206. Bimczok, D., Rau, H., Wundrack, N., Naumann, M., Rothkotter, H.J., McCullough, K., Summerfield, A., 2007, Cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate T cells. Vet Res 38, 597-612.


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Bimczok, D., Wrenger, J., Schirrmann, T., Rothkotter, H.J., Wray, V., Rau, U., 2009, Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells. Appl Microbiol Biotechnol 82, 321-331. Bonifaz, L., Bonnyay, D., Mahnke, K., Rivera, M., Nussenzweig, M.C., Steinman, R.M., 2002, Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. J Exp Med 196, 1627-1638. Brown, G.D., Gordon, S., 2001, Immune recognition. A new receptor for beta-glucans. Nature 413, 36-37. Brown, G.D., Taylor, P.R., Reid, D.M., Willment, J.A., Williams, D.L., Martinez-Pomares, L., Wong, S.Y., Gordon, S., 2002, Dectin-1 is a major beta-glucan receptor on macrophages. J Exp Med 196, 407-412. Carrasco, C.P., Rigden, R.C., Schaffner, R., Gerber, H., Neuhaus, V., Inumaru, S., Takamatsu, H., Bertoni, G., McCullough, K.C., Summerfield, A., 2001, Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties. Immunology 104, 175-184. De Koker, S., Lambrecht, B.N., Willart, M.A., van Kooyk, Y., Grooten, J., Vervaet, C., Remon, J.P., De Geest, B.G., 2011, Designing polymeric particles for antigen delivery. Chem Soc Rev 40, 320-339. Devriendt, B., Verdonck, F., Summerfield, A., Goddeeris, B.M., Cox, E., 2010, Targeting of Escherichia coli F4 fimbriae to Fcgamma receptors enhances the maturation of porcine dendritic cells. Vet Immunol Immunopathol 135, 188-198. Goodridge, H.S., Wolf, A.J., Underhill, D.M., 2009, Beta-glucan recognition by the innate immune system. Immunol Rev 230, 38-50. Gordon, S., 2002, Pattern recognition receptors: doubling up for the innate immune response. Cell 111, 927-930. Huang, H., Ostroff, G.R., Lee, C.K., Specht, C.A., Levitz, S.M., 2010, Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded beta-Glucan Particles. MBio 1. Inumaru, S., Kokuho, T., Denham, S., Denyer, M.S., Momotani, E., Kitamura, S., Corteyn, A., Brookes, S., Parkhouse, R.M., Takamatsu, H., 1998, Expression of biologically active recombinant porcine GM-CSF by baculovirus gene expression system. Immunol Cell Biol 76, 195-201. Jones, G.W., Rutter, J.M., 1972, Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by Escherichia coli in piglets. Infect Immun 6, 918-927. Jones, K.S., 2008, Biomaterials as vaccine adjuvants. Biotechnol Prog 24, 807-814. Lunney, J.K., Walker, K., Goldman, T., Aasted, B., Bianchi, A., Binns, R., Licence, S., Bischof, R., Brandon, M., Blecha, F., et al., 1994, Overview of the First International Workshop to Define Swine Leukocyte Cluster of Differentiation (CD) Antigens. Vet Immunol Immunopathol 43, 193-206. Moon, H.W., Bunn, T.O., 1993, Vaccines for preventing enterotoxigenic Escherichia coli infections in farm animals. Vaccine 11, 213-200. O'Hagan, D.T., Singh, M., 2003, Microparticles as vaccine adjuvants and delivery systems. Expert Rev Vaccines 2, 269-283. O'Hagan, D.T., Singh, M., Ulmer, J.B., 2006, Microparticle-based technologies for vaccines. Methods 40, 10-19.


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Perez, V.L., Van Parijs, L., Biuckians, A., Zheng, X.X., Strom, T.B., Abbas, A.K., 1997, Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6, 411-417. Pescovitz, M.D., Lunney, J.K., Sachs, D.H., 1984, Preparation and characterization of monoclonal antibodies reactive with porcine PBL. J Immunol 133, 368-375. Reid, D.M., Montoya, M., Taylor, P.R., Borrow, P., Gordon, S., Brown, G.D., Wong, S.Y., 2004, Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions. J Leukoc Biol 76, 86-94. Rescigno, M., Winzler, C., Delia, D., Mutini, C., Lutz, M., Ricciardi-Castagnoli, P., 1997, Dendritic cell maturation is required for initiation of the immune response. J Leukoc Biol 61, 415-421. Rice, P.J., Kelley, J.L., Kogan, G., Ensley, H.E., Kalbfleisch, J.H., Browder, I.W., Williams, D.L., 2002, Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-D-glucans. J Leukoc Biol 72, 140-146. Robinson, M.J., Sancho, D., Slack, E.C., LeibundGut-Landmann, S., Reis e Sousa, C., 2006, Myeloid C-type lectins in innate immunity. Nat Immunol 7, 1258-1265. Ross, G.D., Thompson, R.A., Walport, M.J., Springer, T.A., Watson, J.V., Ward, R.H., Lida, J., Newman, S.L., Harrison, R.A., Lachmann, P.J., 1985, Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1. Blood 66, 882-890. Rutter, J.M., Jones, G.W., 1973, Protection against enteric disease caused by Escherichia coli--a model for vaccination with a virulence determinant? Nature 242, 531-532. Saito, H., Yoshioka, Y., Uehara, N., Aketagawa, J., Tanaka, S., Shibata, Y., 1991, Relationship between conformation and biological response for (1----3)-beta-D-glucans in the activation of coagulation factor G from limulus amebocyte lysate and host-mediated antitumor activity. Demonstration of single-helix conformation as a stimulant. Carbohydr Res 217, 181-190. Schwartz, R.H., 2003, T cell anergy. Annu Rev Immunol 21, 305-334. Sonck, E., Devriendt, B., Goddeeris, B., Cox, E., submitted, Beta-glucans induce the maturation of porcine dendritic cells. submitted to Clin Vaccine Immunol. Sonck, E., Stuyven, E., Goddeeris, B., Cox, E., 2009, Identification of the porcine C-type lectin dectin-1. Vet Immunol Immunopathol 130, 131-134. Soto, E.R., Ostroff, G.R., 2008, Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery. Bioconjug Chem 19, 840-848. Taylor, P.R., Brown, G.D., Reid, D.M., Willment, J.A., Martinez-Pomares, L., Gordon, S., Wong, S.Y., 2002, The beta-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J Immunol 169, 3876-3882. Taylor, P.R., Tsoni, S.V., Willment, J.A., Dennehy, K.M., Rosas, M., Findon, H., Haynes, K., Steele, C., Botto, M., Gordon, S., Brown, G.D., 2007, Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol 8, 31-38. Trinchieri, G., Sher, A., 2007, Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 7, 179-190.


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Van den Broeck, W., Cox, E., Goddeeris, B.M., 1999, Induction of immune responses in pigs following oral administration of purified F4 fimbriae. Vaccine 17, 2020-2029. Van der Stede, Y., Cox, E., Verdonck, F., Vancaeneghem, S., Goddeeris, B.M., 2003, Reduced faecal excretion of F4+-E coli by the intramuscular immunisation of suckling piglets by the addition of 1alpha,25-dihydroxyvitamin D3 or CpG-oligodeoxynucleotides. Vaccine 21, 1023-1032. Zimmerman, J.W., Lindermuth, J., Fish, P.A., Palace, G.P., Stevenson, T.T., DeMong, D.E., 1998, A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J Biol Chem 273, 22014-22020.

Chapter 9 : General discussion and future perspectives

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9.1. Introduction During the last decades, considerable amounts of antibiotics were used in animal production, both as

therapeutic and as growth-promoting agent (Casewell et al., 2003). Because of the risk for human public

health resulting from the transmission of antibiotic resistance genes from animal to human microbiota,

the use of growth promoting antibiotics in food animals was completely banned in Europe in 2006.

Alternatives for antimicrobial growth performing promoters have been used, often based on research

funded by manufacturers or distributors of alternative products, and those with the best effect on

performance are currently used as feed additive (Huyghebaert et al., 2011; Millet and Maertens, 2011).

However, also independent studies are required to assess the effectiveness of these alternatives in

enhancing performance or health status (Millet and Maertens, 2011). -glucans have the capacity to act

as immunostimulants and are thus of particular interest as alternative nutritional additives. This thesis is

an independent study of the immunomodulating effect of -glucans in pigs. The aims of this thesis were

to determine which effect different commercial -glucan preparations have on porcine leukocytes and

dendritic cells (DCs), the primary cells of the immune system. As -glucan immunomodulatory effects are

attributed to different -glucan receptors, with dectin-1 as the most important one, the first aim of this

thesis was the identification of this receptor in the pig.

9.2. The expression pattern of porcine dectin-1

Before the discovery of dectin-1, the immunostimulating effects of -glucans were mainly attributed to

activation via the complement receptor 3 (CR3) (Thornton et al., 1996; Vetvicka et al., 1996). Also

lactosylceramide and scavenger receptors were mentioned as mediators of -glucan induced immune

effects (Iwabuchi and Nagaoka, 2002; Kniep and Skubitz, 1998; Vereschagin et al., 1998; Wakshull et al.,

1999). Since Gordon and Brown identified dectin-1 as a -glucan receptor (Brown and Gordon, 2001),

most of the studies focused on and attributed immune recognition and immunomodulatory effects of -

glucans to this receptor. Therefore, dectin-1 is generally accepted as the most important receptor for -

glucans. Since this receptor was not yet identified in the pig, the first goal of this thesis was its

identification in the pig (Chapter 4). Two major and one minor isoform of porcine dectin-1 were

detected. The first two receptors are similar to the two major isoforms dectin-1A and -B in human,

mouse and bovine (Heinsbroek et al., 2006; Willcocks et al., 2006; Willment et al., 2001). The full length

dectin-1A consists of a cytoplasmic region on the N-terminal end, a transmembrane region, a stalk region

and a single C-terminal carbohydrate recognition domain (CRD), while dectin-1B is the stalkless variant of


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dectin-1A. Both isoforms have been described in human and mouse to be responsible for the -glucan

induced immune responses (Heinsbroek et al., 2006; Willment et al., 2001), suggesting that only those

isoforms possess the structures necessary for-glucan binding and –signaling. The third dectin-1 isoform

in the pig has yet to be identified, but has the same length as dectin-1E in humans. Human dectin-1E

resembles dectin-1A, but has a deletion in the transmembrane and stalk region.

With exception of the esophagus and the liver, we demonstrated mRNA expression of the three dectin-1

isoforms throughout the whole gastrointestinal tract. We didn’t determine which cell types are

responsible for dectin-1 expression in the digestive tract, but in blood, neutrophils and monocytes

primarily expressed dectin-1A and to a lesser degree, dectin-1B. In humans, both major isoforms of

dectin-1 appeared to be differentially expressed in granulocytes (expressing dectin-1A and dectin-1B)

and monocytes/macrophages (expressing only dectin-1B) (Willment et al., 2001). However, when surface

expression of dectin-1A was explored in humans in more detail using a dectin-1A specific monoclonal

antibody (mAb), this isoform could also be detected in monocytes (Willment et al., 2005). This illustrates

that one should be careful to extend expression at the mRNA level to expression of the protein at the cell

surface and that also monoclonal antibodies against both porcine dectin-1 isoforms should be developed

to have certainty about expression of both isoforms by porcine cells.

9.3. different immunostimulating effects on leukocytes

In pigs, numerous in vivo studies have been performed to determine whether -glucans could be used as

alternative for antibiotics in response to an inflammatory challenge, although with varying success. The

discrepancies between the different studies were at least partly due to the use of varying doses of non-

characterized -glucans. Moreover, most of these -glucan preparations weren’t tested in standardized

assays to analyze their immunomodulatory capacities, making it difficult to speculate about cell-specific

interactions with -glucans and the contribution of individual cell populations to the -glucan induced

immune response. In vitro assays can be developed for that purpose. At the same time, such tests could

offer information about the characteristics a -glucan should have to induce an immune response. Our

results demonstrated that the nature and the dose of a -glucan, as well as the cell type it targets,

determine the induced immune response and therefore should be taken into account when evaluating

the effectiveness of a -glucan preparation (Chapter 5). We tested the dose-dependent effect of -

glucans differing in solubility (soluble or particulate), molecular weight and structure (branched or

unbranched). With exception of laminarin and scleroglucan, all the -glucans used in our study were

particulate. We confirmed in pigs what was demonstrated before in human and mice, namely that


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particulate -glucans with a high molecular weight have a higher stimulatory effect than soluble -

glucans (Ishibashi et al., 2002). Laminarin is a soluble -glucan with a low molecular weight. On the other

hand, scleroglucan has a very high molecular weight and forms a gel when dissolved at high

concentrations. Gel forming -glucans have a triple helical structure in solution, a highly ordered solution

conformation, while laminarin has a single helical solution conformation (Mueller et al., 2000). Although

it has been demonstrated that -glucans with a triple helical configuration as well as a high molecular

weight have been regarded as powerful immunomodulators (Falch et al., 2000; Maeda et al., 1988;

Tabata et al., 1981), this is not the case for scleroglucan in the pig. Gas chromatographic analysis has

indicated that scleroglucan also contains portions of (1,4)-linked glucose residues (Adams et al., 2008a).

As dectin-1, and possibly other -glucan receptors as well, do not interact with mixed-linkage polymers,

characterized by alternating regions of (1,3),(1,4) and/or 1,6) linkages (Adams et al., 2008a), this

demonstrates that also in the pig, a sufficient long (1,3) backbone is important for immune recognition.

Another possible explanation is that the mixed-linkage backbone structure results in structures that are

not optimal for -glucan receptor interaction.

The particulate -glucans in our study were either long, unbranched -glucans (curdlan and the glucan

from Euglena gracilis) or long -(1,3)-glucans with -(1,6) branches (Macrogard, the glucan from

Saccharomyces cerevisiae and zymosan). From our results, it seems that both branched and unbranched

-glucans have stimulatory capacities in pigs, although this stimulatory activity is dose- and particularly

for the unbranched -glucans, cell type dependent. Curdlan, for example, didn’t induce reactive oxygen

species (ROS) in both monocytes and neutrophils, while it did strongly stimulate lymphocyte

proliferation particularly at higher concentrations. The glucan from Euglena gracilis, on the other hand,

gave a completely different profile. While this -glucan induced a strong ROS production in both

monocytes and neutrophils with increasing concentration, it stimulated lymphocyte proliferation rather

moderately. Structural related information of the tested -glucan preparations was provided by

literature and the product suppliers (Table 5.1a), while information on the amount and their

monosaccharides composition (glucosamine, mannose and glucose) was given by HPAEC-analysis (Table

5.1b). Both unbranched -glucans didn’t differ in structure and were solely composed of glucose-units.

However, we didn’t have information about the presence of non-sugar components nor about the length

of the polymer, although we can suspect that the glucan from Euglena gracilis has a much longer

backbone than curdlan given the higher molecular weight of the former one. It is possible that the high

molecular weight of the glucan from Euglena gracilis, favors recognition by monocytes and neutrophils


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and that more -glucan receptors are bound with increasing concentration. As curdlan has a lower

molecular weight, this would mean that the smaller curdlan has a less favorable structure for binding

one or several -glucan receptors, explaining the very low amount of ROS production by neutrophils and

monocytes. However, curdlan does stimulate proliferation of PBMC especially at increasing

concentrations (200 and 800 µg/ml), making the previous hypothesis, at least for curdlan, not very

plausible. To our opinion, the cell-dependent stimulatory capacity of curdlan and differences in

stimulatory capacity between both unbranched -glucans can’t be attributed to differences in molecular

weight and must have a non-structural related cause. As curdlan is extracted from the cell wall of the

gram-negative bacteria Alcaligenis faecalis, curdlan can also contain other components than -(1,3)-

linked oligosaccharides, having an effect on the immunostimulatory activity of curdlan. In the cell wall of

bacteria, -glucans are situated in the peptidoglycan layer, a layer which itself is surrounded in gram-

negative bacteria by a lipopolysaccharide (LPS) containing outer membrane (Kamio and Nikaido, 1976).

We and others found quite high amounts of LPS in curdlan and it is possible that also other non-glucose

units could have contaminated curdlan during the extraction process. It is not known whether LPS

contamination could result in lower neutrophil or monocyte induced ROS production. However, in

comparison with most of the other -glucans, PBMC stimulated with curdlan displayed high IL-10

concentrations and low TNF- concentrations, a typical anti-inflammatory cytokine profile of PBMC

stimulated with -glucans together with LPS (Engstad et al., 2002; Hetland et al., 2000; Vereschagin et

al., 1998). Also preliminary experiments in our lab demonstrated that co-incubation of PBMC with -

glucans and LPS resulted in a decreased TNF- and an increased IL-10 expression. As it has been

demonstrated that a similar cytokine profile is correlated with reduced ROS production of both

monocytes and neutrophils, it is possible that the lack of ROS production after curdlan stimulation is due

to the LPS contamination (Gougerot-Podicalo et al., 1996; Reglier-Poupet et al., 1998).

While the stimulatory capacity of unbranched -glucans increases with increasing concentrations, the

stimulatory capacity of (1,6)-branched glucans has a concentration optimum. Although all branched -

glucans were extracted from Saccharomyces cerevisiae, this concentration optimum differed between

zymosan and the other two -glucans, Macrogard and the glucan from Saccharomyces cerevisiae, and

could be attributed to non--glucan components present in the zymosan-preparation. Indeed, zymosan

is rather a crude extract of (1,3)-(1,6)-glucans with non-uniform branches and backbone units and also

the presence of mannan and proteins. To our knowledge, both Macrogard and the glucan from

Saccharomyces cerevisiae are much purer extracts and contain no proteins, while the percentage of


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mannose-units in both preparations is smaller than the mannose content in zymosan. Also the presence

of non (1,3)- or (1,6)-linked glucose-units can explain the lower stimulatory capacity of zymosan. As

previously mentioned, -glucan receptors and more specifically dectin-1 don’t recognize non--linked

carbohydrate polymers (Adams et al., 2008a).

It has been shown that the presence of (1,6)-branches increases the affinity of -glucans for their

receptor, and thereby their biological activity, compared to unbranched -glucans (Adams et al., 2008a;

Mueller et al., 2000). However, in our study, these findings are only true for low concentrations of both

Macrogard and the glucan from Saccharomyces cerevisiae. As a lot of cell debris was seen after 72h of

incubation and as confirmed by the MTT assay, this is probably due to their cytotoxic activity at higher

concentrations. Both Macrogard as well as the glucan from Saccharomyces cerevisiae have (1,6)-linked

branches, but Macrogard has a frequency of one (1,6)-branch over 10 to 20 unbranched (1,3)-linked

gluco-oligosaccharides while the branching frequency of the glucan from Saccharomyces cerevisiae is

lower (1:30). As both -glucans have a similar stimulatory capability, we suspect that both branching

frequencies fall within optimal range for the recognition by -glucan receptors. To determine the optimal

distance between two successive (1,6)-linked branches along the (1,3)-linked backbone, it would be

interesting to remove (1,6)-side chain branches from, for instance, Macrogard and to determine the

immunostimulatory effect on leukocytes. Another intriguing question is whether the length of the side

chains is important for recognition by -glucan receptors. However, as the number of glucose-units in

the (1,6)-branches of the glucan from Saccharomyces cerevisiae were unknown, we couldn’t speculate

about the importance of the length of the side chains for its stimulatory capacity.

9.4. Does dectin-1 play a role in the -glucan induced activity of leukocytes? In Chapter 1, we demonstrated mRNA expression of dectin-1 in neutrophils and monocytes, the cell

types we used to investigate potential immunostimulatory effects of different -glucan preparations. As

dectin-1 has been shown to be the most important mediator of -glucan induced immune effects in

mouse and human, we wanted to investigate whether this was also the case in pigs. Laminarin has been

shown to be one of the most effective inhibitors of dectin-1 (Herre et al., 2004a). Therefore, we repeated

the experiments described in Chapter 5 in the presence of laminarin. To investigate the role of dectin-1

in the -glucan induced ROS production of both neutrophils and monocytes, we pre-incubated these

cells with an excess of laminarin (1 mg/ml) during 1 hour, after which the -glucans were added to the

cells and ROS production was measured (Chapter 6). As ROS production didn’t change in both cell types

in the presence of laminarin, dectin-1 doesn’t seem to be involved in -glucan induced ROS production


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or in any case is not the most important route for ROS production. Although binding of laminarin to

porcine dectin-1, and thus inhibition of this receptor, was never demonstrated, it was also recently

described in humans that dectin-1 doesn’t have a role in either the uptake of zymosan or the activation

of ROS production by this -glucan preparation in neutrophils as well as monocytes (van Bruggen et al.,

2009). However, in another study, they did report dectin-1 as the major-glucan receptor involved in

phagocytosis of zymosan and in ROS production by human neutrophils, although their results

demonstrated that inhibition of dectin-1 could not completely reduce neutrophil function (Kennedy et

al., 2007). As we demonstrated that ROS production induced by other -glucans also didn’t change in the

presence of laminarin, this effect is not unique to zymosan and as a result, it cannot be attributed to

impurities in the preparation. It’s not clear why dectin-1 is not the major -glucan receptor involved in

ROS production. At the same time, these results demonstrate that most likely one or more other -

glucan receptors are involved in the activation of the immune cells. In humans, Van Bruggen et al. (2009)

found an indispensable role for CR3 in phagocytosis as well as ROS production of unopsonized zymosan,

as blocking antibodies against this receptor (anti-CD11b or anti-CD18) did completely inhibit the

response of neutrophils towards zymosan (van Bruggen et al., 2009). It is possible that also in the pig, -

glucan induced ROS production is mediated by CR3. It has been demonstrated that CR3 is also expressed

on porcine neutrophils and monocytes (Summerfield and McCullough, 2009), although further

experiments have to point out whether the receptor is involved in -glucan induced ROS production in

both cell types. The minor or even lacking role of dectin-1 in -glucan induced neutrophil and monocyte

ROS production in both human and pig, is in contrast to what has been published for murine cells

(Brown, 2006; Brown et al., 2003). A possible explanation for these discrepancies are the differences in

the carbohydrate recognition domain of dectin-1 between species, which can possibly lead to differences

in -glucanrecognition. At the amino acid level, the extracellular domain (stalk + carbohydrate

recognition domain (CRD)) of dectin-1A in the pig showed a similarity of only 58% with murine dectin-1,

while the identity between human and mice was only 61%. The similarity between pig and human was

73%. However, several studies claimed -glucanrecognition by recombinant human dectin-1. Moreover,

although -glucan binding to dectin-1 was never demonstrated in the pig, Trp221 and His223 are highly

conserved in all the dectin-1 homologues in the different species and as both amino-acids have been

shown to play a crucial role in -glucan binding, we can assume that -glucans will also bind dectin-1 in

the pig. Although these preliminary results indicate that -glucan induced ROS production in the pig is

independent from dectin-1, these results need to be confirmed by blocking monoclonal antibodies


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against porcine dectin-1. To determine whether porcine dectin-1 really can bind -glucans, a cell line

should be transfected with porcine dectin-1 and -glucan binding should be evaluated using a

BIAcoreT100TM surface plasmon resonance instrument (Uppsala, Sweden).

Besides ROS production, also the effect of dectin-1 inhibition on PBMC proliferation and cytokine

production was investigated. Therefore, laminarin and -glucans were added at the same time to the

cells. It was surprising that the -glucan induced PBMC proliferation was downregulated in the presence

of laminarin. However, with this type of test, one cannot elucidate whether laminarin acts directly on T-

cells or whether laminarin suppresses the monocyte-mediated T-cell response. Hereto, monocytes as

well as T-cell subsets should be positively selected and the experiment should be repeated with those

purified cell populations. If dectin-1 has an inhibiting effect on T-cell function, several hypotheses are

possible. First of all, in humans, dectin-1 expression was identified on a subset of CD4+ T-cells

(CD3lowCD4low) as well as B-cells and it was suggested that dectin-1 on these cells may also be involved in

mediating interactions with other lymphocytes through recognition of an unidentified endogenous

ligand (Willment et al., 2005). However, we couldn’t demonstrate dectin-1 expression on T-cells, making

this hypothesis very unlikely with respect to the pig. Second, in human and mice, it has been

demonstrated that dectin-1, besides his role in -glucanrecognition, can also bind to T-cells (Ariizumi et

al., 2000; Hermanz-Falcon et al., 2001; Willment et al., 2001), leading to T-cell activation and

proliferation (Ariizumi et al., 2000; Grunebach et al., 2002). Moreover, Grunebach (2002) demonstrated

that dectin-1 expressing HeLa cells induced an upregulation of several activation markers in CD8+ T

lymphocytes, suggesting that dectin-1 may act as a costimulatory molecule for T-cell activation. In our

study, it is possible that laminarin blocked dectin-1 and thereby its costimulatory capacity resulting in a

reduced proliferation. However, -glucan induced cytokine production (IL-1, IL-6, TNF- and IL-10) was

not affected by laminarin, making the previous hypothesis questionable. Moreover, Willment et al.

(2001) demonstrated that the T- cell binding capacity of dectin-1 isn’t blocked by laminarin, suggesting

that the T-cell binding site on dectin-1 is different from the -glucan binding site. Although these findings

stem from experiments with human dectin-1 transduced cell lines and the results were never confirmed

in primary cells, it demonstrates that laminarin may possibly act on dectin-1 independent regulation of T-

cellproliferation and cytokine production.

9.5. The effect of different -glucan preparations on DC maturation

Based on their immunomodulating potential in humans and mice, -glucans are very often included as

dietary component in pig feed (Kogan and Kocher, 2007). Currently, it’s not completely understood how


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-glucan uptake should occur in the gastrointestinal tract. We didn’t see dectin-1 mRNA expression in

porcine enterocytes. In human intestinal epithelial cell lines (INT407 and Caco-2 NFB), although mRNA

expression was detected, dectin-1 was not expressed on the surface of both cell lines (Volman et al.,

2010a). Also in mice, dectin-1 expression was not detected on enterocytes, although it was

demonstrated that a subpopulation of intestinal epithelial cells, probably M-cells, could actively

internalize -glucans and transport them to the underlying mucosal tissue where they can be

phagocytosed by macrophages and DCs (Rice et al., 2005). In a more recent study, however, a new

model of -glucan uptake has been proposed, where dietary -glucans first activate intestinal

leukocytes, such as DCs, which in turn increase the cellular activation of enterocytes (Volman et al.,

2010b). NFB activation in small intestinal enterocytes allows the secretion of cytokines which are

favorable in alerting the immune system, resulting in an increased resistance against pathogens (Volman

et al., 2010b). In this model, intestinal APCs are directly activated by the -glucans as they can sense and

take up -glucans in the lumen by extending their dendrites through the intestinal epithelium (Rescigno

et al., 2001). However, until today, there is no clear answer whether first enterocytes or intestinal

dendritic cells are activated after -glucan consumption. Either way, dendritic cells will come into

contact with -glucans either indirect after passage through the intestinal epithelial layer or direct by

extending their dendrites through the epithelial layer. Upon phagocytosis, the immature DCs will

undergo phenotypic and functional changes and at the end of this transition process, immature antigen-

capturing DCs will develop into mature APCs. Although intestinal DCs are presumed to be the in vivo

target for -glucan activity, they are not easily accessible for in vitro studies. In contrast, monocyte-

derived DCs (MoDCs) are a well-established DC-model in swine research (Bimczok et al., 2007; Pilon et

al., 2009).

As laminarin failed to induce DC maturation, the conclusions we made in Chapter 5 about the effect of

solubility on immunostimulation were also confirmed in MoDCs (Chapter 7). All particulate commercial

-glucan preparations affect one or more characteristics typical for maturation, and this is partly caused

by their particulate nature. However, Macrogard, zymosan and curdlan affect maturation more than the

glucan from Euglena gracilis. As mentioned previously, Macrogard and zymosan are both -(1,3)-(1,6)-

glucans extracted from Saccharomyces cerevisiae. Although both preparations contain small amounts of

mannose-units, possibly having an additional stimulating effect through the mannose receptor

(McCullough and Summerfield, 2009; Taylor et al., 2002), Macrogard contains proportionally more

glucans than zymosan (see Table 5.1b). As Macrogard stimulated DCs are more activated than after


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stimulation with zymosan, these results demonstrate that -(1,3)-(1,6)-glucans and their recognition by

one or several -glucan receptors, are the determining factor for the DC stimulatory capacity in both

preparations. Which -glucan receptors mediate the DC maturation inducing effects of -glucans in the

pig, could not yet be determined. However, dectin-1 mRNA was highly expressed in immature MoDCs.

Furthermore, in contrast to monocytes and neutrophils, it has been demonstrated in human DCs that

dectin-1 does play an important role in -glucan-recognition (Gringhuis et al., 2009; van Bruggen et al.,

2009). Although it’s too soon to speculate and dectin-1 blocking tests need to be done, it is possible that

also in the pig dectin-1 plays a more important role in the recognition and the response of DCs to -

glucans than of neutrophils and monocytes. As a matter of fact, it has been shown that IL-4 and GM-CSF,

cytokines which are required for the in vitro differentiation of MoDCs, induce both upregulation of

dectin-1 in mice macrophages and that this affects the ability of these cells to respond to curdlan and

zymosan with respect to TNF-IL-6 and IL-12p40 production (Rosas et al., 2008; Willment et al., 2003).

It is possible that in our study, both cytokines contribute to the high expression levels of dectin-1 mRNA

and that they have an additive effect on the -glucan induced cytokine production.

The presence of -(1,6) branches contributes possibly to the stimulatory activity of -glucans as

Macrogard stimulated MoDCs are much more mature than MoDCs stimulated with the glucan from

Euglena gracilis. Curdlan however, also an unbranched -(1,3)-glucan, has much higher

immunostimulating capacities than the glucan from Euglena gracilis and as previously described for the

leukocytes, this again is probably caused by the high LPS contamination. We had only LPS-contaminated

curdlan to our disposal, but Ferwerda et al. (2008) conducted their experiments with LPS-free curdlan. In

human monocyte-derived macrophages, they investigated the synergistic effect of curdlan and LPS on

TNF-and IL-10 production (Ferwerda et al., 2008). TNF- as well as IL-10 production was increased and

they could inhibit the synergistic effect on TNF-, but not IL-10 production, by a neutralizing dectin-1

antibody. These results demonstrate that in human macrophages, at least for TNF- production, curdlan

exert his effects through dectin-1 and that curdlan in combination with LPS induces a synergistic

signaling between dectin-1 and TLR-4 (Ferwerda et al., 2008). Besides TNF- and IL-10, it was shown that

IL-6 and IL-23 production were also mediated through dectin-1 (LeibundGut-Landmann et al., 2007;

Rogers et al., 2005), while the production of all these cytokines was enhanced after co-ligation of dectin-

1 and TLRs (Dennehy et al., 2009). In our results, we demonstrated that although most of the -glucans

could induce IL-1, TNF-, IL-6, IL-12/IL-23p40 and IL-10 production, the highest production of all these

cytokines was found after stimulation with the LPS contaminated curdlan, suggesting that also in the pig


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co-ligation of multiple pattern recognition receptors (PRRs) can occur and that possibly also dectin-1 is

involved. Combination of -glucans together with LPS and other PRR ligands gives a more accurate

picture of the real situation in the intestine, and should be considered when evaluating the effectiveness

of a -glucan preparation as dietary component.

It is well known that the spectrum of cytokines produced by DCs modulate the polarization of the T-cell

response. Although we didn’t evaluate qualitative T-cell responses yet, the DC cytokine profile allows us

to make some assumptions about the outcome of an immune response. We found high secretion levels

of TNF- and IL-8 in the supernatant of all -glucan stimulated MoDCs. TNF- and IL-8 serve both as

chemo-attractants for innate and adaptive immune cells such as neutrophils and T-cells, respectively,

underlining the role of DCs in linking innate and adaptive immunity (Gesser et al., 1996). It has been

demonstrated in humans that a culture of curdlan stimulated DCs with naive CD4+ T-cells sustained their

differentiation towards IL-17 as well as IFN- producing Th cells (Agrawal et al., 2010). Expression of IFN-

has been shown to occur by both Th17 and Th1 cells, while expression of IL-17 occurs exclusively by Th17

cells (Agrawal et al., 2010; Dhodapkar et al., 2008). In the pig, we demonstrated that stimulation of

MoDCs with curdlan gave rise to high levels of the pro-inflammatory cytokines IL-6, IL-12/IL-23p40 and

IL-1, cytokines which can, together with TNF-, direct the T-cells towards a Th1 or Th17 response

(Veldhoen et al., 2006). The p40 subunit is shared by both IL-12 (p35/p40 heterodimer) and IL-23

(p19/p40 heterodimer). While IL-12 is responsible for differentiation into Th1 responses, IL-23 has been

closely associated with Th17 responses (Agrawal et al., 2010; Maloy and Kullberg, 2008). Although we

measured high amounts of IL-12/IL-23p40 after stimulation of MoDCs with curdlan, the p40 subunit

alone gives few information about either IL-12 or IL-23 bioactivity, and as no commercial ELISA kits are

available to measure porcine IL-12p35 or IL-23p19, it is difficult to speculate about its contribution to

possible Th1 or Th17 responses. However, unlike ours, the experiments in human DCS were performed

with LPS free curdlan, resulting in low amounts of IL-10 in the supernatant of DC/T-cell co-cultures

(Agrawal et al., 2010). It is possible that the LPS contamination in the curdlan preparation could favor

regulatory T cell (Treg) responses, characterized by high IL-10 levels. To clarify these issues, cytokine

determination in the supernatant of curdlan stimulated DC/T-cell co-cultures is needed. Moreover, it

would be interesting to compare curdlan preparations with and without LPS to determine the purely -

glucan-effect and the synergistic effect of -glucans and LPS on the stimulation of DCs and the outcome

of the immune response.


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9.6. Are -glucans potential candidates for DC targeting?

Although the precise mechanism of -glucan uptake by the gastrointestinal system still has to be

elucidated, -glucan recognition is mediated through different receptors present on several immune

cells, including DCs. Different -glucan preparations have a demonstrated DC-activating ability in the pig

resulting in DC maturation, antigen presentation and costimulatory molecule expression. -glucans form

the major cell wall component of yeast and in our experiments, both Saccharomyces cerevisiae extracts

(Macrogard and zymosan) have DC immunostimulating properties. This suggests that -glucan rich yeast

shells have the potential to be exploited as an oral antigen delivery system. As vertebrates lack the

appropriate glucanases, an additional advantage is that -glucans aren’t digested in the gastrointestinal

tract, so the encapsulated antigen is protected against degradation.

Oral vaccination with F4 fimbriae results in an intestinal immune response that is protective against an

F4+ ETEC infection (Van den Broeck et al., 1999). This is a remarkable observation since soluble antigens

mainly induce tolerance (McGhee et al., 1992; Strobel and Mowat, 1998). However, to get protection

against postweaning F4+ ETEC infections, vaccination needs to be done during the suckling period,

making effective vaccination difficult due to the presence of F4-specific maternal antibodies in the milk

(Rutter and Jones, 1973). Another drawback of this immunization strategy is that the required dose to

get protection is rather high. These obstacles underline that a more efficient F4 formulation is needed

and in this respect -glucan rich yeast shells may offer a valuable alternative.

Previous experiments with ova revealed that at an equal ratio of 1 mg ova and 1 mg of yeast shells, a

quasi 100% encapsulation was obtained. For encapsulation of F4, we started with the same amounts of

antigen versus yeast shell and obtained a encapsulation efficiency of only 10-30%. To increase loading

efficiency of the F4 antigen into the yeast shells, a number of strategies could be followed. First, by using

polyarginine as complexing agent instead of polyethylenimine (PEI), the cationic charge density within

the yeast shells could be increased (Jain et al., 2005), leading to an enhanced electrostatic interaction

between the F4 antigen and the yeast shells. Second, by increasing the pH of the F4 solution during

encapsulation, the overall charge of the F4 fimbriae becomes more negative. As the overall charge of the

pH independent polyarginine remains unaffected, this would result in a higher electrostatic interaction

between the F4 and the polyarginine modified yeast shells.

Although encapsulation efficiency still has to be optimized, we demonstrated that F4-loaded yeast shells

are more efficiently internalized and induce increased maturation compared to the soluble F4 fimbriae

(Chapter 8). As previously mentioned for the MoDCs, this can be partly explained by their particulate


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nature in combination with the recognition of -glucans by different -glucan receptors. However, as

expected, within the 24h incubation time, no difference was seen in maturation capacity between the

hollow and the F4-loaded yeast shells and this is probably due to the fact that F4 isn’t released yet from

the yeast shells. Indeed, it has been described that DC internalized particles are retained for up to 24h

within DC macropinosomes, vesicles characterized by a mildly acid pH (Rescigno et al., 1997). As a result,

antigen degradation is prevented, allowing retention of the antigens during migration of DCs from the

site of inflammation/infection to the afferent lymph node (Rescigno et al., 1997). To investigate whether

yeast shells are efficient systems for the delivery of the F4 antigen to DCs, presentation of the antigen to

CD4+ and CD8+ T-cells needs to be evaluated by means of an F4-specific T-cell proliferation assay.

However, preliminary experiments showed no additive effect on the antigen presenting capacity of DCs

stimulated with F4 loaded yeast shells in comparison with hollow yeast shells. Probably, these results

could be explained by the still too low encapsulation of F4 in the yeast shell.

9.7. Main conclusions and future perspectives

-glucans have varying immunomodulating effects on porcine leukocytes and DCs. Variability between

the different -glucan preparations can be attributed to a combination of physicochemical properties

like solubility, molecular weight and the presence of -(1,6)-branches. So far, no role for dectin-1 has

been established in the pig, although mRNA expression was both found in leukocytes and DCs.

The issues discussed above have highlighted important questions that should be addressed. First of all,

although dectin-1 mRNA is expressed in different blood cell types, it’s still unclear whether dectin-1 is

expressed on the cell surface of pigs. Furthermore, although laminarin has been described in mice to be

the most effective dectin-1 inhibitor (Herre et al., 2004b), inhibition studies with laminarin on porcine

leukocytes didn’t reduce -glucan induced stimulation. To investigate more directly, if any, the role of

dectin-1 in the pig in -glucan stimulation, monoclonal antibodies against porcine dectin-1 should be

developed. By developing mAbs directed against the -glucan binding site of dectin-1, these mAbs could

be used to inhibit -glucan binding. At the same time, they let us determine whether laminarin is an

effective inhibitor of porcine dectin-1. Differences in stimulatory capacity between soluble and

particulate -glucans could maybe be explained by differences in their specific interaction with dectin-1

or other -glucan receptors. Therefore, porcine dectin-1 should be recombinantly expressed to study its

affinity for structurally different particulate as well as soluble -glucans. Interactions between

recombinant porcine dectin-1 and -glucans could be studied by surface plasmon resonance (SPR). This


153

technique will also allow us to determine whether laminarin is an effective dectin-1 inhibitor. Moreover,

Another possible explanation of the inability of soluble -glucans to activate leukocytes and MoDCs,

could be due to their inability to cross-link membrane -glucan receptors and could be investigated by

immobilization of soluble -glucans to a polystyrene matrix. Furthermore, it has been shown in mice,

that dectin-1 can also function as an actin-dependent phagocytic receptor for zymosan (Hernanz-Falcon

et al., 2009). With this respect, it would be interesting to investigate whether alterations in porcine

dectin-1 expression occur after interaction of the receptor with -glucans and if these alterations vary

between soluble and particulate -glucans. The inability of laminarin to inhibit -glucan induced

stimulation in porcine leukocytes could also be mediated by -glucan receptors other than dectin-1. By

studying the interaction between -glucans and membrane fractions of monocytes/neutrophils, followed

by identification of the binding receptors through structural proteomics, other -glucanreceptors could

be identified.

Another intriguing question remains which influence contaminating components have on -glucan

induced stimulation. In our experiments, there was high variation in stimulatory capacities between

curdlan and the other unbranched -(1,3)-glucan, Euglena gracilis and these variations were probably

caused by the high LPS contamination in curdlan. Therefore, it would be interesting to compare the

stimulatory capacity of the LPS-contaminated curdlan with the LPS-free curdlan (Ferwerda et al., 2008)

or to block LPS in curdlan with polymyxin. -glucan preparations could also be contaminated with

mannan, having possibly an additional stimulating effect through the mannose-receptor. Mannose-units

could be removed by mannosidase. Another strategy to avoid contaminants are synthetic -glucans.

Synthetic -glucans give an additional advantage that the composition of the structure is completely

known and controlled, improving the studying of the interaction between -glucans and their receptors

(Adams et al., 2008b).

In conclusion, -glucans have been shown to induce immunomodulatory effects in vitro.

Immunomodulation however, is strongly dependent of the nature and the dose of the -glucan and

should be taken into account when evaluating the effectiveness of a -glucan preparation. Moreover,

one should be aware that other non -glucan components can have an impact on the -glucan induced

immune response. Although -glucans can’t replace antibiotics to control post-weaning problems, they

should make part of an integral health management system and they may be beneficial in periods of

stress or immunosuppression.


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Summary

Because of economics, the pig industry has largely evolved to early weaning the last decades. As a result,

weaning disorders have become one of the most important problems in pig husbandry leading to high

rates of mortality and significant economical losses. In order to cope this problem, a wide range of

antibiotics has been included in pig feed either at sub-therapeutic level, acting as a growth promoter, or

at therapeutic levels, to treat disease. Unfortunately, because of their long term and extensive use,

antibiotics have resulted in a strong selection of antibiotic resistant bacteria. As genes encoding for this

resistance can also be transmitted to human microbiota causing a serious risk for human health, the use

of in-feed antibiotics has completely been banned in the EU. Alternative approaches to control post-

weaning problems should enhance the natural defense mechanisms of animals. In contrast to antibiotics,

such immunomodulators aim to increase the immune system of an individual and to evoke a rapidly

response to invading pathogens.

-(1,3)-(1,6)-glucans have the capacity to act as immunomodulants and are thus of particular interest as

alternative nutritional additives. However, as their use in pig industry is frequently based on research

funded by the manufacturer of those products, the risk increases that the credibility of -glucan

preparations isn’t always ensured. This thesis is an independent study with the aim to determine the

effect of different commercial -glucan preparations on porcine leukocytes and dendritic cells (DCs),

important cells of the immune system. In addition, as -glucans are frequently assumed to induce direct

receptor-mediated effects, another aim of this thesis was to determine whether dectin-1, described as

the most important -glucan receptor in human and mouse, is also present in the pig and which role this

receptor plays in the -glucan induced immune effects.

Chapter 1 reviews the literature on the different -glucan receptors, but focuses largely on dectin-1 as

being the most important one.

In Chapter 2, the current knowledge on -glucans and their immunomodulating effects is reviewed.

Binding to various -glucan receptors activates the innate immune response, resulting in the secretion of

a range of cytokines that can in turn enhance the adaptive immune response against pathogens.

However, their effect on the immune system is strongly dependent on different physicochemical and

structure-related parameters as well as on the applied dose. In a final section, possible mechanisms of -

Summary

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glucan uptake after oral administration are described. Although orally -glucans are already intensively

used in humans and mammals, it is still unknown how orally applied -glucans modulate systemic and

intestinal immune parameters.

In Chapter 3, different strategies regarding the use of -glucans in oral immunization are discussed.

Besides their application as adjuvants in classic vaccine-formulations,-glucans have also potential

applications as antigen-carrier.

Chapters 4 to 8 present the research on -glucans performed in this thesis.

Following questions were addressed:

1. Is dectin-1, which has been described as the most important -glucanreceptor in different species,

also expressed in pigs? In which tissues and on which blood cell types is this receptor expressed?

2. Which effects have different -glucanpreparations on porcine leukocytes and the maturation of

porcine dendritic cells? Are these effects dose-dependent? Which parameters are important for the

immunomodulating effects of -glucans? Could the observed effects in leukocytes be attributed to

dectin-1?

3. Could the immunomodulating character of -glucans be exploited for enhanced antigen delivery?

Although recognition of -glucans is thought to be mediated by a combination of cell surface receptors,

only dectin-1 has been clearly demonstrated to play a role in the cellular responses induced by these

carbohydrates. Therefore, a first aim in this thesis was to identify and characterize dectin-1 in the pig

(Chapter 4). Two major dectin-1 isoforms were identified, differing from each other by the presence of a

stalk region separating the carbohydrate recognition domain from the transmembrane region. Also a

minor isoform of dectin-1 was identified which showed a deletion in the transmembrane and stalk

region. mRNA transcripts were detected in the stomach, the small intestine, colon and rectum, the

spleen, the mesenteric lymph nodes and the lungs. The transcript was not expressed in the liver, kidneys,

the bladder, the heart, the brains and the skin.

There is a considerable body of published research detailing the biological effects of -glucans but

unfortunately, the literature is inconsistent and often contradictory. This has mainly been due to the use

of varying doses of non-characterized -glucans. The heterogeneity in experiments makes it difficult to

draw conclusions about the immunomodulating potency of -glucans and this can partly explain the

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discrepancies on their efficacy. Therefore, a following aim was to test the dose-response of seven

different -glucan preparations on leukocytes of the pig (Chapter 5). We looked at lymphocyte

proliferation, reactive oxygen species (ROS) production by monocytes and neutrophils, and cytokine

production by peripheral blood monomorphonuclear cells (PBMC). Soluble -glucans (laminarin,

scleroglucan) showed almost no activity in contrast to particulate -glucans. From the particulate -

glucans, both branched (Macrogard, the glucan from Saccharomyces cerevisiae and zymosan) and

unbranched -glucans (curdlan and the glucan from Euglena gracilis) have stimulatory capacities in pigs,

although this stimulatory activity is dose- and particularly for the unbranched -glucans, cell type-

dependent. Furthermore, all -glucans, with exception of laminarin, induced the production of the pro-

inflammatory cytokines IL-1 and TNF- as well as the production of the anti-inflammatory IL-10,

emphasizing the immunomodulatory capacity of -glucans. However, IL-10 production was particularly

produced after stimulation with curdlan, while this -glucan induced lower TNF- levels and no ROS

production. As this -glucan was the only shown to be contaminated with high amounts of

lipopolysaccharide (LPS), this suggests that also the influence of non--glucancomponents are important

in the observed immune effects.

In Chapter 6, we wanted to know whether the effects on porcine leukocytes, described in Chapter 5,

could be attributed to dectin-1, assumed to be the most important -glucan receptor in humans and

mice. Therefore, we blocked -glucan binding to dectin-1 with laminarin, a specific dectin-1 inhibitor,

and the effect of this blocking was investigated on the -glucan induced ROS production by monocytes

and neutrophils, as well as on proliferation and cytokine production by PBMCs. We suspect that other

receptors than dectin-1 are involved in -glucan-recognition by, and -signaling in monocytes as well as

neutrophils, as laminarin couldn’t block ROS production by both cell types. The role of dectin-1 in -

glucan induced lymphocyte proliferation is less clear, as laminarin could only inhibit proliferation induced

by all -glucans, after stimulation with 200 µg/ml. For the other concentrations, the inhibiting effect of

laminarin was -glucan dependent. Although the specific role of dectin-1 in pigs still has to be elucidated,

our results demonstrate that dectin-1 is probably not the major -glucan receptor in pigs.

In a next chapter (Chapter 7), we investigated the effect of different -glucan preparations on the

phenotypical and functional maturation of monocyte-derived dendritic cells (MoDCs). The results of this

study demonstrate that there is a large variation in terms of DC maturation inducing properties between

Summary

162

the different -glucan preparations. The soluble laminarin failed to induce DC maturation, while all

particulate -glucan preparations affected one or more parameters characteristic for maturation, most

likely at least partly caused by their particulate nature. However, Macrogard, zymosan and curdlan

affected maturation more than after stimulation with the glucan from Euglena gracilis, suggesting that

the presence of -(1,6) branches possibly contributes to the stimulatory activity of Macrogard and

zymosan, while the LPS contamination in curdlan probably induces an additional effect on the

maturation. This additional effect is particularly reflected in the cytokine secretion profile, as significant

higher expression levels of IL-1, IL-6, IL-10 and IL-12/IL23-p40 were detected in the supernatant of DCs

stimulated with curdlan. As the cytokine profile of DCs influences the outcome of the ensuing immune

response and thus may prove valuable in intestinal immunity, this chapter demonstrates that a careful

choice is necessary when -glucans are used as dietary supplement.

Given the maturation inducing properties of -glucans, we wanted to investigate in Chapter 8 whether

-glucan rich yeast-shells could function as a potential carrier to target antigen to DCs. As model antigen,

we used F4-fimbriae, purified from F4 enterotoxigenic Escherichia coli (ETEC). The results in this study

demonstrated that F4-loaded yeast shells are efficiently taken up by DCs and induce their maturation.

Although still preliminary, these results demonstrate that yeast shells are a promising candidate for the

delivery of F4 but that further studies have to be undertaken to determine whether yeast shells can

actually be used in DC targeting.

The final chapter (Chapter 9), presents the general discussion and conclusions. The research part of this

thesis clearly demonstrates that -glucans have varying immunomodulating effects on porcine

leukocytes and DCs. Variability between the different -glucan preparations can be attributed to a

combination of physicochemical properties like solubility, molecular weight and the presence of -(1,6)-

branches. So far, no role for dectin-1 has been established yet in the pig, although mRNA expression was

both found in leukocytes and DCs. Furthermore, we demonstrated that LPS contamination can have

additional effects on the stimulatory activity of -glucans. As LPS is abundantly present in the intestine,

their effect on -glucan-induced immunomodulation has to be taken into account when evaluating the

effectiveness of dietary -glucan preparations.

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Samenvatting

Omwille van economische factoren worden biggen op jonge leeftijd gespeend. Infecties die gepaard

gaan met het spenen vormen één van de grootste problemen in de varkenshouderij en hebben hoge

sterftecijfers en significante economische verliezen tot gevolg. Om deze problemen te bestrijden,

werden in het verleden verschillende soorten antibiotica toegevoegd aan het varkensvoeder, enerzijds

subtherapeutisch, om de groei te bevorderen, en anderzijds op therapeutisch niveau, om ziektes te

behandelen. Overvloedig en langdurig gebruik van antibiotica werken echter het ontstaan van antibiotica

resistente bacteriën in de hand. Doordat genen die coderen voor deze resistentie kunnen doorgegeven

worden naar de humane darmflora en dus een gevaar vormen voor de volksgezondheid, werd het

gebruik van antibiotica als groeibevorderaar in veevoeder volledig verboden in de EU vanaf 2006.

Alternatieven voor antibiotica zijn gericht op het verhogen van het natuurlijke afweersysteem van het

dier. Zulke immunomodulatoren hebben als doel het immuunsysteem van een individu te versterken

waardoor het in staat is om snel te reageren op pathogene indringers.

Door hun immunomodulerend vermogen zijn -(1,3)-(1,6)-glucanen uitzonderlijk interessant als

alternatief voeder additief. Onderzoek naar -glucaanpreparaten wordt echter dikwijls gefinancierd door

de producenten van de preparaten waardoor de geloofwaardigheid soms in het gedrang komt. Deze

thesis is een onafhankelijke studie die het effect van verschillende commerciële -glucaanpreparaten

nagaat op leukocyten en dendritische cellen (DCs) van het varken. Omdat aangenomen wordt dat

verschillende receptoren verantwoordelijk zijn voor de -glucaan geïnduceerde effecten, wilden we

bovendien nagaan of dectine-1, welke in mens en muis beschreven wordt als belangrijkste -

glucaanreceptor, ook aanwezig is in het varken, en welke rol deze receptor speelt in-glucaan

geïnduceerde immuuneffecten.

Hoofdstuk 1 geeft een overzicht van de verschillende -glucaanreceptoren waarbij de nadruk gelegd

wordt op dectine-1 als meest belangrijke receptor.

Hoofdstuk 2 beschrijft de huidige kennis over -glucanen en hun immunomodulerende effecten. Binding

van -glucanen aan verschillende -glucaanreceptoren activeert de aangeboren immuunrespons, wat

resulteert in de productie van cytokines, die op hun beurt de adaptieve immuunrespons tegen

pathogenen versterken. Hun effect op het immuunsysteem is echter sterk afhankelijk van verschillende

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164

fysicochemische en structuur-gerelateerde parameters, alsmede van de toegediende dosis. In een

laatste deel worden de mogelijke mechanismen van -glucaan opname na orale toediening beschreven.

Hoewel -glucaanpreparaten reeds beschikbaar zijn in mens en dier voor orale toediening, is het nog

onbekend hoe deze systemische en intestinale parameters van het immuunsysteem moduleren.

In Hoofdstuk 3 worden verschillende strategieën besproken met betrekking tot het gebruik van -

glucanen bij orale immunisatie. Naast de toepassing als adjuvant in de klassieke vaccin formuleringen,

hebben -glucanen ook potentiële toepassingen als antigeen-carrier.

Hoofdstukken 4 tot 8 omvatten de experimenten uitgevoerd in dit proefschrift.

Volgende vragen warden gesteld:

1. Wordt dectin-1, welke beschreven wordt as de belangrijkste -glucaanreceptor in verschillende

species, ook geëxpresseerd in varkens? In welke weefsels en op welke bloed celtypes wordt deze

receptor tot expressive gebracht?

2. Welke effecten hebben verschillende -glucaan preparaten op porcine leukocyten en de maturatie

van porcine dendritische cellen? Zijn deze effecten dosis-afhankelijk? Welke parameters zijn

belangrijk voor de immunomodulerende effecten van -glucanen? Kunnen de waargenomen

effecten in leukocyten toegeschreven worden aan dectine-1?

3. Kan het immunomodulerend karakter van -glucanen aangewend worden om toelevering van

antigenen te verhogen?

Hoewel -glucanen waarschijnlijk door een combinatie van cel oppervlakte receptoren herkend worden,

is enkel de rol van dectine-1 bewezen in de cellulaire respons die tot stand komt na stimulatie met deze

carbohydraten. Daarom was een eerste doelstelling in dit proefschrift de identificatie en karakterisatie

van dectine-1 in het varken (Hoofdstuk 4). De 2 grootste dectine-1 isovormen werden geïdentificeerd.

Deze verschillen van elkaar door de aanwezigheid van een steelregio die het carbohydraat

herkenningsdomein van de transmembraan regio scheidt. Ook een derde, kleinere dectine-1 isovorm

werd geïdentificeerd, welke een deletie vertoonde in de transmembraan en steelregio. mRNA

transcripten werden gedetecteerd in de maag, de dunne darm, het colon en rectum, de milt, de

mesenteriale lymfeknopen en de longen. Het transcript werd niet tot expressie gebracht in de lever, de

nieren, de blaas, het hart, de hersenen en de huid.

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165

Er bestaan veel wetenschappelijke publicaties die de biologische effecten van -glucanen beschrijven,

maar helaas is de literatuur dikwijls tegenstrijdig. Dit heeft vooral te maken met het gebruik van

uiteenlopende dosissen van niet-gekarakteriseerde -glucanen. Door de experimentele heterogeniteit is

het moeilijk om conclusies te trekken omtrent het immunomodulerend vermogen van -glucanen en dit

kan deels de verschillen in effectiviteit verklaren. Een volgende doelstelling van dit doctoraat was

bijgevolg het nagaan van de dosisrespons van zeven verschillende -glucaanpreparaten op leukocyten

van het varken (Hoofdstuk 5). We evalueerden het effect op lymfocytenproliferatie,

zuurstofradicaalproductie door monocyten en neutrofielen, en cytokineproductie door

monomorfonucleaire cellen uit het bloed. Oplosbare -glucanen (laminarine, scleroglucaan) vertoonden

bijna geen activiteit in tegenstelling tot partikelvormige -glucanen. Zowel vertakte (Macrogard, het

glucaan van Saccharomyces cerevisiae en zymosan) als onvertakte -glucanen (curdlan en het glucaan

van Euglena gracilis) stimuleren leukocyten van het varken, hoewel deze stimulerende activiteit dosis-,

en vooral voor de onvertakte -glucanen, cel type afhankelijk is. Verder gaven alle -glucanen,

uitgezonderd laminarin, aanleiding tot productie van zowel pro-inflammatoire (IL-1 en TNF-) als anti-

inflammatoire cytokines (IL-10), waardoor het immunomodulerend vermogen van -glucanen benadrukt

wordt. IL-10 werd echter vooral geproduceerd na stimulatie met curdlan, terwijl dit -glucaan minder

TNF- en geen zuurstofradicaalproductie tot gevolg had. Dit -glucaan was echter als enige

gecontamineerd met grote hoeveelheden lipopolysaccharide (LPS) zodat ook de invloed van niet -

glucaancomponenten waarschijnlijk een belangrijke rol spelen in de waargenomen immuun effecten.

In Hoofdstuk 6 wilden we nagaan of de immuunrespons door varkensleukocyten, zoals beschreven in

Hoofdstuk 5, kon verklaard worden door de aanwezigheid van dectine-1, welke beschouwd wordt als de

belangrijkste -glucaanreceptor in mens en muis. Hiertoe blokkeerden we -glucaanbinding aan dectine-

1 met laminarin, een specifieke dectine-1 inhibitor, waarna het effect van deze blokkering werd

nagegaan op de -glucaan geïnduceerde zuurstofradicaalproductie door monocyten en neutrofielen.

Daarnaast werd ook de invloed op proliferatie en cytokine productie door monomorfonucleaire cellen uit

het bloed nagegaan. Omdat laminarin zuurstofradicaalproductie door zowel monocyten als neutrofielen

niet kon blokkeren, vermoeden we dat andere receptoren dan dectine-1 betrokken zijn in -

glucaanherkenning en –signalisatie in beide celtypes. De rol van dectine-1 in -glucaan geïnduceerde

lymfocytenproliferatie is minder duidelijk. Hoewel laminarin, in tegenstelling tot

zuurstofradicaalproductie, proliferatie kon inhiberen, was dit effect voor alle -glucanen enkel duidelijk

Samenvatting

166

na stimulatie met 200 µg/ml. Voor de andere concentraties, was het inhiberend effect van laminarine, -

glucaan afhankelijk. Hoewel de specifieke rol van dectine-1 in varkens nog opgehelderd moet worden,

tonen onze resultaten aan dat dectine-1 waarschijnlijk niet de belangrijkste -glucaanreceptor is in

varkens.

In een volgend hoofdstuk (Hoofdstuk 7), onderzochten we het effect van verschillende -

glucaanpreparaten op de fenotypische en functionele maturatie van monocyt-afgeleide dendritische

cellen (MoDC). De resultaten van deze studie tonen aan dat het DC maturerend vermogen sterk

afhankelijk is van het -glucaanpreparaat. Het oplosbare laminarin gaf geen aanleiding tot mature DCs,

terwijl alle partikelvormige -glucaanpreparaten wel een invloed hadden op 1 of meerdere maturatie

parameters. Dit effect is hoogstwaarschijnlijk en minstens gedeeltelijk te wijten aan hun partikelvormige

aard. Macrogard, zymosan en curdlan hebben echter een grotere invloed op DC maturatie dan het

glucaan van Euglena gracilis. Dit doet vermoeden dat de aanwezigheid van -(1,6)-vertakkingen bijdraagt

aan het stimulatorisch vermogen van Macrogard en zymosan, terwijl de LPS contaminatie in curdlan

waarschijnlijk een bijkomend effect heeft op maturatie. Dit bijkomend effect wordt vooral weerspiegeld

in het cytokine secretie profiel aangezien beduidend hogere gehaltes van IL-1, IL-6, IL-10 en IL-12/IL-

23p40 werden gedetecteerd in het supernatans van DCs gestimuleerd met curdlan. Omdat het cytokine

profiel van DCs de uitkomst van de immuunrespons kan bepalen en dus belangrijk is voor intestinale

immuniteit, toont dit hoofdstuk aan dat een voorzichtige keuze van -glucanen noodzakelijk is bij hun

gebruik als veevoeder supplement.

Aangezien -glucanen DC-maturatie kunnen bevorderen, wilden we onderzoeken in Hoofdstuk 8 of -

glucaanrijke gist-omhulsels kunnen fungeren als een potentiële carrier om antigen selectief te richten

naar DCs. Als modelantigen maakten we daarvoor gebruik van F4-fimbriae, opgezuiverd van F4

enterotoxigene Escherichia coli (ETEC). De resultaten in deze studie tonen aan dat F4-geladen gist

omhulsels efficiënt worden opgenomen door DCs en hun maturatie kunnen veroorzaken. Hoewel hieruit

blijkt dat gistomhulsels veelbelovende kandidaten zijn voor de aflevering van F4, moeten verdere studies

uitwijzen of gist omhulsels wel degelijk kunnen gebruikt worden in DC targeting.

Het laatste hoofdstuk (Hoofdstuk 9) bevat de algemene discussie en de conclusies. Het experimenteel

gedeelte van deze thesis toont duidelijk aan dat -glucanen een wisselend immunodulerend effect

hebben op varkens leukocyten en DCs. De variabiliteit tussen de verschillende -glucaanpreparaten kan

Samenvatting

167

toegeschreven worden aan een combinatie van fysicochemische eigenschappen zoals oplosbaarheid,

moleculair gewicht en de aanwezigheid van -(1,6)-vertakkingen. Tot dusver kon geen rol voor dectine-1

vastgesteld worden, hoewel mRNA expressie werd gevonden in zowel leukocyten als DCs. Verder

toonden we aan dat LPS contaminatie bijkomende effecten kan hebben op het stimulatorisch vermogen

van -glucanen. Omdat LPS overvloedig aanwezig is in de darm, is het belangrijk om ook hiermee

rekening te houden wanneer de effectiviteit van -glucaanpreparaten wordt beoordeeld.

Curriculum Vitae

168

Curriculum Vitae

Eva Sonck werd geboren op 2 mei 1981 te Gent. Haar secundaire opleiding volgde ze in de richting

Wetenschappen Wiskunde op het Sint Bavo Humaniora te Gent. In 1999 begon ze aan de studies

industrieel ingenieur biochemie aan het KaHo Sint Lieven te Gent waar ze afstudeerde in 2003. Daarna

studeerde ze verder aan de Universiteit Gent en behaalde in 2005 het diploma van Bio-ingenieur in de

Cel- en gen biotechnologie met onderscheiding. Na een eerste werkervaring als

kwaltiteitsverantwoordelijke in de chocolaterie “Neuhaus”, koos ze toch voor het onderzoek. In mei

2006 startte ze aan het laboratorium voor Immunologie aan de Faculteit Diergeneeskunde. In januari

2007 ontving ze een doctoraatsbeurs van het instituut voor de aanmoediging van Innovatie door

wetenschap en technologie in Vlaanderen (IWT- Vlaanderen), waar ze zich gedurende 4 jaar verdiepte in

het effect van (1,3)-glucanen op leukocyten en dendritische cellen. Dit onderzoek werd uitgevoerd

onder leiding van Prof. Dr. E. Cox en Prof. Dr. B. Goddeeris en leidde tot dit proefschrift. Eva Sonck is

auteur en mede-auteur van verscheidene wetenschappelijke publicaties in internationale tijdschriften.

Publications

169

Publications

Sonck E., Devriendt B., Goddeeris B., Cox E., 2011. Beta-glucans induce the maturation of porcine

dendritic cells. Clin. Vacine Immunol., submitted.

Stuyven E., Sonck E., Deprez P., Cox E., Goddeeris B. Effect of beta-glucans in horses. In preparation.

Sonck E., Stuyven E., Goddeeris B., Cox E., 2010. The effect of beta-glucans on porcine leukocytes. Vet.

Immunol. and Immunopathol. 135 : 199–207.

Sonck E., Stuyven E., Goddeeris B., Cox E., 2009. Identification of the Porcine C-type lectin dectin-1. Vet.

Immunol. and Immunopathol. 130: 131–134.

Melkebeek V., Sonck E., Verdonck F., Goddeeris B., Cox E., 2007. Optimized FaeG expression and a

thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG

DNA vaccine in pigs. Clin. Vacine Immunol. 14(1): 28-35.

Abstracts and posters

170

Abstracts and posters

Sonck E., Stuyven E., Goddeeris B., Cox E.. The effect of beta-glucans on porcine leukocytes. 3rd EVIW

2009 (European Veterinary Immunology Workshop), Berlin, Germany, 10-13/09/2009 (abstract and

poster presentation).

Sonck E., Stuyven E., Goddeeris B., Cox E.. Identification of the porcine C-type lectin Dectin-1. Toll 2008,

Lisbon, Portugal, 24-28/09/2008 (abstract and poster presentation).

Stuyven E., Sonck E., Goddeeris B., Cox E.. Effect of beta-glucans in horses. Toll 2008, Lisbon, Portugal,

24-28/09/2008 (abstract and poster presentation).

Melkebeek V., Sonck E., Goddeeris B., Cox E. . Optimization of the FaeG expression and the use of

adjuvantia enhance priming by an FaeG DNA vaccine in pigs. DNA vaccines 2007, Malaga, Spain, 23-

25/05/2007 (abstract).

Cox E., Melkebeek V., Sonck E., Goddeeris B. Optimalisation of an FaeG DNA vaccine to prime an F4-

specific immune response in piglets. DNA vaccines 2004, Monte Carlo, Monaco, 17-19/11/2004 (abstract

and poster).

Dankwoord

171

Dankwoord

De laatste pagina’s van mijn doctoraat… misschien wel de belangrijkste pagina’s. Een uitgelezen kans om

een speciaal woordje van dank te richten tot iedereen die me geholpen heeft om dit werk tot een goed

einde te brengen. Want een doctoraat is niet zomaar een thesis… het is het resultaat van 5 jaar werken,

zoeken, zuchten, soms vloeken, en soms juichen…

Allereerst wil ik mijn beide promotoren, Prof. Dr. Eric Cox en Prof. Dr. Bruno Goddeeris, bedanken dat ze

me 5 jaar, in alle vrijheid, onderzoek hebben laten uitvoeren. Professor Cox, na mijn master eindwerk

twijfelde ik eraan of onderzoek wel mijn ding is, maar na het voltooien van dit doctoraat, ben ik er toch

van overtuigd! Bedankt voor het verbeteren van de teksten en voor de steun en het vertrouwen als ik

het eventjes niet meer zag zitten. En u heeft gelijk: je doctoraat is wat je er zelf van maakt !

Bert, je liet me kennis maken met de wonderlijke wereld van de DCs. Bedankt voor het helpen bij het

uitwerken van mijn experimenten, voor je kritische opmerkingen en voor het ontzettend grondig

verbeteren van mijn doctoraat. Ik sta nog altijd versteld van alle kennis die je in huis hebt. Ik wens je veel

succes toe in je verdere wetenschappelijke carrière!

Ook de overige leden van de examen- en leescommissie wil ik bedanken voor hun constructieve

opmerkingen en suggesties.

Frank, bedankt voor alle hulp bij het schrijven van mijn IWT-voorstel. Ik herinner me nog levendig de

week voor de deadline… zonder jou was ik er waarschijnlijk niet in geslaagd om een goed projectvoorstel

af te leveren!

Herman, bedankt voor je hulp bij de live-cell imager!

En dan… alle bureaugenootjes. Gedurende die 5 jaar heb ik bij heel wat toffe mensen samen gezeten.

Kristien, jij was de eerste met wie ik niet enkel een bureau, maar zelf een heel labo mocht delen. In al die

jaren was je niet enkel een toffe collega, maar ben je ook een goeie vriendin geworden. Bedankt voor

Dankwoord

172

alle serieuze en minder serieuze babbels… en natuurlijk… je ‘vettige’ lach. Het kon niet anders, of ik

begon mee te lachen als jij weer eens je lachsalvo afvuurde…

Edith,… mijn -glucaanmaatje, hoewel we meermaals op die moleculen gevloekt hebben, zijn we er toch

beiden in geslaagd! Ik ben ontzettend content dat jij mijn partner in ‘crime’ was. Bedankt voor alle hulp

en steun… en natuurlijk onze vriendschap die hieruit ontstaan is!

Tine, onze zotte doos… je bent niet op je mondje gevallen, maar dat maakt het juist zo leuk om met jou

in de bureau te zitten! Ook met jou ben ik goed bevriend geraakt en ik hoop dat die vriendschap nog lang

mag duren.

Kris, ook al zit je niet meer bij ons, je hebt toch lang deel uitgemaakt van ‘den toffen bureau’. Bedankt

voor alle toffe babbels en natuurlijk je zin voor orde en netheid! Gelukkig dat jij er was om onze bureau

regelmatig te herorganiseren. Ik wens je veel succes met je gezin en je nieuwe job!

Philippe,… op jouw hulp kon ik ook altijd rekenen. Was het niet met de varkens, dan was het met mijn

computer! Chapeau dat je het uithoudt tussen al die vrouwen. Eén groot voordeel: je kan waarschijnlijk

al een goed mondje meespreken over zwangerschappen en bevallingen!

Kim, je bent onze nieuwe aanwinst in de bureau… en een goede aanwinst! Veel succes met je onderzoek,

en… GO voor die DCs! Toch zalig hoe je altijd uit de lucht komt vallen!

Trouwens, bureaugenootjes, jullie zijn GOE BEZIG!!!

Rudy, merci voor alle hulp bij de varkens,.. we waren een goed team, en ’t was dikwijls door jou dat het

bloed trekken zo goed lukte!

Denise, we konden goed opschieten met elkaar! Bedankt voor die 1001 dingen!

Griet, Simon en Maaike, bedankt voor de hulp bij al het labowerk. Maaike, ik denk dat je een

supergespierde rechterduim hebt! Ook merci voor die laatste PCR’kes!

Pieter, veel hulp heb ik je nog niet gevraagd, maar dat zal binnenkort wel veranderen .

Annelies, Michaela en Vesna, jullie kon ik dikwijls als hulplijn gebruiken als ik met iets in de knoop zat.

Bedankt voor al jullie kritische opmerkingen! Annelies en Michaela, het VEOS verhaal is nu grotendeels

voor mij, maar jullie hebben al schitterend werk geleverd!

Delfien, veel succes met je onderzoek en met je schaapjes! En hopelijk heb je in de toekomst wat meer

geluk met je stagiairs

Dankwoord

173

Gosia, succes met het assistentschap en je doctoraat, het is een zware taak!

Johanna, Maryam, Ut, Bakr and Pedro… our foreign people in the lab, it shouldn’t be easy to work with

all these crazy, Belgian people . Good luck with your Phd, and Maryam, hopefully you can really start

your life in Canada!

En dan de ‘Hermanologie’… Nina, Maria, Céline, Thary en Korneel, jullie zijn een toffe bende! Door die

dunne muurtjes hebben we heel wat interessante weetjes opgevangen . Nina, toch grappig hoe klein

de wereld soms is… veel succes met je doctoraat en met Guillaume! Korneel, zoals beloofd, krijg jij ook

een speciale vermelding… merci voor de hulp bij de gellekes! Schitterend hoe jij gebeten bent door die

onderzoeksmicrobe…. Have fun at night!

Sarah, Ann, Mieke en Gert, merci voor alle administratieve beslommeringen. Ann, bedankt dat je alles zo

vlug regelde als ik holderdebolder weer iets kwam bestellen. Sarah, bedankt voor de laatste regelingen

van mijn doctoraatsverdediging!

Dirk, dingen die je bij een computer nog nooit meegemaakt had, kwam je bij mij tegen… merci!

Klaartje, merci voor alle interesse die je toonde in mijn doctoraat. Elke keer wou je wel een update van

de stand van zaken . Je bent er eentje uit de duizend…

Ook mijn andere vrienden wil ik graag bedanken voor al die toffe BBQ’tjes, feestjes, uitstapjes,… hoewel

ik de laatste maanden heel wat minder plezier gemaakt heb, begin ik nu aan een inhaalmanoeuvre!

De meest dierbare mensen houd ik tot het einde… Moeke en vake, merci voor het warme nest dat jullie

ons gegeven hebben. Elke keer kon ik bij jullie terecht en elke keer stonden jullie me bij met woord en

daad! Het is door jullie dat ik hier vandaag sta. Na mijn 1e jaar industrieel ingenieur hadden jullie dit

waarschijnlijk nooit gedacht… .

Broertje, we hebben altijd een goeie band gehad… Je moet meermaals gedacht hebben: augurken,

varkens, pralines… waar gaat ‘diene kleinen aap’ nu weer aan beginnen . Ook Salie, de kleine Kobe en

Lotte wil ik bedanken, het waren altijd toffe familiemomenten!

Annemie en Marc, merci dat ik ook altijd op jullie kon rekenen!

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174

En dan last but not least… sjoeke,.. je hebt afgezien met mij de laatste tijd! Wonderbaarlijk hoe jij me

telkens weer verzekerde dat alles goed kwam… Ik ben jaloers op je eeuwige optimisme! Merci ook voor

de lay-out…, hoe perfect ik het ook wilde, je hebt niet 1x geklaagd… Ik kan me geen betere man

voorstellen…

Amélieke, mijn hartediefje… ik ben ontzettend blij dat ik jouw mama mag zijn!

Documents

Immunomodulation of porcine leukocytes and dendritic cells ...and as most important one, dectin-1 (Battle et al., 1998; Brown and Gordon, 2001). Dectin-1 is a microbial sensor or pattern